1996 — 2000 |
Perozo, Eduardo A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Architecture of Potassium Channels @ University of Virginia Charlottesville
DESCRIPTION (Adapted from the investigator's application): The long term goal of this project is to elucidate the relationship between the molecular structure and the functional behavior of voltage-dependent potassium channels. This will be accomplished by applying a new approach using electron paramagnetic resonance (EPR) spectroscopy to the study of the structure and dynamics of Shaker K+ Channels. K+ channels play a key role in a variety of cellular processes. They are directly involved in the generation and control of electrical potentials in nerve and muscle cells, the regulation of cell volume, the response of endocrine cells to their environment (insulin secretion) and the control of the heart rate. Consequently, efforts to understand K+ channel structure and function relate directly to human health and disease. High-resolution structural information in membrane proteins has been very difficult to obtain, mainly because of the limited sources of pure protein and the problems involved with their crystallization. The strategy we will pursue overcomes these difficulties. It is based on the replacement of native residues by cysteine using site-directed mutagenesis methods and subsequent labeling with a sulfhydryl-specific nitroxide spin label. Information on the structure and dynamics of the spin-labeled mutants can be obtained using EPR spectroscopy, which only requires a few micrograms of pure protein. To our advantage, the functional properties of each mutant can be studied by expression in Xenopus oocytes with conventional electrophysiological techniques. With these methods the investigators will 1) Obtain experimental information about the folding and topology of the Shaker K+ channel. 2) Obtain a set of intra-molecule and inter-molecule distances in order to constraint three-dimensional models of the channel. 3) Study voltage- dependent conformational changes correlated with channel activation and inactivation. Studies on the structure of K+ channels are both timely and relevant. Information on the structure of the super-family of voltage-dependent channels has been basically limited to their amino acid sequences and scattered information about their topology. New developments in EPR spectroscopy together with the availability of purified material in over-expressing systems will provide valuable information about the structural dynamics of these membrane proteins.
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0.922 |
1998 — 2018 |
Perozo, Eduardo A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
High Resolution Structural Dynamics of K Channels @ University of Virginia Charlottesville
DESCRIPTION (provided by applicant): In response to membrane potential depolarization, voltage-dependent potassium channels undergo a series of conformational changes from a non-conducting state (closed) to an activated (conducting) state. K+ channel function has been associated with such basic cellular functions as the regulation of electrical activity, signal transduction and osmotic balance. In higher organisms, K+ channel dysfunction may lead to uncontrolled periods of electrical hyperexcitability, like epileptic episodes, myotonia and cardiac arrhythmia. Consequently, efforts to understand K+ channel structure, function and dynamics relate directly to human health and disease.The continuing long-term goal of this project is to further understand the molecular mechanisms of gating in voltage-dependent channels, by focusing on the analysis of K+ channel gating. This understanding encompasses two interrelated processes, the protein rearrangements that lead to channel opening and the energy transduction events that convert external stimuli (voltage, ligand binding, etc) into protein motion. Specifically we will address the following key questions: What are the molecular entities determining channel activity? How energy (in the form of specific ligand binding or transmembrane electric field) is transduced into protein motion? How different parts of the channel interact to define open channel activity? We plan to study these problems by combining site-directed spin labeling/EPR spectroscopy and electrophysiological methods with classical biochemical and molecular biological procedures. This particular strategy has proven very successful over the previous application period, leading to direct structural determinations of KcsA, the Streptomyces K+ channel, the types of molecular movements underlying its gating mechanism and structural information on the role of the selectivity filter in gating. We intend to continue these structure-function studies while extending them using new experimental approaches like Double Quantum Resonance FT-EPR. In addition, we will focus our attention on a newly characterized six-transmembrane segment (6TM) channel from Methanococcus janschii (which we have named KchV-O). This channel contains a bona fide S4 segment and is ideally suited to study the structure and dynamics of the voltage-sensing domain and voltage-dependent gating mechanisms. This proposal should open new experimental avenues that will contribute to our understanding of biologically important events such as electrical signaling, signal transduction and ion channel gating.
|
0.958 |
2001 — 2008 |
Perozo, Eduardo A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structural Dynamics of Mechanosensitive Channels
DESCRIPTION (provided by applicant): Mechanosensitive (MS) channels are oligomeric membrane proteins that respond to changes in bilayer tension by catalyzing the transfer of ions and other solutes across the membrane, fulfilling a major role in the response of living organisms to mechanical stimuli. These channels are considered to function as mechano-electrical switches in such diverse physiological processes as touch, hearing, proprioception, turgor control in plant cells and osmoregulation in bacteria. The overall, long-term goal of this project is to understand the molecular mechanism of gating in prokaryotic mechanosensitive channels. Although the recent determination of the MscL and MscS crystal structures has dramatically improved our knowledge of this class of molecules, a number of mechanistic questions remain to be solved. This is particularly true for the molecular events underlying channel gating. In this respect, we plan to experimentally address several fundamental questions: What regions of the channel form the gate(s) and how do they move to produce gating? What is the physical basis of the energy transduction steps, starting with transbilayer tension and culminating in protein motion? Where in the molecule does mechanical transduction occur? What are the structures of the key functional states? The fact that MscL and MscS can be activated by pressure gradients both in native membrane and after reconstitution in pure lipid systems indicates that these channels are gated directly by tension transmitted through the bilayer. Therefore, establishing the physical principles underlying the energy transduction steps in these proteins will require studying the role of protein-lipid interactions in this process. The approach we plan to pursue combines reporter-group spectroscopic techniques (spin labeling/EPR, Fluorescence) and electrophysiological methods with classical biochemical and molecular biological procedures. Functional studies will be targeted to understand the physical basis of energy transduction in mechanosensitive channels. Information on the topology, secondary, and tertiary structure of MscS and MscL will be obtained from EPR analysis of spin labeled mutants. The data will be interpreted to generate backbone models of the different stages of the gating pathway in each type of channel. This proposal opens up a new experimental avenue that will contribute to the understanding of biologically important events such as ion channel gating, nociception and signal transduction.
