Robert Fairman - US grants

Fairman
MCB-9817188:


Coiled coils are motifs which involve the interaction of sets of a-helices
and are stabilized largely through hydrophobic amino acids that line the
interface of the structure. This study focuses on a natural four-chain
coiled coil model motif from Lac repressor to quantitate sequence-structure
relationships in a systematic fashion. A mutational approach is used to
study the effects of different amino acid sidechains at specific positions
in the Lac repressor coiled coil. Experimental strategies involve
synthesis of peptides by solid-phase synthetic methods, purification by
reversed-phase chromatography, and biophysical characterization. Coiled
coil stability is measured by the oligomeric states during temperature or
chemical denaturation, peptide concentration titrations using circular
dichroism spectropolarimetry and analytical ultracentrifugation. Using
such approaches, a series of experiments will systematically explore the
relationship between coiled coil stability and several defining
characteristics of coiled coils. Sequences chosen for study will test the
relationship between helix length and stability, the role of hydrophobic
amino acids on stability and oligomerization, the importance of side-chain
interactions at other heptad positions on tetramer stability, and the
ability to design simplified sequences based on rules so learned.

With an understanding of the determinants that define coiled coil
structure, it will be possible to design novel structures that may be of
use in nano-engineering, particularly for the synthesis of long
"nanoropes", and with appropriate modifications, may even be converted into
conducting materials such as "nanowires." This project is well-suited for
an undergraduate laboratory since the synthesis and characterization of
each peptide described herein is a project unto itself and can be
accomplished easily within the constraints of a one-year senior research
project. Important skills that will be learned by undergraduate students
include rigorous quantitative methods in protein biochemistry using
biophysical methods and cross-cutting interdisciplinary methods that span
from biology and chemistry to physics and mathematics.

9970203


Abstract


We are requesting funds to purchase a circular dichroism (CD) spectropolarimeter. This instrument is an important tool in biochemistry for studying regular structure in proteins and nucleic acids and has been used extensively as a tool to help define how proteins fold. This
instrument is also used to study spectroscopic properties of other biomolecules, such as light-harvesting assemblies as described in this proposal.

Research in Dr. Fairman's and Dr. Akerfeldt's laboratories focuses on the use of synthetic peptides as model systems to study important protein structures. Dr. Fairman's research program involves a study of how amino acid sequences dictate the stability and specificity of protein structures. His work uses the coiled coil structural motif, a simple protein structural motif involving interactions between alpha-helices, first described at the molecular level by Frances Crick and Linus Pauling in the early 1950's. Information gained from these studies will be used to design self-assembling polymers towards the creation of new nanoengineering tools. Dr. Akerfeldt's research tries to understand how protein domains cooperate to define functions in proteins. Calbindin d28k is used as a model system to study the cooperative interactions of motifs designed to bind and store calcium. A reductionist approach is applied by synthesizing each individual structural motif, characterizing its structure and affinity for calcium, and then reconstructing the full activity of the protein by combining these synthetic peptides back together. Dr. de Paula's research involves understanding the mechanism of aggregation of the light-harvesting molecules, porphyrin and chlorophyll. These systems are promising nano-sized materials for light-harvesting applications. Dr. White's research involves the study of protein-RNA interactions focusing on investigating the interaction between yeast ribosomal protein L30 and its RNA transcript, L30 RNA. The existence of secondary structure in peptide fragments of the L30 proteins that retain RNA-binding ability will be examined. CD will be used to study the RNA-protein complex for exploring conformational changes upon complex formation.

In addition to the use of this instrument for specific research programs, the instrument will also play an important role in the training of students in both the Biology and Chemistry Departments at Haverford College. In the Biology Department, the instrument is used in a junior level laboratory course, required of all majors, focusing on the study of secondary structure and stability in fibrinogen. In addition, plans are being developed to use the instrument both in a sophomore level biochemistry course and a junior level Chemistry laboratory course. Thus, a major goal for the use of this instrument beyond our research is to train students in interdisciplinary approaches to science. This instrument will help emphasize a recent commitment by Haverford College to promote interdisciplinary education.

