Lila M. Gierasch - US grants

The overall objective of the proposed research is to develop and test a model for the role of the signal sequence in protein secretion, particularly in the initial encounter between the nascent protein and membrane components. The importance of signal sequence conformation and membrane-binding properties will be investigated through the study of synthetic signal peptides. The sequences of these signal peptides will be chosen from secreted proteins of E. coli in which mutations have been selected or created within the signal region. The in vivo functions of these mutants will be correlated with the conformational preferences and membrane interactions of their signal peptides. As a model is developed for the role of the signal peptide in the secretion process, signal sequence mutants will be designed to test the model in vivo and via the physical properties of the isolated peptides. Methods to be used to study the conformations of the signal peptides include circular dichroism, nuclear magnetic resonance, infrared spectroscopy, and X-ray diffraction. A range of environments will be examined: bulk solvents, micelles, small unilamellar vesicles, or multilamellar vesicles. The membrane-binding properties of the signal peptides will be studied by tensiometry at air-water and lipid-water interfaces and by vesicle binding/fusion assays. Results of the proposed research should shed light on critical aspects of protein secretion both in prokaryotes and eukaryotes, and should also provide insight into membrane-peptide interactions in general.

Continuing studies are proposed in the area of sequenceconformation relationships in polypeptides. We will use synthetic model peptides and will examine their conformations by several physical methods, with nuclear magnetic resonance serving as the principal tool. The various peptide sequences to be studied in the next project period are of intermediate-length (5 to 40 residues) and are selected from native systems wherein structure and function appear to be directly influenced by conformational behavior of short linear polypeptide sequences. Each case therefore explores a facet of the fundamental question of importances of local interactions in proteins and peptides. Furthermore, in each case reverse turns play a critical role as conformational determinants. The conformational properties of the different sequences will be examined in a variety of environments that are illustrative of biological microenvironments and are likely to favor adoption of particular conformations. Specifically, we propose to: (1) extend our basic work on reverse turns, with emphasis on: a) importance of sequence and of environment on likelihood of gamma turns; and b) key interactions of turns in hydrophobic environments (for example, specific interactions with water); (2) investigate the conformational behavior of geneticallycharacterized "foldons" from the bacteriophage P22 tail spike endorhamnosidase; (3) test a conformational hypothesis put forth by Berzofsky and coworkers for immunodominance of particular protein sequences in activation of T cells; (4) test the hypothesis that exons encode polypeptide sequences with structural roles in the gene product; (5) determine conformations of several conformationallyconstrained analogues of gonadotropinreleasing hormone (GnRH), compare these results to molecular dynamics calculations and use the findings to propose a bioactive conformation of GnRH itself; (6) design and test potential inhibitors of C3/5 convertase, a critical protease in the complement cascade.

Continued studies are proposed to elucidate the mechanisms by which targeting sequences (including signal sequences and organellar localization sequences) facilitate correct localization of nascent polypeptide products. The emphasis of this proposal is the development of sequence/function correlations. We will use chemically synthesized targeting sequences and a number of biophysical methods to analyze them: circular dichroism (CD), nuclear magnetic resonance (NMR), infrared spectroscopy (IR), and surface tensiometry; peptide/lipid interactions will be probed in micelle, vesicle and multilayer systems. Our working hypothesis, supported by our results on signal sequences, is that there are intrinsic properties of targeting sequences that confer upon them the ability to interact productibly with cellular components and to mediate the process of protein localization. These properties will be assessed by biophysical and biochemical study of the isolated targeting sequences, of targeting sequences linked to portions of their cognate protein products, and of targeting sequences in combination with other cellular components (e.g., membranes, SRP, export factors). The importance of the context in which a targeting sequence is presented will also be investigated. Through collaborative interactions, our results will be integrated with genetic and cellular biological characterization of the targeting sequences.