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0.958 |
2006 — 2009 |
Perozo, Eduardo A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
High Resolution Sructural Dynamics of K Channels
DESCRIPTION (provided by applicant): In response to membrane potential depolarization, voltage-dependent potassium channels undergo a series of conformational changes from a non-conducting state (closed) to an activated (conducting) state. K+ channel function has been associated with such basic cellular functions as the regulation of electrical activity, signal transduction and osmotic balance. In higher organisms, K+ channel dysfunction may lead to uncontrolled periods of electrical hyperexcitability, like epileptic episodes, myotonia and cardiac arrhythmia. Consequently, efforts to understand K+ channel structure function and dynamics relate directly to human health and disease. The continuing long-term goal of this project is to further understand the molecular mechanisms of gating in voltage-dependent channels, by focusing on the analysis of K+ channel gating in prokaryotic systems. Specifically we will address the following key questions: What are the molecular entities determining channel activity? How energy (in the form of specific ligand binding or transmembrane electric field) is transduced into protein motion? How different parts of the channel interact to define open channel activity? We plan to study these problems by combining spectroscopic techniques (EPR NMR and Fluorescence), X-ray crystallography and electrophysiological methods. This particular strategy has proven very successful over the previous grant period, leading to i) Direct structural determinations of the types of molecular movements underlying KcsA gating mechanism and structural information on the role of the selectivity filter in gating;and 2) A structural analysis of the native conformation of KvAP, the voltage-dependent channel from Aeropyrum pernix. We intend to continue these structure-function studies while extending them using new experimental approaches. In addition, we will focus our attention on a newly characterized hyperpolarization-activated six-transmembrane segment (6TM) channel from Methanococcus janschii (MVP). This proposal should open new experimental avenues that will contribute to our understanding of biologically important events such as electrical signaling, signal transduction and ion channel gating.
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0.958 |
2008 |
Perozo, Eduardo A |
R24Activity Code Description: Undocumented code - click on the grant title for more information. |
Midwest Center For Membrane Protein Structural Dynamics
[unreadable] DESCRIPTION (provided by applicant): Membrane proteins play an essential role in controlling the movement of material and information in and out of the cell, in determining the flow and use of energy, as well as in triggering the initiation of numerous signaling pathways. To fulfill these roles, conformational and interaction dynamics exert a dominant influence on their functional behavior, for it is the interplay between structure and dynamics what ultimately defines their function. The Midwest Center for Membrane Protein Structural Dynamics (MMPSD) is proposed as a highly interactive, tightly integrated and multidisciplinary effort focused on elucidating the relationship between structure, free energy landscapes, dynamics and function. [unreadable] [unreadable] The MMPSD will be organized around multidisciplinary project teams with investigators from institutions clustered geographically inn [sic] the Midwest to maximize true interactive collaborations and an efficient exchange of ideas. These teams will study major mechanistic questions associated with membrane protein function as it relates to three major areas: energy transduction in signaling (ion channels and receptors) energy interconversion (transporters and pumps) and chemo-transduction pathways (membrane-embedded proteases and phosphatases). Our ultimate goals is to decode the general mechanistic principles that govern protein movement and its associated fluctuation dynamics by dissecting and analyzing the molecular and dynamical bases of these functions at an unprecedented and quantitative level, as well as exploiting this information to engineer altered and novel activities into membrane protein frameworks to rationally evolve new functions. To accomplish its goals, the MMPSD will develop in parallel a set of tools, concepts and reagents to: 1) Determine time-averaged structures of "Archetype" membrane proteins using Chaperone-assisted crystallization methods; 2) Apply state of the art spectroscopic methods (Magnetic Resonance, Fluorescence) to follow conformational changes and dynamics of the determined structures; and 3) Design and implement novel computational approaches to link static and dynamic data with function. Four core facilities will feed and interconnect with the individual projects in a highly interactive way. The cores will support the research in the Center by providing service and expertise in four critical areas: Membrane protein expression, the establishment of chemical synthesis capabilities for probes and detergents, the generation of a large variety of crystallization chaperones and other target binders, and generation of a pipeline of novel membrane targets through metagenomics approaches. [unreadable] [unreadable] All of the information, tools and new reagents/targets will be shared with the research community at large through the "membrane protein dynamics gateway", a state of the art web page and a series of scientific meetings open to the public. [unreadable] [unreadable] [unreadable]
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0.958 |
2009 — 2010 |
Perozo, Eduardo A |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Structural Basis For K+ Channel Slow Inactivation
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Analysis of the molecular identity of the pH sensor in the prokaryotic potassium channel KcsA identified several key mutants that stabilized the KcsA inner bundle gate in the fully open conformation at neutral pH, a fact that makes possible the crystallographic analyses of KcsA in a variety of functional states. As a first approach, we planned to determine the crystal structure of open KcsA via analysis of several of these constitutively open KcsA mutants. Additional mutations in the selectivity filter were designed towards the determination of a fully conductive and an inactivated form of the channel. A preliminary crystal structure of one of these mutants in complex with a Fab has provided tantalizing new information regarding the mechanism of gate opening in KcsA and a likely path towards the fully open state.