The atomic force microscope (AFM) can image samples with a resolution of 2 nanometers laterally and 0.2 nanometers vertically, and can also measure extremely small forces (from 100 piconewtons up to 1 micronewton). Laser tweezers extend this force range below 1 piconewton and also allow manipulation of objects on length scales of 50 nanometers. A fluorescence microscope permits imaging of fluorescent tagging molecules, which, in addition to their typical applications, will be used to insure that the objects being studied with AFM and laser tweezers are indeed those of interest, rather than artifacts or contamination. This instrument cluster will be used for a variety of experiments including:

1) the study of novel self-assembling biomaterials based on the coiled-coil protein folding motif
2) the response of both mature and immature T lymphocyte cells to infected cells
3) the effect of naturally-occurring modifications of the microtubule surface on motor protein traction
4) basic experiments to characterize electron transfer in DNA.


With support from the National Science Foundation, an atomic force microscope, a fluorescence optical microscope, and a laser tweezers workstation will be purchased and combined into a single instrument with powerful imaging, manipulation, and force measurement capabilities. The instrument will be the first to integrate these three technologies using commercial components, and should pave the way for other researchers. Auxiliary equipment for vibration isolation, temperature regulation, and the formation of micropipettes, all of which are required for optimal performance and for the force measurements, will also be purchased.

Haverford College has a strong commitment to excellence in the education of undergraduates. At least four graduating students per year will use this instrument cluster for a senior research project. The interdisciplinary nature of the work will spark exciting interactions between the students and also between their mentors.

The objective of this project is to explore the relationship between sequence and structure for very long coiled coils, such as found in myosin, using both protein folding and design approaches. Three-pronged approach will be used to accomplish the goal. First, synthetic genes that encode copolymers of 14-amino acid blocks will be constructed, whose sequences are based on de novo, minimalist-design principles. These genes will be cloned into expression vectors to make dimeric coiled coils that range from 70 residues to greater than 1,000 residues per helix. Specifically, this system will be used to test the role of intermediates in the assembly of long coiled coils, such as monomeric helix formation and nucleation of specific helix pairing interactions to dictate proper phasing of helices. After expressing and purifying these designed proteins, their structures will be characterized using circular dichroism, analytical ultracentrifugation, and single molecule techniques such as atomic force microscopy and laser tweezing. Second, to complement these design studies, a myosin coiled-coil rod domain will be used as a model system for folding studies involving segment swapping between designed and natural sequences. In addition, the myosin coiled coil will be used to help develop biophysical protocols for studying designed coiled coils, using the instruments described above. Finally, these synthetic peptide blocks will be used to make long copolymers for the study of other coiled coil topologies and higher order assembly to form fibrils. Long copolymers are generated by forming staggered helical structures that act as templates for their own head-to-tail self-assembly. Peptides will be synthesized and purified in the laboratory and then characterized using the same biophysical techniques described above.

The overall goal of this research is to understand the basic relationship between protein sequence and structure. It is still not possible to predict protein structure and function from first principles, mainly because it is still not understood how proteins balance the major chemical forces in attaining their three dimensional shape. Two approaches have been applied to study this problem, defining the fields of protein folding and design. The two questions are the inverse of one another: scientists in the field of protein folding ask, "Can we predict the structure of a protein given its amino acid sequence?" and those who work on protein design ask, "Can we predict what sequence of a protein will result in a target structure?" Both of these strategies will be used in the study of coiled coils. These structural motifs, predicted to be in 1/3 of all proteins, involve the interaction between two or more alpha-helices. The modular design of the experiments will allow students to make significant achievements over the course of a summer experience and an academic year working towards a senior thesis project. This cohesive program in design, along with a strong modular component, should provide a rewarding experience for students interested generally in interdisciplinary sciences, including elements of biochemistry and biophysics.

The objective of this project is to explore factors that regulate the polymerization of short peptides into a-helical polymeric structures. An improved understanding of such biopolymer processes will contribute to the advancement of a number of different active fields of research, including: (1) elucidating general protein folding rules and how they relate to polymerization; (2) understanding how such processes can lead to misfolding and aggregation; and (3) designing novel biomaterials. The ability of these helical polymers to form depends on designed staggered interactions between helical peptides, whose structure is based on the well-known coiled-coil structural motif. The peptide sequence will be mutated to test the role of the hydrophobic interaction in polymer assembly and structure. Mutations will also be made to test regulation of polymerization by metal binding and a novel photosensitive switch capable of inducing local structural changes. One particular sequence variant made for this project was discovered to form polymers containing either a-helical or b-strand conformations. The investigator will test the role of glutamines in influencing the propensity to form these two polymer structures. Circular dichroism and Fourier transform infrared spectroscopy will be used to quantify secondary structure content and stability of the polymers. The size and shape of the polymers will be tracked by analytical ultracentrifugation, atomic force microscopy, and dynamic light scattering.