Signal sequences play a central role in the membrane targeting and translocation of nearly all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes, yet major questions remain about the molecular details of their involvement in these processes. The present application has as its long-term goal elucidating the conformations and interactions of signal sequences as they participate in the steps that make up the export pathway, namely, targeting of a nascent chain to the appropriate membrane, initiation of interactions with lipids and proteins of the membrane-resident translocation apparatus, and translocation of the polypeptide chain across the membrane. We seek a fundamental physical-chemical understanding of how signal sequences may take part in these steps. We thus propose to: l) determine the conformation of a signal sequence in a lipid bilayer; 2) determine the ability of a signal sequence, in the absence of other components of the secretory apparatus, to mediate topologically specific interactions of its mature passenger with a lipid bilayer; 3) determine the conformation of a signal sequence upon binding to SecA; 4) determine the conformation of a signal sequence upon binding to the signal recognition particle (SRP); 5) map the region of SecA that is involved in signal sequence binding and use this information, in combination with structural and biochemical analyses of SecA, to develop a detailed model for SecA function; 6) map the region of SRP that is involved in signal sequence binding and use this information, in combination with structural and biochemical analyses of SRP, to develop a detailed model for SRP function; 7) as systems become available, use the same methods applied to SecA and SRP to explore the interactions of signal sequences with other proteins of the secretion pathway. In order to accomplish these aims, we will use physical methods, including nuclear magnetic resonance experiments (e.g., measurement of transferred nuclear Overhauser effects) that enable determination of the conformation of a signal peptide bound to a membrane or to a protein of the export pathway. We will also employ biochemical approaches such as cross-linking and limited proteolysis to define the peptide-binding regions of the SecA and SRP54 proteins that recognize the signal sequence in bacteria and mammals, respectively. Since all serum antibodies, digestive enzymes, and peptide hormones are secreted, as are many other physiologically important proteins, this research will shed light on several biomedical problems.

The projects proposed address two areas where fundamental studies are critically needed: the mechanism of folding of predominantly beta-sheet proteins, and the mechanism of action of the Hsp70 family of molecular chaperones. The specific aims that will be pursued are: 1) Based on the model for folding emerging from studies in the previous grant period, we will use cellular retinoic acid binding protein I (CRABP I), a predominantly beta- sheet protein with an ill-defined hydrophobic core, to develop principles about the relation between amino acid sequence and the folding of beta sheet proteins. We seek to understand how hydrophobic interactions influence CRABP I folding, how important local sequence conformational preferences are in CRABP I folding, what residue interactions specify the topology of CRABP I, and what the nature of intermediate states is for this beta-rich protein. To address these aims, we will carry out sequence and structure analysis of CRABP I and its family members; perform protein engineering experiments to dissect the contributions of specific residues and groups of residues in folding; assess the influence of local sequences by study of peptide fragments and by kinetic analysis of sub-millisecond folding events; and explore the dynamics of CRABP I using experimental and computational methods under conditions where the protein is stable and under unfolding conditions. 2) We will carry out structure-function studies of two highly homologous members of the Hsp70 family of moleuclar chaperones, DnaK, the E. coli representative of this family, and BiP, the mammalian representative that resides in the lumen of the endoplasmic reticulum. We seek to understand how ATP binding to the N-terminal domain of Hsp70s leads to reduced affinity of substrate binding to the C-terminal domain, and how two highly homologous Hsp70s have the common ability to recognize unfolded substrates, yet show differential sequence preferences in their substrates. To address these aims, we will analyze in detail mutants that have impaired allosteric communication, using NMR structure determination and biochemical assays. We will compare structures with and without substrate bound. We will incorporate tryptophan and cystein residues in strategic locations, attach fluorophores to the Cys residues, and measure interdomain movements by fluorescence energy transfer-based distance. Our studies of the folding of a predominantly beta- sheet protein will contribute to an improved understanding of the origin of amyloidogenic diseases, such as Alzheimer's, bovine spongiform encephalomyelitis, and Huntington's, which appear to arise from misfolding events. Elucidation of the mechanism of action of the Hsp70 family of molecular chaperones will enhance our understanding of how organisms respond to heat shock and stress, as well as the origins of diseases postulated to arise from defects in Hsp70-facilitated protein folding, such as cystic fibrosis and p53-related cancers.

9871451
Kaltashov
A high resolution magnetic sector mass spectrometer will be purchased under this Major Research Instrumentation award. The instrument includes a high-resolution, high-transmission efficiency, and high m/z range double focusing mass analyzer. The mass spectrometer is equipped with a standard electron impact/chemical ionization ion source, as well as easily interchangeable fast atom bombardment, electrospray ionization and field desorption/ionization sources. The configuration also includes two collision cells and a linked scan unit providing tandem capabilities. Major users of the instrument include groups from the Department of Polymer Science and the Department of Chemistry. The instrument will also benefit a large group of minor users from other departments such as Biochemistry and Molecular Biology, Veterinary and Animal Science, Department of Chemical Engineering, Food Science. It will also be used by researchers from Amherst College and Mt. Holyoke College. The 16 research groups that will be users of the mass spectrometer include 22 post-doctoral fellows, 82 graduate students and 33 undergraduate students.
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The high resolution magnetic sector mass spectrometer purchased with the Major Research Instrumentation award will become a part of the University of Massachusetts' Mass Spectrometry Center. Because of its high resolution, unmatched mass accuracy and a variety of ionization modes, the new instrument will greatly expand the analytical capabilities offered by the Center.
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With this award from the Chemistry Research Instrumentation and Facilities (CRIF) Program, the Department of Chemistry at the University of Massachusetts in Amherst will acquire a CCDbased X-ray diffractometer. This equipment will enhance research in a number of areas including the following: a) synthesis of novel olefin polymerization catalysts; b) biomimetic solid-state syntheses of inorganic/organic composites; c) crystal structures of stable radicals and related precursor substances; d) studies of metallobiomolecules and synthetic model approaches; e) crystal engineering using functionalized diaminotriazines; and f) the design and construction of solid state materials using 'crystal engineering'.