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0.914 |
2009 — 2012 |
Perozo, Eduardo A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structural Basis For Transmembrane Mg2+ Transport
DESCRIPTION (provided by applicant): Structural basis for transmembrane Mg2+ transport Abstract Mg2+ plays an essential role in a variety of cellular functions, as enzymatic cofactor, regulator of lipid-derived second messengers and promoter of genomic stability, among other functions. In this proposal we will focus on the CorA Mg2+ transporter/channel, which functions as the primary Mg2+ uptake system for Eubacteria and Archaea. The structure of the CorA ortholog from Thermotoga maritima has been recently determined at medium resolution, revealing a funnel-shaped homopentamer with 2 transmembrane (TM) helices and a large, mostly helical extracellular region. The overall, long-term goal of this project is to understand the molecular mechanism of Mg2+ transport and regulation in prokaryotic mechanosensitive channels. Although the recent determination of the CorA crystal structures has dramatically improved our knowledge of this class of molecules, a number of mechanistic questions remain to be solved. This is particularly true for the molecular events underlying channel/transport gating. In this respect, we plan to experimentally address several fundamental questions: Is CorA a coupled transporter of an ion channel? What regions of CorA form the gate(s) and how do they move to produce gating? What is the physical basis of the energy transduction steps, starting with Mg2+ binding and culminating in protein motion? What are the structures of the key functional states? The approach we plan to pursue combines reporter-group spectroscopic techniques (spin labeling/EPR, Fluorescence) X-ray crystallography and electrophysiological methods with classical biochemical, genetic and molecular biological procedures. Functional studies will be targeted to understand the physical basis of energy transduction in CorA. Information on the topology, secondary, and tertiary structure of CorA and structurally-similar orthologs will be obtained from EPR analysis of spin labeled mutants. The data will be interpreted to generate backbone models of the different stages of the gating pathway in each type of channel. This proposal opens up a new experimental avenue that will contribute to the understanding of Mg2+ homeostasis in prokaryotes with particular emphasis o the mechanisms of ion translocation and gating, and signal transduction. PUBLIC HEALTH RELEVANCE: Understanding of CorA structure and function relates directly to health and disease, not only as key element in the most basic aspect of cellular function but due to its relationship to the mechanism of mitochondrial Mg++ homeostasis in eukaryotic cells. This is relevant because of the known role of mitochondria in apoptosis. CorA is also a virulence factor in prokaryotes and thus an important potential antibiotic target.
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0.958 |
2010 |
Al-Shawi, Marwan Khalid Bowie, James U (co-PI) [⬀] Nakamoto, Robert K. [⬀] Perozo, Eduardo A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Core D1: Membrane Protein Expression/Purification
The main role of the Protein Core will be to produce proteins for each of the Bridge and Pilot Projects. Furthermore, the Core will be accessible to investigators outside the consortium who are seeking help to develop methods for high yield expression of specific membrane proteins, detergent solubilization optimization, and purification of protein targets. The emphasis of the Core will be preparation of homogeneous functional proteins for use in studies of structure, dynamics and mechanism. Core D1 members will cooperate in using their collective expertise to develop and optimize new expression protocols for proteins used by the Consortium. The Core will utilize its expertise and technologies already in place to provide a wide range of expression systems, both prokaryotic and eukaryotic expression in cells or cell-free, to accommodate the specific requirements of each protein target. Because none of the proposed studies can be accomplished without protein, we will use our technologies from the onset of this project to provide samples for the Consortium as soon as possible. In general, membrane proteins present challenges at every step of the way to structural determination. The synthesis and processing of these proteins is complex and often involves specific folding factors or chaperones [1, 2]. Expression in bacteria is favored because of the low cost to grow large culture volumes by fermentation, the potential for high yields, the very fast growth rate, and the simplicity and flexibility of expression systems. However, intrinsic differences in how proteins are processed often prevent the expression of adequate amounts of protein with the proper fold, and it may not be possible to isolate and purify sufficient quantities in a homogenous state. For these reasons, the Protein Core will employ a range of expression systems including preparative scale cell-free systems, which should provide a high probability that any selected protein target can be produced. In addition, we describe high throughput screening methods to further enhance and optimize expression. Because the Consortium emphasizes the study of proteins for which a high resolution structure is already accomplished, such proteins have already been successfully expressed at high levels and homogeneous purified protein prepared. We will take advantage of the information already available, but in some cases targets are proposed for study, in particular mutant forms, that have not been expressed at high levels. Furthermore, investigators may require the protein target in a specific environment or state. For example, the protein may be part of a complex, which requires expression of multiple proteins, some of which do not express in bacteria. This example may require simultaneous expression of all the components in a mammalian or insect cell for proper processing. The Protein Core will apply a range of expression strategies taking into consideration several needs: the quantity, the multimeric state, the level of purification, and the modifications required for manipulations. Of foremost importance is the functionality of the protein. For this reason, the Protein Core will work closely with all investigators to assure that the activity of each target is properly assessed.
|
0.958 |
2010 — 2013 |
Perozo, Eduardo A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
High Resolution Structural Dyanmics of K Channels
DESCRIPTION (provided by applicant): In response to membrane potential depolarization, voltage-dependent channels undergo a series of conformational changes from a non-conducting state (closed) to an activated (conducting), finally stabilizing in a non-conducting inactivated state. K+ channel function has been associated with such basic cellular functions as the regulation of electrical activity, signal transduction and osmotic balance. In higher organisms, K+ channel dysfunction may lead to uncontrolled periods of electrical hyperexcytability, like epileptic episodes, myotonia and cardiac arrhythmia. Consequently, efforts to understand K+ channel structure function and dynamics relate directly to human health and disease. The continuing long-term goal of this project is to further understand the molecular mechanisms of gating in voltage-dependent channels, by focusing on the analysis of K+ channel gating in prokaryotic and eukaryotic systems. Specifically we will address the following key questions: What are the atomic structures of the key conformations that determine channel activity? What are the molecular bases of gating in the 5s-to-ms regime? What is the mechanism of voltage sensing in voltage sensing domains? And how different parts of the channel interact to define open channel activity? We plan to study these problems by combining spectroscopic techniques (EPR and NMR), X-ray crystallography electrophysiological and computational methods. We intend to continue these structure-function studies by investigating proven model systems like the H+ activated channel from Streptomyces lividans and KvAP, the voltage-dependent channel from from Aeropyrum pernix, while extending them using new experimental approaches. In addition, we will focus our attention on the voltage sensing domain from the Ciona intestinalis-Voltage-Sensor-containing Phosphatase (Ci-VSP) and the hyperpolarization-activated six-transmembrane segment (6TM) channel from Methanococcus janschii (MVP). This proposal should open new experimental avenues that will contribute to our understanding of biologically important events such as electrical signaling, signal transduction and ion channel gating. PUBLIC HEALTH RELEVANCE: Understanding of K+ channel structure and function relates directly to health and disease, not only as key element in the most basic aspect of cellular function but due to its relationship to the mechanism of electrical excitability, cancer, and hormone secretion (diabetes) in humans. This is particularly relevant because of the known role of K+ channel dysfunction in a variety of pathological conditions. Some K+ channels are also involved as a virulence factor in prokaryotes and thus represent an important potential antibiotic target.