Although focusing on basic research questions, this project will also impact a different project in the lab whose goal is to create bio-electronic materials, involving faculty at Haverford and at U. Pennsylvania. The work described here will be driven primarily by undergraduates, since Haverford College is an undergraduate institution. Students fulfill major course credit by writing a thesis proposal and a final thesis paper, presenting their work at group meetings and to the Department as a whole, while carrying out their experiments. Elements of this research program have been developed by the investigator for a junior laboratory course to be taught to chemistry and biology majors, and ideas relevant to biomaterial design are being developed for inclusion in a junior-level lecture course in chemical biology and biochemistry. The investigator has a long-standing commitment to improving access to research for under-represented groups. In addition to a long-term commitment to an existing outreach program for science and writing for K-12 students from Philadelphia, The PI regularly reserved space in his research lab over the summer for at least one student self-identified as belonging to an under-represented group. In a newer direction, the investigator has also hosted a high school teacher and student from Philadelphia to work in his lab this past summer, in which they learned to synthesize and purify peptides critical for the goals of this project.

Supramolecular directed self-assembly (DSA) has the potential for realizing well-defined and well-regulated macromolecular structures with self-limiting dimensions and designed functionality. This project seeks to develop an understanding of the basic science governing DSA of functional monomers, and the photonic and electronic properties of the resulting structures. The research plan is based on close interplay between molecular modeling and design, synthesis of building blocks and scaffolds, spectroscopic characterization of self-assembly under varying conditions, and structural and physical characterization of the resulting assemblies using a variety of scanned probe and electron microscopies, and electrical measurements under controlled illumination and temperature. The focus of these studies will be materials made from the assembly of porphyrins into one-dimensional structures. Related to the chlorophylls found in photosynthetic organisms, porphyrins can form structures that are photophysically and photochemically active. Studies will focus on the self-organization of porphyrins, and their self-assembly with the aid of peptide scaffolds to give control over the geometry and photoelectronic properties of the final assemblies. The underlying mechanisms of the photoelectronic properties will also be explored, to evaluate possible use of the building blocks for devices, such as conducting nanowires, solar cells, and chemical sensors.

With this RUI award, the Office of Multidisciplinary Activities and the Organic and Macromolecular Chemistry Program are supporting the research of Professors Karin S. Akerfeldt, Robert Fairman, and Walter Fox Smith, of the Departments of Chemistry, Biology, and Physics (respectively) at Haverford College. This collaborative team is studying the phenomenon of molecular self-assembly, by which smaller molecules spontaneously assemble into structurally-defined aggregates. Mastery of the rules for designing molecular assemblies, by controlling the structure and chemical properties of the basic building blocks, would permit the creation of nanometer- to micron-size assemblies with desired shapes, reactivity, and photonic or electronic properties. Such control would represent a powerful approach to the design and manufacture of devices big and small, with atomic-scale precision in the relative placement of components. Postdoctoral fellows will be given the opportunity to co-teach courses with experienced faculty at Haverford, providing excellent training for careers that involve teaching and research. Aspects of the collaborative research efforts will be exported to workshops for high school teachers, and high school students will be hosted for six-week summer research experiences.

The objective of this project is to study the molecular and chemical role of the amino acid, glutamine, in the process of misfolding and aggregation of proteins containing long glutamine repeats. This aberrant process, resulting in the formation of large fibrils, is a general molecular problem common to all organisms and can lead to metabolic and structural deficiencies. Recently, the process of this aggregation has come under scrutiny since the intermediates formed during fibril growth can themselves persist in the organism and interfere with normal cellular processes. Stretches of glutamine repeats fold into a b-sheet structure, which then acts as a template for aggregation by the further addition of polypeptide strands to form a long filament. Individual filaments can further associate to form multi-filament fibrils. The glutamines are thought to stabilize these structures through mechanisms involving hydrogen bonding and hydrophobic interactions, although the details of these interactions and their respective roles during the self-assembly process are still controversial. The PI has developed a peptide model system that can form a prototypical b-sheet, called a b-hairpin, that can be studied using a variety of biophysical methods to probe the role of the glutamines in fostering b-sheet, filament, and fibril formation. Three specific goals are: (1) finding conditions to stabilize intermediates in the fibril assembly pathway so that the role of glutamine interactions on growth and stability can be assessed; (2) measuring the effects of amino acid mutations on these processes to probe chemical mechanism; and (3) isotope-labeling of specific glutamines to probe directly for stable hydrogen bonding in the various intermediates. The outcome of these studies will help to understand a fundamental problem in protein misfolding and to test a variety of structural models that have been proposed for proteins containing poly-glutamine repeats.