The X-ray diffractometer allows accurate and precise measurements of the full three dimensional structure of a molecule, including bond distances and angles, and it provides accurate information about the spatial arrangement of the molecule relative to the neighboring molecules.

9908399

Abstract

This project involves the upgrade of a 500 MHz NMR spectrometer to provide state-of-the-art capabilities in order to support the research of a group of users in the Department of Chemistry at University of Massachusetts, Amherst. The major research projects will be facilitated by the availability of the new instrumentation. This research will include determination of the structure of the peptide-binding domains of the molecular chaperones DnaK and BiP; determination of bound conformation of signal sequences upon interaction with their receptors; determination of the structure of an SRP-binding RNA fragment alone and in complex with a polypeptide; studies of the mechanism of folding of the predominantly beta-sheet protein, cellular retinoic acid biding protein; structural analysis of myoglobin mutants as models for guanylate cyclase; conformational studies of model helical peptides; determination of the active site geometry of metalloproteins; determination of the structure and dynamics of membrane proteins, including E. coli chemotactic receptors and colicin A; studies of domains of the E. Coli chemotactic receptor that are involved in adaptation; and determination of the structures of ribosomal proteins and the RNAs to which they bind. Additional projects will further utilize the remaining time on this instrument.

We request funds to purchase a state-of-the-art 600 MHz nuclear magnetic resonance spectrometer to support the research of a group of NIH- sponsored major users (Gierasch, Decatur, Maroney, Thompson, Weis, Zimmermann), whose research will require 85% of the instrument time. The remaining time of the instrument will be allotted to minor users (Gross, Hong, Martin, Rotello, Krejsa) and to maintenance and implementation of new experiments by the NMR Facility Manager and Associate Manager. The major research projects that will be facilitated by the availability of the requested instrumentation include: determination of the structure of the peptide-binding domains of the molecular chaperones DnaK and BiP; determination of the bound conformation of signal sequences upon interaction with their receptors; determination of the structure of an SRP-binding RNA fragment alone and in complex with a polypeptide; studies of the mechanism of folding of the predominantly beta-sheet protein, cellular retinoic acid binding protein; structural analysis of myoglobin mutants as models for guanylate cyclase; conformational studies of model helical peptides; determination of the structure and dynamics of membrane proteins, including E. coli chemotactic receptors and colicin A; studies of domains of the E. coli chemotactic receptor that are involved in adaptation; and determination of the structures or ribosomal proteins and the RNAs to which they bind. Minor projects that will benefit from the availability of this instrumentation include: studies of bound conformations of EGF receptor fragments upon interactions with actin; structural characterization of difficult sequences for peptide synthesis; structural studies of domain of T7 RNA polymerase; structural studies of models for flavin cofactors and their environmental perturbations; and correlation of crosslinker structure with physical and chemical properties of nylons.

Signal sequences play a central role in the membrane targeting and translocation of nearly all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes. This project has as its goal the elucidation of the conformations and interactions of signal sequences as they participate in the steps that make up the export pathway, namely, recognition of a nascent or newly synthesized secretory protein, targeting of the protein to the appropriate membrane, initiation of interactions with lipids and proteins of the membrane- resident translocation apparatus, and translocation of the polypeptide chain across the membrane. We seek a fundamental physical-chemical understanding of how signal sequences may take part in these steps. In the next project period, we will focus on the roles of signal sequences in early steps in export-recognition and targeting. To this end, we will continue studies using synthetic signal peptides and a battery of biophysical and biochemical methods to elucidate the nature of signal sequence binding by two proteins that serve as the first point of recognition of a protein that is destined for the export pathway, namely the signal recognition particle (SRP), which recognizes secretory proteins in eubacteria and targets them to the endoplasmic reticulum membrane, and SecA, the central player in membrane insertion of the polypeptide chain in bacteria, and to relate the findings to the mechanism of protein export in bacteria and mammals. In our studies, we will use Ffh, the E. coli homologue of the mammalian SRP54, as our SRP model. Fluorescence, circular dichroism, nuclear magnetic resonance, and site-specific mutagenesis will be used to determine the conformation of signal peptides upon binding to Ffh and to SecA, and, correspondingly, the structural basis of the protein's ability to bind signal peptides. Furthermore, we will explore the influence of binding to signal sequences and other ligands on the function of Ffh and SecA. Notably, we will explore the relationship of the 4.5S RNA to the structural stability and functions of Ffh. Since all serum antibodies digestive enzymes, and peptide hormones are secreted, as are many other physiologically important proteins, this research will provide important fundamental insight into several biomedical problems. The bacterial secretory apparatus is also a target for new antibiotics, the design of which will be aided by detailed understanding of the key components.

