Axel Brunger, Ph.D. - US grants

X-ray crystallography of biological macromolecules is an increasingly important tool to understand structure, function, and control. Developments in genetics, data collection, and computer hardware have produced an unprecedented growth of crystallographic studies. X-ray crystallography produces large amounts of data and is entirely dependent upon the availability of powerful computers and sophisticated algorithms for the interpretation of raw data. After crystallization, data collection and phasing, a large variety of computational procedures are required in order to solve and refine the structure. These procedures include methods of phasing, density modification, chain tracing, refinement, and correction of errors in structures. Recent publication of partially erroneous structures has drawn everyone's attention to the difficulties involved at these stages of the crystallographic structure determination pathway. It is the goal of this proposal to develop a comprehensive, machine-independent, and user-friendly program system for all computational aspects of macromolecular crystallography. Previous experience with the program X-PLOR will proved a framework for the proposed software development. The specifications of the proposed software development are open to discussion at a workshop that has been organized by the principal investigator and that has been attended by 30 leading researchers in crystallography. The propose software will significantly reduce the time required to obtain structures and will increase the reliability of crystallographic structure determination.

9317832 Brunger Computational approaches are important for structure determination by X-ray crystallography and solution NMR spectroscopy, studies of macromolecular structure/function relationships, protein folding, structure prediction, and drug design. The proposed workstation cluster will enable us to extend the limitations of these methods and to apply them to more difficult and important biological problems. The workstation cluster will also serve as a platform for testing advanced algorithms for the parallelization of our programs. The main computational tool for the proposed projects is the program X-PLOR (Brunger, 1992b). We routinely use X-PLOR for structure determination and refinement of X-ray and solution NMR structures, as well as for free energy perturbation calculations, structure prediction and certain aspects of structure-based drug design. It is proposed to acquire a workstation cluster which provides the most cost-effective and flexible solution to satisfy our needs. X-PLOR performs very efficiently on the proposed workstations. We intend to primarily use the cluster in a mode where multiple and independent jobs are run independently on the processors. Thus, we can readily make use of the proposed cluster without extensive software development.

Our long-range goal is to develop methodologies for drug design based on free energy calculations. We wish to obtain a better understanding of the physical principles that govern the interaction between drugs and proteins. To this end we plan to carry out free energy calculations on HIV-1 protease complexed with various inhibitors for which crystal structures and binding constants are available to us. Particular emphasis will be placed on parameterization of the force field and sampling of the conformations of the protein/inhibitor complex and of the free inhibitor. Macroscopic approaches that have been suggested recently by various groups to estimate free energies of binding will be tested and their utility for data base searches evaluated. Our efforts will include the reverse transcriptase protein of HIV as soon as appropriate high-resolution data on inhibitor complex become available.

X-ray crystallography and solution nuclear magnetic resonance spectroscopy of biological macromolecules have become important tools for understanding biological phenomena at the atomic level. The number of structures solved continues to increase rapidly due to advances in techniques such as molecular cloning, crystallization screening, data collection, and data analysis. Despite these successes, fundamental questions remain about the solution of difficult structures and about the adequate description of thermal motion and solvation of macromolecules. After data acquisition and reduction, a large variety of computational procedures are required to solve and refine a crystal or solution NMR structure. In the previous grant period we have extended the X-PLOR program by incorporating most aspects of phasing and phase improvement in X-ray crystallography. New refinement techniques were developed for both crystal and solution structures. Some of these developments were implemented by design of a high-level computer language. This high-level language allows one to easily modify existing algorithms and to even design new ones without an intervening programming step. It is proposed to pursue the following specific aims: 1. Continue the software development in order to make X-PLOR a nearly complete package for structure determination. My laboratory has recently undertaken several experimental crystallographic projects. This involvement in structure solution will enable us to test and to further develop X-PLOR by application to real problems. 2. Collect highly accurate phases for selected cases obtained by multi-wavelength anomalous diffraction and develop improved models of solvation and conformational variability. 3. Develop protocols for crystallographic refinements at low to medium resolution using all available experimental information in a reduced-variable conformational space. 4. Continue development of molecular replacement methods in the case of flexible molecules. 5 . Develop improved methods for heavy atom searches for multiple isomorphous replacement and multi-wavelength anomalous diffraction. 6. Develop improved protocols for structure determination of RNA molecules by solution NMR.

The following three projects have benefited from experiments performed at the Cornell High Energy Synchrotron Source. -Lambda phage lysozyme lysozyme from lambda phage, like other lysozymes, is capable of hydrolyzing bacterial cell-walls. However, the mechanism by which this hydrolysis is accomplished differs dramatically from that of other lysozymes (e.g. T4 - and hen egg-white lysozyme). We have recently solved the structure of the enzyme in the presence of an inhibitor to 2.7 Angstrom, by molecular replacement. The structure provides insight into the unique mechanism employed by this enzyme for hydrolyzing the peptidoglycan layer of bacterial cell-walls. - fungal homoserine dehydrogenase Homoserine dehydrogenase from fungal sources has been shown to be an excellent target for antifungal agents. Since this 370 residue enzyme lacksany methionines, we have pursued the multiple isomorphous replacement method for determining the three-dimensional structure. Optimized anomalous derivative data collected at the CHESS F2 beam-line has resulted in a partial tracing of the structure. Unfortunately, severe non-isomorphism between crystals has hampered rapid progress. - AAC(6')-Ii AAC(6')-Ii is a bacterial enzyme responsible for bacterial resistance against aminoglycoside antibiotics. Two MAD datasets of a selinomethionine derivative of this enzyme were collected at the CHESS F2 beam-line. We have successfully solved the structure, and are currently refining the atomic model. The significance of this structure determination considering the problem of antibiotic resistance is obvious.

protein structure function; synaptosomes; exocytosis; nerve /myelin protein;

Abstract Not Provided.