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0.958 |
2010 — 2014 |
Perozo, Eduardo A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Core B/C: Information/Dissemination
The overarching goal of the Membrane Protein Structural Dynamics Consortium (MPSD) to break new ground toward a comprehensive understanding of membrane protein systems requires a new organizafional model permitting a fight integrafion of structural, dynamical and funcfional data together with theory, modeling and simulafions. Communication plays a key role in this organization. The crucial step is to ensure that all scientific results, new technologies, and novel advances are communicated in real time and in a user-friendly form among the members of the Consortium, as well as to the broader community. No matter how great the science, if it's not communicated it has little benefit to the goals of MPSD. For this reason, the combined Information/Dissemination (B/C) Core is a central element of a robust strategy toward productivity and success. In the proposed organizafional scheme, the Website is the central conduit for the dissemination of information relating to the Consortium. It will focus on describing ongoing research- both progress and problems, ongoing educational activities, as well as providing a direct link to the databases, technologies, and services from the Scientific Resource Cores. In addition, it will chronicle the set of activities that contribute to promoting communications and the dissemination of knowledge, expertise and training in membrane biophysics to the scientific community beyond the researchers and institutions within. These include the Annual retreat with all the Center members. Scientific conferences and workshops, a E-Newsletter that will interface with the Press to relate the activities within the MPSD and articulate our vision and progress.
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0.958 |
2010 — 2014 |
Perozo, Eduardo A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Core a: Administrative
We have organized the Consortium Administrative Core based on the premise that productive collaborations do not necessarily require physically proximity. However, to nurture and maximize their collective output the proper leadership and administrative structure must be in place. We recognize a need to set in place an effective environment to foster the full engagement of each investigator and their staffs in the total science picture and to provide the scope of resources that not only facilitates, but also enhances individual research. Thus, the key motivation behind the organization of the present administrative structure is efficient integration. This integrafion will be key to our hwo major goals: promofing the highest quality science and fostering breakthroughs in the projects that bind us together, as well as maximizing the Consortium's impact by disseminafing advances in membrane protein structure and dynamics within and beyond its virtual walls.
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0.958 |
2010 |
Perozo, Eduardo A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Bridging Project 8: Dynamics of Ion Permeation &Conformational Coupling in Kcsa
11.2. BACKGROUND AND SIGNIFICANCE A number of independent pieces of evidence point to the selectivity filter as a region with a great deal of influence over the gating behavior of a channel, not only in regards to C-type inactivation but also in terms of on-off transitions during activation gating. The effect of certain permeant ions on gafing is have been well documented [1-4]. Ions with long occupancy fimes (Rb+, Cs+, NH4+) tend to stabilize the open state through a "foot in the door" effect on the gate, yet the only region of channel-ion interacfion occurs at the selecfivity filter. Additionally, channels seem to populate sub-conducting states on the way to the open state [5-7], and these sub-conducting states show altered selectivity. Unnatural amino acid mutagenesis targeted to the signature sequence of an inward rectifier K channel revealed dramatic consequences upon rapid gating transitions [8], again, pointing to the selectivity filter as a contributor to the gating process. Structurally, there is eariy evidence of subtle conformational changes in regions flanking the selectivity filter, and these changes appear only under conditions that favor channel opening [9]. KcsA undergoes C-type inacfivation similar to other biologically important K+ channels [10, 11]. After a transition to acidic pH, the lower gate at the inner-helical bundle opens and imparts conformational changes around the selectivity filter. This conformational wave leads to C-type inactivation. Nonetheless, a demonstration of the role of the selectivity filter in influencing activation gating requires additional structural approaches. Three recent developments in the Perozo lab have opened an interesting window of opportunity to further analyze the role of the different moving parts of a channel on gating. First, as stated above, we have identified a C-type inactivation mechanism in KcsA, found and characterized KcsA mutants around the selectivity filter.