This research has been developed as a set of projects suitable for undergraduates since the primary research efforts will be undertaken by students working to complete their thesis requirements in biology. Students engaged in this work will gain training in interdisciplinary research. This necessitates collaborative work and students will be working closely with colleagues in the Chemistry Department and collaborators at the University of Pennsylvania. Work on this project has led to the creation of lectures in protein folding and misfolding for an introductory course, more advanced lectures for an upper-level course in protein structure and function, and the development of a 7-week primary-literature-based course on protein misfolding and aggregation. The PI has a long-standing commitment to improving access to research for under-represented groups. In addition to a long-term commitment to an existing outreach program for science and writing for K-12 students from Philadelphia, he has regularly reserved space in his research lab over the summer and during the academic year for at least one student self-identified as belonging to an under-represented group. More broadly, as the director of the HHMI-sponsored programs at Haverford College, the PI is responsible for awarding student interdisciplinary research fellowships and funding internal and external research opportunities for students belonging to under-represented groups, administering several outreach programs, and supporting faculty development seminars and workshops to enhance the overall research program in the sciences.

Bryn Mawr College and Haverford College are collaborating to prepare STEM teachers for high need school districts. An objective is to determine whether purposefully integrating a broad range of existing campus student-support and civic engagement structures together with a strong scholarship program and an ambitious publicity/recruitment campaign will increase the number of students who become precollege STEM teachers. Additional new initiatives designed to enhance teacher success include professional development and induction components. The program is developing a model for STEM teacher education that is compatible with the goals and structure of a liberal arts education: students complete a rigorous disciplinary major during their four years of undergraduate study and then complete their education requirements during a fifth year. Bryn Mawr and Haverford Colleges are collaborating with three local, secondary schools, and with the Math Science Partnership of Greater Philadelphia/21st Century Partnership Network. Nine Noyce Scholars are receiving two-year scholarships during the senior and fifth year. The program also is providing post-certification mentoring and professional development support during their first two years of teaching. The project's merit involves investigating the program's impact on encouraging STEM majors in a liberal arts setting to pursue careers in teaching. The project has the potential to serve as a model for the development of STEM teachers within the context of a highly selective liberal arts college.

This award from the Major Research Instrumentation (MRI) program will be used to purchase four significant instruments to support a new core imaging facility at Haverford College, a selective liberal arts college with a diverse student body. The new instruments funded by this grant are a transmission electron microscope, a scanning electron microscope, a confocal microscope, and a fluorescence-activated cell sorting (FACS) system. This facility will serve the research and educational missions of the Biology Department and will also be an important resource for faculty and students in the Physics and Chemistry Departments. These instruments will significantly enhance the research capabilities of the faculty, most with external funding to support their research programs. Since the focus of the Haverford Biology Department is in cell and molecular biology, access to these imaging tools will benefit research projects in areas such as biomaterials research, nanotechnology, developmental biology and embryogenesis, neurobiology, immunology, and stem cell biology. Notably, the faculty collaborate frequently in their research, bringing together skills and expertise to develop synergies that advance science in new directions and serving as role models in students' training. Students are immersed in the process of doing science, fostering their ability to think broadly and from interdisciplinary perspectives. Students will use these instruments for their course work and their senior thesis projects, and will also develop state-of-the-art skills that will be helpful in for a career path in STEM (science, technology, engineering, and mathematics).