Abstract Not Provided.

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. The proposed experiments involve the characterization of small protein crystals. The protein in question is a fairly large (~500 kDa) complex of two different proteins. The crystals stain blue in the presence of crystal violet dye, indicating that they are composed of protein. However, the crystals grow only as small plates, with dimensions of approximately 100-200 microns on the plate face and 50 microns measured orthogonal to the face. The crystals are to be studied to determine extent of diffraction, twinning and mosaicity. Potential improvements in cryo-conditions will also be examined. The crystals have been grown in a variety of different substrates. The effect of these substrates upon the diffraction properties will be studied as well. Determination of space group and the collection of a native set for these crystals would be the ultimate goal.

mass spectrometry

X ray crystallography

Over the last 4.5 years of this Merit award (R37 MH63105) we have achieved a number of milestones: (1) the development of a single vesicle fusion assay that mimics certain properties of spontaneous and calcium triggered vesicle fusion observed in neuronal cultures; (2) the atomic resolution structure of the complex between the calcium sensor synaptotagmin-1 and the neuronal SNARE complex, revealing an unexpected calcium-independent interface that we believe forms the foundation for the process of calcium-triggered synaptic vesicle fusion; (3) the near-atomic resolution structure of the complex between NSF, SNAPs, and SNAREs that has revealed clues how NSF is capable of disassembling the SNARE complex. Moreover, we have studied the molecular mechanism of complexin-1 and Munc18. We found that complexin-1 inhibits spontaneous release and activates calcium-triggered vesicle fusion in our synthetic system, consistent with complexin's function as observed in neuronal cultures. However, we found no effect of Munc18 on intrinsic fusion rates, suggesting that its primary function is to facilitate SNARE complex formation. The specific aims for the next 5 year period are as follows: (1) Decipher the molecular mechanism of NSF-mediated SNARE disassembly. We plan to determine structures of NSF and of the complex of NSF, SNAREs and SNAPs upon hydrolyzing ATP by using single- particle cryo-EM. (2) Study how complexin and synaptotagmin-1 cooperate in fast synchronous release. Our recent work revealed a conserved, calcium-independent interface between synaptotagmin-1 and the neuronal SNARE complex. Following up on this result, we plan to investigate the interplay between synaptotagmin-1, and complexin-1, and the neuronal SNARE complex at the atomic level. We plan to crystalize this supercomplex, along with functional studies in neuronal cultures in order to test the new interfaces that we may discover in the crystal structure. (3) Investigate the role of Munc13. We will test the hypothesis that Munc13 is a facilitator for efficient SNARE complex formation. (4) Establish a hybrid fusion assay to study the fusion kinetics of purified endogenous synaptic vesicles obtained from mice brains. We will extend our single vesicle fusion assay to use purified synaptic vesicles in combination with synthetic plasma membrane mimicking ?acceptor? vesicles. We hypothesize that different pools of synaptic vesicles may result in different fusion kinetics.

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. Clostridial neurotoxins (CNTs), such as botulinum (BoNT) and tetanus (TeNT) neurotoxins, are the causative agents of the neuroparalytic diseases tetanus and botulism. CNTs impair neuronal exocytosis by specific proteolysis of SNARE proteins once inside the neuron, resulting in the clinical manifestations of flaccid and spastic motor paralysis. CNTs bind with high specificity at neuromuscular junctions. The molecular details of the toxin-cell recognition have been elusive. We reported the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15-[unreadable] resolution. On binding, a helix is induced in the luminal domain that binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. The mechanism by which a CNT properly identifies and cleaves its target SNARE once inside the neuron involves one or more regions of enzyme-substrate interaction remote from the active site (exosites). Our studies provide the basis for the development of preventive vaccines or inhibitors against these neurotoxins for bio defense, as well as design of modified neurotoxins with different target specificities for clinical applications. In addition, this work is a paradigm for protease inhibitor development in general since proteases represent major challenges for drug development due to the inherent flexibility of these enzymes.

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. The transmembrane transport of neurotransmitters is of fundamental importance for proper signaling between neurons. The transport processes are mediated by distinct classes of membrane transport proteins that have key roles in controlling the neurotransmitter concentration in the synaptic cleft. Overall, these transporters can be classed as intracellular vesicular transporters that are responsible for sequestering transmitters from the cytoplasm into synaptic vesicles, and plasma membrane transporters that are responsible for sequestering released transmitter from the extracellular space. There are three subclasses of intracellular transporters: the vesicular amine transporters (SLC18), the vesicular inhibitory amino acid transporter family (SLC32) and the vesicular glutamate transporters (SLC17). There are two major subclasses of plasma membrane transporter: the high-affinity glutamate transporters and the Na+[unreadable]Cl[unreadable]-coupled transporters. The latter subclass is the largest and includes transporters of dopamine, norepinephrine, glycine and GABA. The main research objective is to improve our understanding of the human vesicular neurotransmitter transporters, by utilizing structural data to perform functional studies. Homology modeling could then be used to produce high-quality structural models for proteins from the same family, allowing us to plan specific functional studies on each member, and possibly enabling the design of small molecules that fit the specific targets (drug design).