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0.958 |
2010 — 2017 |
Perozo, Eduardo A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Membrane Protein Structural Dynamics Consortium
? DESCRIPTION (provided by applicant): To understand membrane proteins, one must ultimately be able to visualize how these complex nanoscale molecular machines move and change their shape atom-by-atom as a function of time while they perform their function. Grounded on the understanding that membrane proteins are dynamic entities that evolved to execute complex sets of movements to perform their functions, what is critically needed is a conceptual movie that captures the essential structural rearrangements underlying function. In spite of recent progress, any particular approach, albeit experimental or computational, is too limited to provide complete information about the transient features associated with such conformational transitions. To make a significant leap forward, the quantitative study of membrane protein dynamics requires a synergistic and multi-disciplinary effort. The main task of this 10-year Consortium is to quantitatively address these issues and provide a basic set of mechanistic principles that relate membrane protein structural dynamics to their function based on a set of membrane protein archetypes. In this proposal, we highlight our recent advances in membrane protein crystallization, spectroscopic, biophysical and modeling techniques. Through highly collaborative partnerships that balance technology incubators (the scientific Cores) with specific projects (Bridging and Pilot projects) we have reached a level of applicability to complex systems unimaginable just a decade ago. However, dynamic information must be quantitatively determined to understand function and this requires the application of both known strategies and methods development. Our proposition remains that a tight integration between structural methods, spectroscopic techniques, functional analyses and computational approaches, is required to provide a deep understanding of these nano-machines and their biological roles. In its Phase II, we find ourselves in an excellent position to expand the number of systems under study, their overall complexity and incorporate new experimental and computational techniques. Accordingly, the MPSDC will continue to be organized around multidisciplinary project teams studying major mechanistic questions associated with membrane protein function in nine archetype systems, spanning a multiplicity of energy transduction mechanisms. Furthermore, the research infrastructure in place for phase II will extend the capacity of the Consortium to make further transforming contributions that should define the fundamental principles governing membrane protein function into the next decade
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0.958 |
2010 |
Perozo, Eduardo A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Bridging Project 4: Transport Cycle in Neurotransmitter Uptake Systems
DAT, NET, and SERT are well established targets for many pharmacological agents that affect brain function [16]. These biogenic amine transporters terminate synapfic transmission by reuptake of the released neurotransmitters from the synaptic cleft back to the presynaptic neuron, coupled to the movement of Na+ down its electrochemical gradient. Drugs that interfere with reuptake profoundly influence behavior and mood. For example, DAT is the primary target for the psychosfimulants cocaine, amphetamine, and methylphenidate [17] whereas inhibitors of SERT are antidepressants (imipramine, fluoxefine) [18]. However, our understanding of the molecular mechanisms whereby these inhibitors exert their effects is sfill at a primifive stage. Our analysis of the LeuT structure [2] has shown intriguingly strong consistency between the structural and funcfional characteristics and our current understanding of the mammalian homologues DAT, SERT, and NET [1]. This is important from a modeling perspective, because computational simulation results are strongly influenced by the quality of the homology models, which in turn depends on the degree of conservafion and similarity between the template and target. Advantages to working with bacterial membrane proteins and bacterial expression systems include easier scale up and increased levels of protein expression, and more limited posttranslational modificafion and thus more homogeneous material as compared to their eukaryotic counterparts. Thus, we propose to use LeuT as an established model system [13] for the proposed studies. To monitor protein conformational changes under native-like conditions, i.e. in proteoliposomes, without the conformational selectivity of crystal lattice forces, we will use the established 8DSL-EPR technique (see [15]). This technique requires site-directed mutafion of native residues to cysteine for the incorporation of a sulfhydryl-specific nitroxide spin label. EPR analysis of the spin labeled proteins yields spectroscopic constraints describing the local environment of a nitroxide probe incorporated at select sites in a protein sequence. These structural constraints are generated from observables such as spin label solvent accessibility, which describes the collisional frequency of the probe with other paramagnetic reagents. Furthermore, dipolar coupling between two spin labels has been shown to be an effective spectroscopic ruler for the determination of global spatial constraints that can provide details of packing interactions and domain movements[19]. Changes in the pattern of these spectroscopic signatures have been shown to correlate with conformafional changes in protein structure [20]. When the labeling sites are selected based on specific hypotheses generated from computafional analyses of dynamics in structurally defined or cognate systems, the approach becomes an incisive tool for determining key functional characteristics in a structure-dynamic context that is interpretable, in turn, in the frame of the computational analysis and simulation and the experimental data for functional properties of the system. The proposed studies aim at a major challenge in structure-function studies of NSS or any transporter family, namely the characterization of the conformational states that constitute the substrate translocation cycle (e.g., see [11]). The absence of crystal structures for multiple conformational states (see above) calls for elucidation of the dynamic nature of transport through the type of combined approach of computational and experimental studies we propose here. In this respect, the integration of the proposed study in this glue grant offers major advantages as it will take advantage of the capabilities and resources in the Cores (see section 4.4, below) and the cognate studies on other membrane protein systems as described throughout this application.
|
0.958 |
2010 |
Perozo, Eduardo A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Bridging Project 2: Structural Dynamics of Abc Transporter
