REPEAT-PROTEINS; STABILITY, FOLDING KINETICS & EVOLUTION
Douglas E Barrick, Associate Professor
Johns Hopkins University, W400 Wyman Park Building, Baltimore, Md 21218
Grant 5R01GM068462-06 from National Institute Of General Medical Sciences
Abstract: Protein folding is the process by which polypeptides adopt their complex, three dimensional structure. In most monomeric proteins, this structure is required for function, and is encoded in the amino acid sequence. Thus, protein folding is the bridge between the gene and its function, and is central to understanding biology. Deciphering the rules by which proteins fold is also critical for understanding a number of genetic diseases that result either from essential gene products that cannot fold to their native state, or from proteins that misfold to a non-native, aggregation-prone complex, forming toxic oligomers or fibers. The research proposed here seeks to understand the folding problem using proteins of a simplified architecture in which a small cluster of secondary structure units (helix, strand, turn) is repeated in a linear array. The extended, modular architecture of repeat proteins allows units of structure to be removed and inserted, providing a detailed mapping of how folding energy is distributed along the polypeptide chain. This direct mapping of the energy landscape allows long-standing questions about protein folding to be addressed, such as the origin and kinetic consequences of cooperativity, existence and specification of kinetic pathways. In addition, the structural similarity of the repeated units allows the contributions of different regions to be compared with great clarity. Here we use two different repeat protein architectures, the ankyrin (a/a) and LRR (¿/non-¿) repeats to explore the structural origins of cooperativity, the role of cooperativity in folding kinetics, and how bulk cooperativity is manifested when unfolding is promoted by a directed force. To rigorously quantify cooperativity and its structural origins, we will take advantage of a recent discovery by us and by other groups that stable arrays can be built of repeats of identical sequence. These "consensus arrays" will be analyzed using an "Ising" statistical model, which quantifies intrinsic versus nearest-neighbor energies. Consensus sequence variants will be used to resolve which types of interactions give rise to the extraordinary cooperativity we have seen in these proteins. Once we have variants in hand that resolve local versus long-range interactions, we will be able to probe how cooperativity influences kinetics and transition state ensembles, developing a kinetic Ising model in the process. Kinetic analysis of these proteins will also provide insights as to how folding proceeds on a genuinely "flat" landscape. These cooperativity variants will also be used to explore the relationship between solution cooperativity and end-to-end forced unfolding. Comparison to natural (nonconsensus) repeat arrays will provide continued insight into the relationships between sequence, stability, and folding in these simple but ubiquitous proteins. Studies will combine standard equilibrium and stopped flow folding with collaborative hydrogen exchange mass spectrometry, atomic force microscopy, and optical tweezer methods. A large number of human diseases including cancers and Alzheimer´s disease are caused by proteins that cannot fold up to their active shapes, or that fold to the wrong shapes, poisoning cells and tissues. The proposed research will use simplified "repeat" proteins to learn the rules of how proteins fold into unique, well-determined structures. These rules will help us to understand the causes of "folding diseases", and will also provide new biomaterials that can be used to diagnose and perhaps ultimately treat human diseases
Keywords: 3-D structure; 3-dimensional structure; 3D structure; ANK Domain; ANK Repeat; Address; Adopted; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer`s; Alzheimer`s Disease; Alzheimers Dementia; Alzheimers disease; Amino Acid Sequence; Ankyrin Repeat; Ankyrin Repeat Domain; Ankyrins; Architecture; Atomic Force Microscope; Atomic Force Microscopy; Biocompatible Materials; Biological Models; Biology; Biomaterials; Body Tissues; Cancers; Cations; Cells; Charge; Complex; Consensus; Consensus Sequence; ConsensusSequence; Data; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Diagnosis; Disease; Disorder; Electrostatics; Elements; Engineering / Architecture; Environment; Equilibrium; Essential Genes; Evolution; Fiber; Force Microscopy; Free Energy; Genes; Genes, Essential; Genetic Condition; Genetic Diseases; H element; Hand; Height; Hereditary Disease; Hydrogen; Kinetic; Kinetics; LRR; LRR