The traditional approaches for treating oligopeptides and proteins in molecular mechanics and molecular dynamics assume that the charges are invariant to conformational changes. These charges are fixed at the beginning and are kept constant throughout the whole simulation. Attempts to use these traditional approaches to explain some experimental dependences of oligopeptide conformation on pH have not been very successful. Our goal was focused on a specific aspect of the folding processes: It attempts to understand how the enviroment affects the conformational preference of a short polypeptide with the sequence: Ac-ETGTKAELLAKYEATHK-NHMe, and vice versa; i.e. how the conformation affects the pK's of ionizable groups. In our work a new approach is presented. It considers explicitly the coupling between the conformation of the molecule and the ionization equilibria at a given pH value. Calculations of the solvation free energy and free energy of ionization of this 17-residue (for which the available NMR and CD experimental data indicate that the conformations containing a right-handed alpha-helix segment at low pH are energetically more favorable) were carried out using a fast multigrid boundary element method (MBE). The MBE method was integrated into ECEPP/3 (Empirical Conformational Energy Program for Peptides) algorithm to compute the coupling between the ionization state and the conformation of the molecule. The results of our calculations using the present method agree quite well with experiments, in contrast with previous applications with standard techniques (using pre-assigned charges at each pH). In particular we have been able to show how the coupling to the conformation leads to different degree of ionization of a given type of residue, for example glutamic acid, at different positions in the amino acid sequence, at a given pH. The results of this study provide a sound basis to discuss the origin of the stability of oligopeptide conformations and its dependence on enviromental conditions. We expect to continue with the development of this approach and further testing of a series of oligopeptides of biological interest.

[unreadable] DESCRIPTION (provided by applicant): We request funding to support the next three Federation of American Society of Experimental Biologists (FASEB) Summer Conferences on "Protein Folding in the Cell," beginning with the 2002 meeting to be held July 27 to August 1, in Saxtons River, VT. The proposed conferences will be the seventh, eighth, and ninth in a series of highly successful FASEB Conferences on this topic. This meeting is unique in its goal of bringing together cell biologists/geneticists and biophysicists/biochemists to tackle the complex problems of protein folding and misfolding and implications for human disease. This was the concept behind the first conference in 1990, and it remains a unique opportunity for scientists from very diverse backgrounds. Most protein folding conferences consider either the theoretical and in vitro aspects of folding or the role of molecular chaperones and protein folding catalysts in the cell. However, the need for interdisciplinary approaches is stronger than ever. The cell biology has progressed to where reaching a mechanistic understanding is crucial, and biological constraints play a critical role in formulating theory and interpreting in vitro data. In addition to examining recent advances in in vitro folding and molecular chaperone mechanism, this meeting will focus on protein translocation across membranes, protein misfolding in diseases, folding of membrane proteins, folding of metalloproteins, newly recognized roles of chaperones in signal transduction, and protein degradation. Misfolding of proteins and potential consequences are of central importance in human disease. Finding therapeutic strategies requires a fundamental understanding of the behavior of the polypeptide chain and the cellular environment and constraints imposed on it. Our goal is to bring together physical and biological scientists in an interactive environment, to promote education and understanding of the approaches, experimental limitations and to explore common interests. The small size and isolated setting of this meeting with all participants residing and taking their meals in close proximity promotes optimal scientific exchange. The conference format will provide extensive arenas for informal discussions. Poster sessions will allow all participants to interact scientifically with other investigators. Young investigators and women will be well-represented among speakers and encouraged to attend the conference.