ATP-binding cassette (ABC) transporters constitute the largest family of transporters {1-4). It includes both exporters and importers of solutes ranging in size from small molecules to entire protein domains. Eukaryotic ABC transporters predominantly extrude hydrophobic molecules (5), while most bacterial ABC transporters import essential nutrients. The functional unit of an ABC transporter consists of two molecular motors with nucleotide (ATP) binding and hydrolysis domains (NBD or ATP cassette), each coupled to a transmembrane domain (TMD) that encodes the determinants of substrate specificity and provides the binding chamber and passageway across the membrane. The molecular organization of the four domains of ABC transporters was gleaned from crystal structures of a number of ABC importers {6-9) as well as the bacterial multidrug efflux systems Savl866(^0) and MsbA {11). A subclass of ABC exporters has been implicated in multidrug resistance (MDR). Human P-glycoprotein (Pgp) and LmrA from Lactococcus lactis are capable of extruding a large variety of drug molecules;the former providing a strategy for tumor cells to evade the toxicity of chemotherapeutic 6rugs{12-14). This Bridging Project aims to answer a major outstanding question in the field, namely to characterize the nature and amplitude of the conformational motions that transduce the ATP energy input to transport of drugs. Recent crystal structures of bacterial ABC exporters, Sav1866 and MsbA {10, 11), along with extensive spin labeling analysis of MsbA in liposomes {15-18) define a blueprint of the conformational changes induced by ATP binding. However, these investigations were carried out in the absence of drug or substrate. The resulting "minimalist" two-state model is not compatible with biochemical analysis of Pgp that identified at least six intermediates in the transport cycle {19). The missing link is an understanding of the conformational dynamics of ABC transporters as they cycle between transport intermediates. The nature of this problem calls for methods capable of investigating the structure of ABC transporters in their native environment with sufficient spatial resolution and dynamic sensitivity to link structure and function. Pgp provides an ideal system for spectroscopic analysis of functional dynamics. In addition to its direct medical significance, a wealth of information has been accumulated describing its interaction with substrates, including a detailed thermodynamic and kinetic analysis {19, 20). Furthermore, Dr. Al-Shawi has already initiated spin labeling analysis of Pgp {20). The recently determined crystal structure of nucleotide-free {apo) Pgp {21) provides an excellent starting point for experimental design and computational studies of the dynamics, and a context to interpret spectroscopic data. Pgp was captured in an inward-facing conformation where the two symmetry-related halves, each consisting of six helices, are packed in V-shaped geometry, resulting in a cavity open to the cytoplasm and the inner leaflet of the bilayer (Fig. IB). The crystal structure also pinpoints putative drug entry portals near the water/membrane interface that allow access to the cavity. This structure was interpreted mechanistically as a pre-transport state ready to bind drugs. This structure provokes a number of important questions. Very likely apo Pgp needs to sample a large conformational ensemble to accommodate the spectrum of transported substrates. One of these conformations is selectively stabilized by contacts in the crystal lattice. Thus, whether the crystal structure captures the most populated conformer in the membrane needs to be tested. Transported substrates stimulate the ATPase activity and their binding is expected to be signaled to the NBDs through induced conformational changes (22). However, virtually no changes were observed in the substrate-bound crystal structure of Pgp further reinforcing concerns of conformational selectivity {23). In light of these questions. Aim 1 focuses on determining whether the crystalized apo structure reflects the average conformation in the membrane and defines the conformational changes induced by various classes of Pgp substrates. We will also determine the accessibility of the cavity and analyze the environments in the putative entry portals following substrate binding. If indeed the apo state is open to the cytoplasm, ATP binding and hydrolysis are predicted to lead to substantial structural rearrangements. The blueprint of these can be gleaned from the nucleotide bound structures of MsbA and Sav1866 (Fig. 1A). The two NBDs form the canonical ATP sandwich;the TMDs undergo alternating access whereby the cavity closes to the cytoplasm and the inner bilayer leaflet and opens to the extracellular side. Underiying this reconfiguration are large distance changes on the cytoplasmic side and extensive repacking of transmembrane helices. To create the extracellular opening, a twisting motion repacks the TM helices changing the identity of the swapped helices between the two halves of MsbA. We generated a fully energy-minimized homology model of human Pgp in an outward-facing conformation based on the AMPPNP containing structure of MsbA {11) (Fig. 1A). Assuming that the new structure of apo mouse Pgp (ABCB1a)(23) represents Pgp in an inward-facing conformation (Fig. IB), large amplitude conformational changes are predicted between these two key states during multi-drug transport (Fig.lC). Aims 2 and 3 propose to test the MsbA-centric model of ATP-induced conformational change in the presence of the various classes of Pgp substrates. The investigations described below will facilitate a molecular description of the multi-drug efflux phenomenon mediated by Pgp. Structural intermediates inaccessible to other methods of analysis will be defined and tested. The structure and dynamics of key functional intermediates and the nature of conformational changes between them may eventually provide templates for the rational design of specific modulators of Pgp function.
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0.958 |
2011 |
Perozo, Eduardo A |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Potassium Channel Selectivity Filter
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The long-term goal of this project is to further understand the molecular mechanisms of gating in voltage-dependent channels, by focusing on the analysis of K+ channel gating in KcsA and KvAP. Analysis of the molecular identity of the pH sensor in the prokaryotic potassium channel KcsA identified several key mutants that stabilized the KcsA inner bundle gate in the fully open conformation at neutral pH, a fact that makes possible the crystallographic analyses of KcsA in a variety of functional states. Additional mutations in the selectivity filter were designed towards the determination of a fully conductive and an inactivated form of the channel and the probing of modal gating events.
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0.914 |
2015 — 2017 |
Perozo, Eduardo A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Information and Dissemination Core
A crucial step in fulfilling the scientific goals of the MPSD consortium is to ensure that all scientific results, new technologies, and novel advances are communicated in real time and in a ?user-friendly? form among both, the members of the Consortium and the broader community. The combined Information/Dissemination (B/C) Core is the central element of a robust strategy toward productivity and success. Over the past four years, and in the proposed organizational scheme of Phase II, the MPSDC Website shall continue to be the predominant conduit for the dissemination of information relating to the Consortium activities, science and extension. In consequence, the website will continue its focus on describing ongoing research- both progress and problems, ongoing educational activities, as well as providing a direct link to the databases, technologies, and services from the Scientific Resource Cores. In addition, the website will chronicle the set of activities that contribute to promoting communications and the dissemination of knowledge, expertise and training in membrane biophysics to the scientific community beyond the researchers and institutions within. These include coordination and advertising of the Annual retreat with all the Center members and the larger community, Scientific conferences and workshops, an E-Newsletter that has related the activities within the MPSD and articulate our vision and progress. So far, we consider our communication strategy to be an unqualified success and as such, propose few if any changes to our approach in the last five years of consortium operation. The Core B/C is designed to accomplish four Aims: Aim 1. Establish and maintain the Consortium Website 1.1 Enable internal exchanges among the investigators of the MPSD Consortium. 1.2 Ensure the dissemination of information and publication originating from the research carried out by the Investigators of the Consortium and about the technology developed and made available by the Scientific Resource Cores. Aim 2. Publish an E-Newsletter about the research in the Consortium. Aim 3. Organize the annual retreat and the scientific workshops. Aim 4. Support education, outreach and diversity.