protein; Learning; Length; Leucine-Rich Repeat; Malignant Neoplasms; Malignant Tumor; Maps; Mass Spectrum; Mass Spectrum Analysis; Methods; Microscopy, Atomic Force; Model System; Modeling; Models, Biologic; Models, Statistical; Molecular; Molecular Disease; Nature; Optics; Pathway interactions; Photometry/Spectrum Analysis, Mass; Poisoning; Position; Positioning Attribute; Primary Senile Degenerative Dementia; Probabilistic Models; Process; Protein Analysis; Protein Array; Protein Structure, Primary; Proteins; Research; Right-Handed Beta-Alpha Superhelix; Role; SEQ-AN; Scanning Force Microscopy; Screening Result; Sequence Analyses; Sequence Analysis; Shapes; Side; Sodium Chloride; Sodium chloride (NaCl); Solutions; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Statistical Models; Structure; System; System, LOINC Axis 4; Testing; Thermodynamic; Thermodynamics; Tissues; Variant; Variation; aberrant protein folding; abnormal protein folding; balance; balance function; dementia of the Alzheimer type; density; disease/disorder; experiment; experimental research; experimental study; gene product; genetic disorder; globular protein; hereditary disorder; human disease; insight; interfacial; laser tweezer; leucine-rich repeat protein; malignancy; neoplasm/cancer; optical tweezers; pathologic protein folding; pathway; poisoned; polypeptide; primary degenerative dementia; protein folding; protein mis-folding; protein misfolding; protein sequence; protein structure; public health relevance; research study; salt; senile dementia of the Alzheimer type; simulation; social role; theories; three dimensional structure
Relevance: A large number of human diseases including cancers and Alzheimer´s disease are caused by proteins that cannot fold up to their active shapes, or that fold to the wrong shapes, poisoning cells and tissues. The proposed research will use simplified "repeat" proteins to learn the rules of how proteins fold into unique, well-determined structures. These rules will help us to understand the causes of "folding diseases", and will also provide new biomaterials that can be used to diagnose and perhaps ultimately treat human diseases
Project start date: 2005-03-01
Project end date: 2013-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-07-070
5R01GM068462-06 (2010): $274950
Sponsored Links Excellgen http://Excellgen.com
REPEAT-PROTEINS; STABILITY, FOLDING KINETICS & EVOLUTION
Douglas E Barrick, Associate Professor
Johns Hopkins University W400 Wyman Park Building Baltimore, Md 212182680
Grant 5R01GM068462-03 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: In all branches of life, there are a variety of proteins that contain short, tandemly repeated sequences. Each repeat-sequence folds up into a closed loop containing helices, beta-strands, or a mix of secondary structure units. Adjacent repeats then pack against each other to create an elongated, linear domain with a regular, modular architecture. These repeat-proteins usually participate in protein-protein interactions, often at the center of important biological processes such as signal transduction, and are often associated with disease states such as cancer and bacterial pathogenesis. In the research outlined here, biophysical studies will be performed on repeat-proteins to better understand their folding and their stability. Our experiments will take advantage of the linear architecture of these proteins to answer questions that are difficult to address with non-repeat proteins. We will determine the distances over which repeats can couple with one another, and measure the end-to-end stability distribution in these proteins. We will use deletion experiments to build an "energy landscape" with single-repeat resolution. We will then examine how this "energy landscape" relates to folding rates by studying the folding kinetics of repeat-protein fragments and by determining which repeats are structured in the rate-limiting steps in folding, and will test models that relate folding rates to various structural and energetic features. Owing to their linear, repeated architecture, repeat-proteins seem likely targets for rapid evolution through insertion and deletion. We will evaluate the outcome of such rearrangements by studying the effects on stability of different types of deletions and duplications. We will test whether the fusion of different types of repeat-proteins can adopt a stable fold. The finding that such rearranged proteins are stable would support the idea that functional diversity can be produced by recombination of genes encoding repeat-proteins. We will also seek support for the hypothesis that repeat-proteins appeared early in protein evolution by computer analysis of protein sequences and structures.