DESCRIPTION (provided by applicant): This project has as its overarching goal elucidation of the roles played by signal sequences in various steps of protein export or integration of proteins into the cytoplasmic membrane. Signal sequences serve as cellular 'zipcodes' in that they carry essentially all of the information necessary to target a protein for export from the cell. Despite considerable progress in our understanding of protein secretion and integration into membranes, the means by which signal sequences on nascent or newly synthesized proteins are specifically recognized, stably bound, and then released at the appropriate time for interaction with membrane components is still poorly understood. The proposed work addresses the question of how these processes are accomplished by the two signal sequence-mediated pathways in E. coli, that involving Ffh, which is homologous to the 54 kDa subunit of the mammalian signal recognition particle (SRP), and that involving SecA, a uniquely prokaryotic protein. To address this goal, we will utilize synthetic peptides corresponding to signal sequences of bacterial proteins as probes. The proposed strategy is to identify the signal sequence-binding sites on Ffh and SecA by cross-linking them to signal peptides; to explore how these binding sites are altered in affinity as the signal sequence is bound and released; and to determine the conformation adopted by a signal sequence bound either to Ffh or to SecA. We hypothesize that both of these proteins have intramolecular sequences that bind to the hydrophobic signal sequence-binding site to protect it in the absence of a nascent or precursor protein. Several diseases (e.g., cystic fibrosis, Alzheimer's, Wolman, and some prion-associated diseases) have been shown to be associated with signal peptide polymorphisms that lead to mislocalization of a secreted protein or to hypersecretion. Elucidation of the mode of recognition, binding, and release of signal sequences by SRP will help efforts to develop therapeutic strategies for these diseases. Additionally, SecA is an ideal target for antibiotics, since it is uniquely present in bacteria. Enhanced understanding of its mode of recognition will provide a basis for development of new antibacterial agents.

Proteins are the basic building blocks of life. They perform many important functions within the cells of each living being. These functions include signaling, metabolism, transport, and reproduction. A protein performs its function by interacting with other proteins or molecules inside the cell. Such an interaction may result in the binding of two molecules to form a complex, or in the separation of such a complex into its components. During the interactions among proteins, each molecule may have to change its three-dimensional shape in order to accommodate the binding with another molecule. This is possible because many proteins are inherently flexible. It is generally accepted that the three-dimensional shape of a protein and its ability to change its shape uniquely determine the protein's biological function.

An understanding of the biological function of proteins in the cell would allow a detailed understanding of the complex processes that happen inside a cell. Such an understanding would also facilitate the design of new drugs to influence these processes, should they be affected in the case of a disease. To gain such an understanding from biological experiments alone is very costly and time-consuming. The ability to accurately simulate interactions among flexible biomolecules therefore promises to facilitate scientific advances in computational drug design and represents an important computational tool to expand our understanding of cellular processes. As a significant contribution towards this objective, the investigators propose to develop a novel computational framework for computationally efficient and biologically accurate docking of flexible proteins.

The problem of protein docking is computationally challenging, even under the simplifying assumption that both bodies are internally rigid. However, ignoring the internal flexibility of a protein is recognized as a shortcoming in current docking approaches. The proposed algorithmic framework uses methods from robotics to effectively analyze and model the internal flexibility of a protein.
Based on this analysis, conformational changes occurring during the docking process can be accommodated effectively. The resulting computational framework can be seen as a new algorithmic foundation for efficient and biologically accurate computational docking of flexible proteins.

Surprisingly little is known about how proteins fold in vivo, yet it is this process, and not the test-tube idealized folding reaction so intensively studied over the past several decades, that is crucial to the fitness of an organism. The fidelity of folding and the stability of proteins in the cell are critical to their functions, their degradation, and their vulnerability to aggregation. Many diseases are now known to arise from defects in protein folding, either because of the loss or alteration of essential protein functions, or because of the build-up of toxic species such as aggregates. We believe that novel methods and creative collaborations will allow us to overcome the daunting technical obstacles that have impeded progress on protein folding in the cell. Focusing first on a small model protein for which we have detailed descriptions of folding in vitro will enable methods optimization. Folding in cellular conditions will be followed in systems of increasing complexity: bacterial protein expression, cell-free biosynthesis, and semi-permeabilized or intact eukaryotic cell expression. The new strategies will reveal how larger proteins of biomedical interest adopt their structures in their cellular context and how this process may go awry. Methodologically, we anticipate placing major reliance on spectroscopic methods, including fluorescence and nuclear magnetic resonance, and using novel labeling strategies to observe the protein under study in the complex cellular milieu. Complementary in-cell imaging methods will be used to insure that observed signals report on relevant phenomena and to reveal novel functionally significant spatial localization patterns. We anticipate that this research will lead to new paradigms for how amino acid sequences encode folding information and that the resulting enhanced understanding of folding in vivo will lead to new strategies for therapeutic intervention in misfolding and aggregation diseases.

This Integrative Graduate Education and Research Traineeship (IGERT) award establishes a novel interdisciplinary training program at the University of Massachusetts-Amherst to address the emerging field of Cellular Engineering. Engineering cellular form and function is the basis for many ventures in the biomedical and biotechnology industries, including design of bioremediation processes, generation of artificial organs/tissues, production of biologics from cell culture, design of new and improved protein-based pharmaceuticals and targeted drug delivery. Students matriculate in one of 12 degree programs with a research focus in one of three interrelated cellular engineering thrust areas: 1) Applied Systems Biology, 2) Cell Delivery and 3) Protein Engineering. Key features include a novel unifying lecture/laboratory course to train both life scientists and engineers/physical scientists in cellular engineering fundamentals, interdisciplinary research involving "supergroup" projects in which students seek out collaboration with a related training laboratory; interactions with industry through the established UMass-Amherst Institute for Cellular Engineering; weekly research seminars with a mentoring component; and formal professional development activities.