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0.958 |
2015 — 2017 |
Perozo, Eduardo A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Administrative Core
The Administrative Core of the Membrane Protein Structural Dynamics (MPSD) Consortium is set-up to provide the necessary leadership and administrative support structure to ensure optimal performance and effective coordination of the MPSD Consortium activities. This Core is the hub of all activities and will provide strong scientific leadership and oversight. The function of the Administrative Core is to achieve five objectives: (1) facilitate interactions among Projects and Cores; (2) provide necessary administrative assistance to MPSD Consortium investigators and participating institutions; (3) manage all MPSD Consortium finances; (4) convene all necessary MPSD meetings, including Executive Committee meetings, monthly scientific meetings, annual External Advisory Committee meetings and annual retreats; and (5) provide day-to-day logistical coordination for the Consortium. However, as one of the key lessons learned in the previous funding period, the leadership provided by the Core cannot come at the cost of micromanaging individual projects or teams. Core A will continue to ensure compliance with all institutional, governmental and NIGMS-specific regulations and requirements, including timely communication and consultation with NIGMS staff. Other critical management objectives have been, during Phase I, to make decisions about allocation (and reallocation) of resources, project expectations and progress, and providing a structure to recruit new talent into the Center. The present scientific line-up planned for Phase II of the consortium should ensure the successful completion of our 10 year scientific plan, securing the highest possible impact of the Center's scientific discovery, education, and public outreach activities.
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0.958 |
2015 — 2017 |
Perozo, Eduardo A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Bridge 4: Dynamics of Ion Permeation and Conformational Coupling in K+Channels
Protein-protein and protein-H2O hydrogen bonds have been implicated in defining selectivity filter dynamics and overall function. In Phase II of this consortium, we will continue to study these events by combining spectroscopic, crystallographic, electrophysiological and computational methods to probe the dynamics of the different gating modes in K+ selective filters. In addition to our basic experimental model (KcsA) we have now have added a second system with remarkable selectivity filter plasticity in regards to overall conformation. The NaK-hERG construct undergoes dramatic structural rearrangements in response to both changes in permeant ions and as a result of clinically relevant mutations (associated with the long QT syndrome). Because of these findings, we are in a position to analyze, at atomic level, a wider universe of conformational dynamics at the selectivity filter through multiple time scales. This unique structural dynamics dataset will allow us to pursue detailed computational analyses to provide a definitive description of the energy landscape that connects each static structure with its conformational pathway and associated functional consequences. We propose a concerted approach using cell free and semi-synthesis of KcsA and NaK-hERG, patch clamp, X- ray diffraction and state of the art spectroscopic methods (NMR and ssNMR) to develop, with the aid of cutting edge computational methods, an understanding of the conformational changes taking place at the selectivity filter when it inactivates and during ion conduction. These overall goals will be carried out through the following specific aims: 1. Determine the molecular motions, overall energetics and role of ion interactions associated with the conformational flexibility of K+ selective filter in KcsA and a NaK-hERG chimera. 2. Establish the role of selectivity filter structural water dynamics on the kinetics of C-type inactivation in KcsA and the energetic rules that connect all key gating intermediates using a combination of experimental and computational approaches. The types of questions we plan to address can be approached only because of the robustness and experimental maturity of the systems at hand (KcsA and NaK-hERG). Estimation of the energy landscape for the universe of conformations together with high bandwidth electrophysiology will allow us to establish the general rules that describe a wide range of motion, dynamics and gating modes associated with the K+ selectivity filter. This data is expected to provide a firm understanding of selectivity filter gating mechanisms in the two model systems and is expected to also apply to other members of the K+ channel superfamily.
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0.958 |
2017 — 2020 |
Perozo, Eduardo A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structural Basis of Mg2+ Homeostasis
Abstract Mg2+ plays an essential role in a variety of cellular functions, as enzymatic cofactor, regulator of lipid-derived second messengers, promoter of genomic stability and marker for bacterial pathogenesis, among other functions. In this proposal we will investigate the molecular basis for gating and divalent permeability of the Mg2+ channel CorA, the primary Mg2+ uptake system for Eubacteria and Archaea and a close homolog for the mitochondrial Mg2+ importer Mrs2p. The structures of the CorA orthologs from Thermotoga maritima and Methanocaldococcus janaschii have been recently determined, revealing a funnel-shaped homopentamer with 2 transmembrane (TM) helices and a large, mostly helical extracellular region. The overall, long-term goal of this project is to understand the molecular mechanism of Mg2+ translocation and homeostasis in prokaryotic. Although the recent determination of the CorA crystal structures and our own functional and spectrospcopic work during the past funding period have dramatically improved our knowledge of this class of molecules, a number of mechanistic questions remain to be solved. This is particularly true for the molecular events underlying ion selectivity and permeation as well as Mg2+ -driven channel gating. In this respect, we plan to experimentally address several fundamental questions: How does CorA select for Mg2+ against a host of monovalent and divalent cations? How does Mg2+ drive gate closing? What is the physical basis of the energy transduction steps, starting with Mg2+ binding and culminating in protein motion? What are the mechanisms that underlie inward rectification? The approach we plan to pursue combines single particle cryo-EM, reporter-group spectroscopic techniques (spin labeling/ CW and DEER EPR,) X-ray crystallography and electrophysiological methods with classical biochemical, genetic and molecular biological procedures. Functional studies will be targeted to understand the physical basis of ion selectivity and energy transduction in CorA. Information on the topology, secondary, and tertiary structure of CorA and structurally similar orthologs will be obtained from cryo-EM and EPR analysis of spin labeled mutants. This data will be computationally interpreted to generate models of the different stages of the gating pathway, w i t h multiple conformers contributing to a highly dynamic open conformation. This proposal aims to continue a new experimental avenue that will contribute to the understanding of Mg2+ homeostasis in prokaryotes with particular emphasis on the mechanisms of ion translocation and gating, and ion selectivity.