DESCRIPTION (provided by applicant): The over-arching goal of this project is to understand the molecular and cellular mechanisms that drive glutamine-repeat (polyQ) protein aggregation. Such polyQ sequences are found in at least twelve human genetic diseases including Huntington's disease and Machado-Joseph disease. It is well established from in vitro experiments that stable intermediates, or oligomers, may be present during the assembly of polyQ proteins into fibrils. Such oligomers have been shown to have cytotoxic and pathogenic properties in cells. We propose that native sequences flanking the polyQ stretches in mutant proteins will greatly influence the distribution of aggregation species in the context of the cell, and that the activities of molecular machinery that mitigate protein aggregation may be influenced by such flanking sequences. A recent study, using crude extracts from a mouse neuroblastoma cell line (2), has identified the presence of oligomers using the newly developed analytical ultracentrifuge with fluorescence detection capability. This technique will be used, combined with FRAP (fluorescence recovery after photobleaching, carried out using a confocal microscope), to probe the aggregation states of polyQ-containing proteins both in vivo and in crude extracts in the worm, C. elegans. The effect of protein flanking sequences will be explored on the relative distribution of oligomers, and its correlation to cytotoxicity. To further assess biological significance for the role of various aggregation states in disease, D. melanogaster will be used to discover how two well-studied chaperones (Hsp104 and Hsp70) might mitigate the effects of intermediate aggregation states, using the same set of fluorescence techniques. Key reagents and fly lines are being developed while the principal investigator is on sabbatical leave in the laboratory of Dr. Nancy Bonini, an HHMI investigator at the University of Pennsylvania. The Bonini laboratory has expertise in the study of the role of several polyQ- containing proteins on fly development and aging (3-8). Preliminary work with C. elegans will be carried out in the laboratory of Dr. Christopher Link, at the University of Colorado, Boulder in the summer of 2012. He has expertise in the use of this animal model to study protein aggregative diseases (9-14). The products of this critical work will be brought back to Haverford, and will provide an important set of tools for undergraduate students to use in their senior research work. Two faculty in the Biology Department at Haverford College already use these animal models for their research, so there is adequate infrastructure support in place, which will allow for a sharing of resources, and provide new synergies in the existing research programs. By involving undergraduate researchers in all aspects of the project, this award will also contribute significantly to the education and training of future generations of biomedical scientists. PUBLIC HEALTH RELEVANCE: Twelve human diseases involve polyQ-driven aggregation, and the pathway to sequestration into inclusion bodies or nuclear inclusions has been shown to be complex, involving the accumulation of toxic oligomers. New biophysical and imaging techniques have been recently developed that allow scientists to probe aggregation both in vivo and in crude extracts, and the PI plans to use fluorescence techniques (using a confocal microscope, and characterization of molecular populations using an analytical ultracentrifuge) to probe polyQ aggregation using D. melanogaster and C. elegans model systems. The ability to mitigate potentially toxic intermediate populations will be explored using chaperones, proteins whose function is to prevent or reduce protein aggregation.

Intellectual Merit
Bacteria that can colonize multi-cellular organisms live in or on their hosts as part of complex communities. They use specific surface proteins to adhere to one another, and to host cells, to form a stable community of bacteria that are not washed away. In this research, mentored undergraduate students, will identify and characterize protein colonization factors and determine how they interact with one another. Hra1 was previously described as a member of the agglutinin family of outer surface proteins which by self-association mediates adjacent bacterial cells to adhere to one another. Hra1 also mediates in vivo binding of Escherichia coli to eukaryotic cells and specifically promotes colonization of nematode worm intestines in small aggregates. There is preliminary evidence that suggests that a second protein, Aap, prevents Hra1 mediated interactions. The inhibitory activity of Aap provides a mechanism for restricting Hra1 mediated associations as bacteria travel towards their intestinal niche. Once bacteria reach a suitable niche however, Aap must be removed for colonization to ensue. This research, will determine the mechanism for Aap removal and thus how Hra1 and Aap work together to ensure that bacteria can bind and disperse in a manner that optimizes colonization of nematode intestines. The project will also identify new surface factors involved in intestinal colonization and determine how, if at all, they interact with known surface proteins. In addition to identifying proteins involved in binding among identical bacteria, the project will focus on uncovering surface proteins that allow different species of bacteria to interact with one another, as a first step to understanding community organization among the many types of bacteria living within a single multi-cellular organism.

Broader Impact
Bacteria are the most abundant life-forms on earth and colonize a wide variety of living and non-living niches by mechanisms that are not very well understood. This project will improve current understanding of how communities of bacteria are established and persist within other organisms, as well as add to knowledge on the structure and function of bacterial surface proteins. The research required to address questions relevant to this project will largely be performed by undergraduate students working at Haverford College and overseen by a principal investigator, who teaches there. The project will provide early opportunities for such students with the hope of increasing their interest and retention in science. Four to eight students, including women and other groups underrepresented in the sciences will be engaged in laboratory research projects each year. Several dozen other students will be enriched by course modules and classroom examples in courses that are developed and taught by the principal investigator. This includes 20-40 students who will participate in inquiry-based laboratory research courses each year that will expose them to skills in microbiology, molecular biology and computational science.