This IGERT has all-female leadership and significant numbers of female faculty participants. Underrepresented students are recruited through the NEAGEP, an NSF-funded project co-led by UMass-Amherst and including ten research-extensive and six minority-serving institutions that collaborate to increase the number of underrepresented students who receive doctoral degrees in science, technology, engineering and mathematics disciplines. This IGERT encourages novel research collaborations in cellular engineering among faculty, creating new bridging programs among departments and providing unique learning opportunities for trainees. Purposeful alignment with the Institute for Cellular Engineering enables substantial interaction with regional cellular engineering companies, significantly broadening student training. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

DESCRIPTION (provided by applicant): Hsp70 chaperones occur in all organisms and essentially all cellular compartments. Among their wide array of essential cellular functions, they facilitate folding of newly synthesized proteins;protect cells from damage such as aggregation that can occur under stress conditions;help to target proteins to extra- cytoplasmic locations;and facilitate assembly and disassembly of macromolecular complexes. All of these functions rely on the ability of Hsp70s to bind unfolded regions of a protein substrate, and to release their substrates upon allosteric binding of ATP. The research proposed focuses on the fundamental molecular mechanism of Hsp70 allostery. The work proposed builds on exciting recent results: We showed in the last project period that both the ATPase domain and the substrate-binding domain (SBD) of the paradigmatic E. coli Hsp70 DnaK undergo major conformational changes upon ATP binding, and we gained understanding of the allosteric remodeling of these domains. Our results led us to a model for interdomain allosteric communication in DnaK that has been validated by a recent structure from the Hendrickson lab of a related chaperone, Sse1, the yeast Hsp110 [Q. Liu and W. A. Hendrickson, Cell 131, 106-1202007)]. Our specific aims are: to refine the current Sse1-based homology model of ATP-bound DnaK and to use this model, together with our knowledge about the ADP-bound state of DnaK, to elucidate the mechanism of allosteric interdomain communication in this Hsp70 molecular chaperone;to assess the generality of results on DnaK and develop general principles about Hsp70 allosteric function;to explore how the allosteric conformational changes in DnaK are modulated by interaction with co-chaperones DnaJ and GrpE. We will utilize new NMR strategies applicable to large molecules in order to analyze both structural and dynamic aspects of the allosteric conformational transitions in Hsp70s upon binding to their ligands and co-chaperones. Complementary data will be provided by time-resolved fluorescence energy transfer and electron spin resonance methods, as well as computational approaches based on sequence analysis, normal mode calculations, and ensemble-based thermodynamic dissection of ligand-mediated energetics. Hsp70s constitute relatively simple allosteric machines. Studying in detail their allosteric interdomain communication will shed light on the broader puzzle of how proteins harness ligand-binding energy to modulate binding and catalytic functions at a distance. PUBLIC HEALTH RELEVANCE Hsp70 molecular chaperones play key cellular roles under normal physiological conditions and enable cells to withstand stress such as heat shock. Hsp70s are known to be anti-apoptotic and up- regulated in tumors;ironically, their up-regulation is protective against neurodegenerative diseases caused by protein misfolding. The intimate involvement of Hsp70s in both normal and disease states has led to their emergence as possible therapeutic targets, but using heat shock proteins in a therapeutic capacity requires that we fully understand their mechanism of action, including how nucleotide modulates substrate binding and how interactions with co-chaperones modulate Hsp70 allostery.