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0.958 |
2018 |
Perozo, Eduardo A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structural Basis of Mg2+ Homeostasis - Equipment Supplement
Project Summary from Parent Award Mg2+ plays an essential role in a variety of cellular functions, as enzymatic cofactor, regulator of lipid-derived second messengers, promoter of genomic stability and marker for bacterial pathogenesis, among other functions. In this proposal we will investigate the molecular basis for gating and divalent permeability of the Mg2+ channel CorA, the primary Mg2+ uptake system for Eubacteria and Archaea and a close homolog for the mitochondrial Mg2+ importer Mrs2p. The structures of the CorA orthologs from Thermotoga maritima and Methanocaldococcus janaschii have been recently determined, revealing a funnel-shaped homopentamer with 2 transmembrane (TM) helices and a large, mostly helical extracellular region. The overall, long-term goal of this project is to understand the molecular mechanism of Mg2+ translocation and homeostasis in prokaryotic. Although the recent determination of the CorA crystal structures and our own functional and spectrospcopic work during the past funding period have dramatically improved our knowledge of this class of molecules, a number of mechanistic questions remain to be solved. This is particularly true for the molecular events underlying ion selectivity and permeation as well as Mg2+ -driven channel gating. In this respect, we plan to experimentally address several fundamental questions: How does CorA select for Mg2+ against a host of monovalent and divalent cations? How does Mg2+ drive gate closing? What is the physical basis of the energy transduction steps, starting with Mg2+ binding and culminating in protein motion? What are the mechanisms that underlie inward rectification? The approach we plan to pursue combines single particle cryo-EM, reporter-group spectroscopic techniques (spin labeling/ CW and DEER EPR,) X-ray crystallography and electrophysiological methods with classical biochemical, genetic and molecular biological procedures. Functional studies will be targeted to understand the physical basis of ion selectivity and energy transduction in CorA. Information on the topology, secondary, and tertiary structure of CorA and structurally similar orthologs will be obtained from cryo-EM and EPR analysis of spin labeled mutants. This data will be computationally interpreted to generate models of the different stages of the gating pathway, w i t h multiple conformers contributing to a highly dynamic open conformation. This proposal aims to continue a new experimental avenue that will contribute to the understanding of Mg2+ homeostasis in prokaryotes with particular emphasis on the mechanisms of ion translocation and gating, and ion selectivity.
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0.958 |
2019 — 2021 |
Perozo, Eduardo A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structural Basis of ?Force From Lipids? Activation in Mechanosensitive Channels
Project Summary/Abstract Mechanosensitive (MS) channels are oligomeric membrane proteins that function as mechano-electrical sensory switches in a wide range physiological processes. These include touch, hearing, proprioception, turgor control in plant cells and osmoregulation in bacteria. Among these, a fundamental class of MS channels responds to changes in the physical properties of the lipid bilayer by undergoing major structural transitions in response to membrane tension, thus fulfilling a major role in the response of living organisms to mechanical stimuli. This has been referred to as the ?force from lipid? principle of mechanosensitivity. The overall, long-term goal of this project is to understand the molecular mechanism of ?force from lipid? gating in mechanosensitive channels. Specifically, we will focus on the MscS family of MS channels found in most prokaryotes and plants. These channels are of fundamental importance in various physiological events, can been engineered for biomedical applications, and display fascinating intramembrane heterogeneity among family orthologs. More importantly, the MscS family Affords us the possibility of studying the functional behavior, high resolution structure and dynamics in the same MS system. Although MscL and MscS channels have been studied extensively and crystal structures have been available in multiple conformations, there are still major mechanistic questions that remain to be solved. This is particularly true for the molecular events underlying channel gating, in light of exciting new preliminary data at the core of this proposal. In this respect, we plan to experimentally address several fundamental questions: What is the physical basis of the energy transduction steps, starting with trans-bilayer tension and culminating in protein motion? What are the structures of the key functional states in its native, bilayer-embedded form? Where in the molecule does mechanical transduction occur? And how? Functional studies will be designed to understand the physical basis of energy transduction. Information on the architecture, dynamics and energetic relationship of MscS (plus other related members of the superfamily) with its surrounding lipid bilayer will be obtained from cryo-EM, EPR analysis of spin labeled mutants and computational methods. The data will be interpreted to generate high resolution structures of the different stages of the gating pathway in each type of channel. We suggest that the advent of new cryo-EM approaches to the analysis of structure and dynamics in membrane proteins in their native environment shall open an exciting new experimental avenue that will contribute to the understanding of biologically important events such as ion channel gating, nociception and signal transduction.
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0.958 |
2021 |
Perozo, Eduardo A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structural Basis of Outer Hair Cell Electromotility At High Resolution
Project Summary The overall, long-term goal of this project is to understand the molecular mechanism of that define the cochlear amplifier in outer hair Cells (OHC). Specifically, we will focus on the voltage-driven motor Prestin, a unique member of the SCL26 family of transporters found in the basolateral membranes of OHCs. Although Prestin has been studied extensively though functional approaches, the basic mechanistic understanding of this fundamental component of the cochlear amplifier remain to be solved. In spite of the richness of the existing functional data, the lack of a high resolution structure is a key missing element in defining its mechanism at a molecular level. This is particularly so for the two fundamental aspects of Prestin?s mechanism of action: the process underlying voltage sensing and the molecular mechanism of electromotility. In light of exciting new preliminary data at the core of this proposal we will be able to study the functional behavior, high resolution structure and dynamics of Prestin as a biological piezoelectric device. To do so, we plan to experimentally address several fundamental questions: What is the physical basis of the energy transduction steps, starting with transmembrane voltage changes and culminating in protein (and ultimately OHC) motion? What are the structures of the key functional states in its native, bilayer-embedded form? Where in the molecule does mechanical transduction occur? And how? What are the physical basis of the Prestin-bilayer interaction? Functional studies will be designed to understand the physical basis of energy transduction. Information on the high resolution structure of functionally relevant conformations, conformational dynamics and energetic relationship of Prestin with its surrounding lipid bilayer will be obtained from cryo-EM, electrophysiology and Fluorescence microscopy experiments. The data will be interpreted to generate high resolution structures of the different stages of the electromechanical transduction. We suggest that the advent of new cryo-EM approaches to the analysis of structure and dynamics in membrane proteins in their native lipidic environment shall open an exciting new experimental avenue. This information will impact our understanding of physiologically important events such as hearing, high frequency amplification and signal transduction.
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0.958 |