Keywords: biochemical evolution, chemical kinetics, molecular dynamics, protein folding, protein protein interaction, protein sequence, thermodynamics
Project start date: 2005-03-01
Project end date: 2009-02-28
5R01GM068462-03 (2007): $226537
5R01GM068462-02 (2006): $233144
Grants awarded to Douglas E Barrick
Repeat-Proteins; Stability, Folding Kinetics & Evolution
Douglas E Barrick, Professor
Biophysicsjohns Hopkins University
Grant 2R01GM068462-05 from National Institute Of General Medical Sciences IRG: MSFB
Abstract: Protein folding is the process by which polypeptides adopt their complex, three dimensional structure. In most monomeric proteins, this structure is required for function, and is encoded in the amino acid sequence. Thus, protein folding is the bridge between the gene and its function, and is central to understanding biology. Deciphering the rules by which proteins fold is also critical for understanding a number of genetic diseases that result either from essential gene products that cannot fold to their native state, or from proteins that misfold to a non-native, aggregation-prone complex, forming toxic oligomers or fibers. The research proposed here seeks to understand the folding problem using proteins of a simplified architecture in which a small cluster of secondary structure units (helix, strand, turn) is repeated in a linear array. The extended, modular architecture of repeat proteins allows units of structure to be removed and inserted, providing a detailed mapping of how folding energy is distributed along the polypeptide chain. This direct mapping of the energy landscape allows long-standing questions about protein folding to be addressed, such as the origin and kinetic consequences of cooperativity, existence and specification of kinetic pathways. In addition, the structural similarity of the repeated units allows the contributions of different regions to be compared with great clarity. Here we use two different repeat protein architectures, the ankyrin (a/a) and LRR (a/non-a) repeats to explore the structural origins of cooperativity, the role of cooperativity in folding kinetics, and how bulk cooperativity is manifested when unfolding is promoted by a directed force. To rigorously quantify cooperativity and its structural origins, we will take advantage of a recent discovery by us and by other groups that stable arrays can be built of repeats of identical sequence. These "consensus arrays" will be analyzed using an "Ising" statistical model, which quantifies intrinsic versus nearest-neighbor energies. Consensus sequence variants will be used to resolve which types of interactions give rise to the extraordinary cooperativity we have seen in these proteins. Once we have variants in hand that resolve local versus long-range interactions, we will be able to probe how cooperativity influences kinetics and transition state ensembles, developing a kinetic Ising model in the process. Kinetic analysis of these proteins will also provide insights as to how folding proceeds on a genuinely "flat" landscape. These cooperativity variants will also be used to explore the relationship between solution cooperativity and end-to-end forced unfolding. Comparison to natural (nonconsensus) repeat arrays will provide continued insight into the relationships between sequence, stability, and folding in these simple but ubiquitous proteins. Studies will combine standard equilibrium and stopped flow folding with collaborative hydrogen exchange mass spectrometry, atomic force microscopy, and optical tweezer methods. A large number of human diseases including cancers and Alzheimer´s disease are caused by proteins that cannot fold up to their active shapes, or that fold to the wrong shapes, poisoning cells and tissues. The proposed research will use simplified "repeat" proteins to learn the rules of how proteins fold into unique, well-determined structures. These rules will help us to understand the causes of "folding diseases", and will also provide new biomaterials that can be used to diagnose and perhaps ultimately treat human diseases
Project start date: 2005-03-01
Project end date: 2013-02-28
The Johns Hopkins Folding Meeting
Douglas E Barrick, Associate Professor
Johns Hopkins University W400 Wyman Park Building Baltimore, Md 212182680
Grant 5R13GM067710-02 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: Over the last two decades, Johns Hopkins has developed a strong presence in the field of protein folding. The common interests of the various groups led to the establishment of the annual Johns Hopkins Protein Folding Meeting in 1996. More recently, Johns Hopkins has had the good fortune to become home to a number of outstanding groups with interests in RNA, including RNA folding. As our RNA and protein communities educated one another, it became clear to us that the fields of protein and RNA folding had much in common, and that better communication could prove synergistic. Thus, beginning in 2001, our gathering was renamed the Johns Hopkins Folding Meeting. It continues to promote open and informal communication of frontier developments on all aspects of the folding problem, including contributions from experimentalists, physically oriented theoreticians, and those interested in computer-based algorithms. This meeting has attracted an international roster of participants representing the most prominent laboratories engaged in folding research. We are requesting support for the next three Johns Hopkins Folding Meetings. These will be held on a biennial cycle, in March of 2003, 2005, and 2007, at the Coolfont Conference Center, located about 2 hours from Baltimore and Washington in Berkeley Springs, West Virginia. We have adopted an organizational structure that includes a steering committee, currently consisting of eight Hopkins PIs involved in protein and RNA folding research, and in which one of two meeting organizers is from outside Johns Hopkins. This structure will ensure that speakers and topics focus on the most recent developments in folding, and avoid repetition from year to year. In selecting participants for the meeting, we will obtain representation from as many labs as possible and maintain a balance between experiment and theory, and between RNA and protein. Participants will also be selected to provide a balance in gender and career level.