The endoplasmic reticulum (ER) is a folding factory for the cell, able to churn out thousands of secretory proteins per second. Misfolding of a number of these proteins results in diseases ranging from cystic fibrosis to liver cirrhosis. While full-length proteins can be studied in vitro using a myriad of biophysical techniques, thus allowing biophysicists to characterize in detail their folding energy landscapes, studying protein folding in the more physiologically relevant ER environment presents daunting technical obstacles. Using recently developed, powerful single molecule fluorescence techniques on nascent polypeptide chains that contain fluorescently-labeled amino acids, we will determine the conformational evolution as a nascent polypeptide chain elongates in the ER and how conformational space is modulated by interactions with the chaperones and modifying enzymes resident in the ER. The goal is to obtain detailed information on the folding landscape of the growing nascent chain, rivaling the data available in test-tube experiments. Co- and post-translational interactions between the ER-resident proteins and nascent chains likely smooth the folding energy landscape, biasing against misfolded and aggregation-prone intermediates. This remodeling of the folding landscape is particularly important for proteins with high contact order, i.e. many contacts between distal parts of the chain. We will focus on an important family of secreted proteins, the inhibitory serpins, which fold into native structures with high contact order. Intriguingly serpin native states are energetically metastable, poised to change to a different structure. Serpins play crucial biological roles by regulating proteases involved in key physiological processes including blood coagulation and inflammation. A number of serpin mutations, associated with diseases such as liver cirrhosis and emphysema (together called 'serpinopathies'), lead to serpin aggregation in the ER. By studying serpin folding in the ER and comparing it to in vitro folding, we will determine how the folding landscape is altered by interactions with ER-resident proteins and by disease-associated mutations. The results of this research will provide unprecedented insight into how serpins misfold and how their energy landscapes differ in vitro and in vivo. This work will also provide a readily accessible experimental toolbox for scientists studying protein folding in the ER as well as a platform for potential therapeutic strategies to treat serpinopathies and other ER folding diseases. PUBLIC HEALTH RELEVANCE: Secretory proteins, many of which are associated with diseases, begin life in the endoplasmic reticulum (ER). By deploying powerful biophysical and cell biological approaches, we will explore the folding energy landscape in the ER of a biomedically significant secreted protein from the serpin family and determine how it is altered by disease-causing mutations.

DESCRIPTION (provided by applicant): Protein structure prediction is one of the great challenges in structural biology. The ability to accurately predict the three-dimensional structure of proteins would bring about significant scientific advances and would facilitate finding cures and treatments for many diseases. We propose a novel computational framework for protein structure prediction. The novelty of the framework lies in its approach to conformation space search. Conformation space search is considered to be the primary bottleneck towards consistent, high-resolution prediction. The proposed approach to conformation space search represents a major conceptual shift in protein structure prediction, made possible by combining insights and algorithms from robotics and machine learning with techniques from molecular biology in an innovative manner. The key innovation comes from the insight that target-specific information can effectively guide conformation space search towards biologically relevant regions. We propose to develop a framework for protein structure prediction that achieves biological accuracy and computational efficiency by guiding conformation space search using target-specific information. The proposed framework exploits two sources of target-specific information: 1) information about the characteristics of the target's energy landscape acquired continuously during search, and 2) spatial restraints about the target's structure obtained from NMR experiments. As search progresses, the continuous integration of these sources of information will tailor conformation space search to the particular characteristics of the target. This tailored conformation space exploration can overcome the current bottleneck, yielding highly accurate and efficient structure prediction. The ability to determine the three-dimensional structures of proteins, which represent the molecular machinery inside every cell, would greatly facilitate finding cures or treatments for many diseases. This research effort will develop of a novel, efficient, and biologically accurate computational approach to determine the three-dimensional structure of proteins.

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. Molecular chaperones play essential roles in cellular maintenance and response to stress by protecting unfolded and misfolded proteins until they can be folded or assembled into complexes and then helping promote successful folding and assembly. They also partner with degradation machinery to manage cellular 'quality control'and prevent accumulation of potentially deleterious misfolded products. Chaperones work together to form a complex proteostasis network that is essential for cell survival. The ubiquitous Hsp70 protein family of chaperones prevents incorrect interactions in unfolded proteins that can lead to misfolding or aggregation by cycles of binding and release of client proteins regulated by ATP hydrolysis and further modulated by co-chaperones such as nucleotide exchange factors (NEFs) and Hsp40s. Understanding cellular roles of Hsp70s requires in-depth elucidation of the Hsp70 allosteric mechanism and interactions with co-chaperones;this is essential in order to develop therapeutic strategies based on modulation of Hsp70 chaperones. The goal of our work at the ACERT facility is to obtain crucial information for a full description of the structural ensembles of the E. coli Hsp70, DnaK, throughout its functional allosteric cycle and upon interaction with co-chaperones. We will determine distance profiles between various spin pairs strategically located throughout the protein. These distance profiles will provide information about the structures populated by DnaK in various nucleotide- or substrate- or co-chaperone-bound states. In addition, we will use double electron-electron resonance (DEER) experiments to identify sparsely populated conformers within these ensembles. These profiles, in conjunction with paramagnetic relaxation enhancement NMR data being collected at U. Mass will enable us to map out the allosteric landscape of an Hsp70.

DESCRIPTION (provided by applicant): We propose to develop a computational model of a complete cellular protein homeostasis (proteostasis) network, including protein synthesis, degradation, folding stability, aggregation, and competing/cooperating chaperoning systems. This project is a collaboration between Lila Gierasch (University of Massachusetts Amherst) and Evan Powers (The Scripps Research Institute). Our model, called FoldEco, aims to describe the balance of biochemical and physical aspects of folding and aggregation, and their impact on the health of the proteins in proteomes in general, and here the proteome of the E. coli cytoplasm, in particular. FoldEco begins with the current extensive knowledge of mechanisms, biochemical circuits, and parameters, and allows for the generation of hypotheses about large-scale, complex protein folding networks under various conditions. We propose now: (1) to advance FoldEco so that it better captures the full complexity of the E. coli proteome as well as physiological processes such as the heat-shock regulatory response that play a role in proteostasis in E. coli; (2) to experimentally interrogate E. coli proteostasis under physiological conditions in order to test and ultimately improve the FoldEco model. As part of our work, we will be addressing the burden placed on the proteome by perturbation of individual proteins, how well the proteostasis network copes with such perturbations, and whether some proteins are particularly vulnerable to such perturbations. We will continue to make the FoldEco model freely available to the broad community through a web-based interface. We already have the FoldEco code in a first generation version and several experimental tests of predictions of the code. Because chaperone networks are conserved across all organisms, this work in the simple model organism, E. coli, will provide insight into protein homeostasis in higher organisms and potentially assist in the development of therapeutic strategies for protein misfolding diseases. Additionally, FoldEco will benefit the biotechnological and pharmaceutical industries where the need to produce functional proteins efficiently is critical. If successful, this work will broadly advance our ability to comprehend complex circuits that play critical roles in health and disease.

? DESCRIPTION (provided by applicant): Protein folding is a process of supramolecular assembly that suffers unavoidable competition from alternative processes including misfolding to dead-end products and intermolecular association that may lead to aggregation. Yet, in order to perform their normal functions most proteins must fold correctly in challenging and complex cellular environments. In vivo, the processes that compete with folding can cause major physiological problems both because a protein cannot function if it does not adopt its native fold and also because the side-products of misfolding are potentially toxic to cells. Protein misfolding and its attendant consequences are implicated in a growing number of diseases, including cystic fibrosis, serpinopathies, and a number of neurodegenerative diseases such as Parkinson's, Huntington's and Alzheimer's. Cells commit substantial resources to facilitate protein folding in complex in vivo environments and to minimize risks of misfolding. Central to how cells cope with the challenges of protein folding are the molecular chaperones and degradation enzymes that together comprise a protein homeostasis network. However, the ability of protein homeostasis networks to cope with proteins that are prone to misfolding (due either to their intrinsic properties, or mutations, or aberrant production) can be exceeded, and it is this situation that underlies many pathologies. The research in this MIRA application seeks to elucidate the interplay between biophysical properties of protein folding and the mechanisms of protein homeostasis networks through two overarching projects: 1) a combined computational modeling and experimental interrogation of protein homeostasis networks in E. coli and in eukaryotic cellular compartments, and 2) structure-function studies of the Hsp70 family of molecular chaperones, which play central roles in protein homeostasis networks in all kingdoms of life. The knowledge and insights gained from this research will shed light on how in-cell protein folding energy landscapes are remodeled by protein homeostasis networks. The understanding and insights provided by this research will help guide future efforts to develop therapeutic strategies to treat protein misfolding-associated diseases.

? DESCRIPTION (provided by applicant): Protein folding is a process of supramolecular assembly that suffers unavoidable competition from alternative processes including misfolding to dead-end products and intermolecular association that may lead to aggregation. Yet, in order to perform their normal functions most proteins must fold correctly in challenging and complex cellular environments. In vivo, the processes that compete with folding can cause major physiological problems both because a protein cannot function if it does not adopt its native fold and also because the side-products of misfolding are potentially toxic to cells. Protein misfolding and its attendant consequences are implicated in a growing number of diseases, including cystic fibrosis, serpinopathies, and a number of neurodegenerative diseases such as Parkinson's, Huntington's and Alzheimer's. Cells commit substantial resources to facilitate protein folding in complex in vivo environments and to minimize risks of misfolding. Central to how cells cope with the challenges of protein folding are the molecular chaperones and degradation enzymes that together comprise a protein homeostasis network. However, the ability of protein homeostasis networks to cope with proteins that are prone to misfolding (due either to their intrinsic properties, or mutations, or aberrant production) can be exceeded, and it is this situation that underlies many pathologies. The research in this MIRA application seeks to elucidate the interplay between biophysical properties of protein folding and the mechanisms of protein homeostasis networks through two overarching projects: 1) a combined computational modeling and experimental interrogation of protein homeostasis networks in E. coli and in eukaryotic cellular compartments, and 2) structure-function studies of the Hsp70 family of molecular chaperones, which play central roles in protein homeostasis networks in all kingdoms of life. The knowledge and insights gained from this research will shed light on how in-cell protein folding energy landscapes are remodeled by protein homeostasis networks. The understanding and insights provided by this research will help guide future efforts to develop therapeutic strategies to treat protein misfolding-associated diseases.