Keywords: RNA, meeting /conference /symposium, protein folding, travel
Project start date: 2003-02-01
Project end date: 2007-01-31
5R13GM067710-02 (2005): $5000
1R13GM067710-01 (2003): $5000
Structure And Mechanism In Intracellular Notch Signaling
Douglas E Barrick, Associate Professor
Johns Hopkins University W400 Wyman Park Building Baltimore, Md 212182680
Grant 5R01GM060001-03 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: Notch signaling is a general transmembrane signal transduction pathway that is essential for cell fate determination, development, homeostasis, and nervous system function in eukaryotes. Genetic lesions in the pathway are linked to human disease states such as cancer and neurological disorders. A central component of this pathway is the cytosolic -1000 residues of the Notch transmembrane receptor; upon activation of the pathway, this receptor fragment is released by a protease, and potentiates downstream signaling events. The activity of this soluble fragment, termed NIC, is modulated by at least nine intracellular effector proteins through what is thought to be direct interactions. Thus, NIC is the hub of a complex network of interactions. The aim of the research proposed here is to elucidate and quantify the molecular mechanisms and structural details of how this network modulates signaling and determines cell fate. Dr. Barrick will identify and characterize the structural and functional domains along NIC through analysis of solution structure and stability of nested and overlapping polypeptides derived from NIC. Structural domain maps will also be determined for intracellular effectors. These domains will then be used to analyze pairwise and higher order interactions. A systematic high-throughput approach will screen qualitatively for all possible pairwise interactions; conventional solution thermodynamic methods will be used to quantify the strength of pairwise interactions. Dr. Barrick will study the effect of existing Notch pathway mutations on structural stability and pairwise interactions, to help connect in vitro results to the biology of the signaling pathway. Multi-protein allosteric interactions among our NIC and effector domains will be identified and quantified; elucidation of such interactions will help to clarify the molecular mechanisms that modulate Notch signaling. Finally, Dr. Barrick will crystallize structural domains and complexes to provide an atomic level of domains and complexes by x-ray crystallography.
Keywords: biological signal transduction, membrane protein, protein protein interaction, protein structure function, ankyrin, binding protein, binding site, gene mutation, mutation, protein sequence, thermodynamics, X ray crystallography
Project start date: 2001-04-01
Project end date: 2006-03-31
5R01GM060001-03 (2003): $237281
1R01GM060001-01A1 (2001): $263876
5R01GM060001-02 (2002): $237592
5R01GM060001-05 (2005): $236636
5R01GM060001-04 (2004): $236967
5R01GM060001-09 (2010): $267828
5R01GM060001-08 (2009): $271305
Sponsored Links Excellgen http://Excellgen.com
2R01GM060001-06A1 (2007): $272560
REPEAT-PROTEINS; STABILITY, FOLDING KINETICS & EVOLUTION
Douglas E Barrick, Associate Professor
Johns Hopkins University W400 Wyman Park Building Baltimore, Md 212182680
Grant 1R01GM068462-01A2 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: In all branches of life, there are a variety of proteins that contain short, tandemly repeated sequences. Each repeat-sequence folds up into a closed loop containing helices, beta-strands, or a mix of secondary structure units. Adjacent repeats then pack against each other to create an elongated, linear domain with a regular, modular architecture. These repeat-proteins usually participate in protein-protein interactions, often at the center of important biological processes such as signal transduction, and are often associated with disease states such as cancer and bacterial pathogenesis. In the research outlined here, biophysical studies will be performed on repeat-proteins to better understand their folding and their stability. Our experiments will take advantage of the linear architecture of these proteins to answer questions that are difficult to address with non-repeat proteins. We will determine the distances over which repeats can couple with one another, and measure the end-to-end stability distribution in these proteins. We will use deletion experiments to build an "energy landscape" with single-repeat resolution. We will then examine how this "energy landscape" relates to folding rates by studying the folding kinetics of repeat-protein fragments and by determining which repeats are structured in the rate-limiting steps in folding, and will test models that relate folding rates to various structural and energetic features. Owing to their linear, repeated architecture, repeat-proteins seem likely targets for rapid evolution through insertion and deletion. We will evaluate the outcome of such rearrangements by studying the effects on stability of different types of deletions and duplications. We will test whether the fusion of different types of repeat-proteins can adopt a stable fold. The finding that such rearranged proteins are stable would support the idea that functional diversity can be produced by recombination of genes encoding repeat-proteins. We will also seek support for the hypothesis that repeat-proteins appeared early in protein evolution by computer analysis of protein sequences and structures.
Keywords: biochemical evolution, chemical kinetics, molecular dynamics, protein folding, protein protein interaction, protein sequence, thermodynamics
Project start date: 2005-03-01
Project end date: 2009-02-28
1R01GM068462-01A2 (2005): $283631