Most proteins conform to the classical view that a polypeptide chain populates a single, stable native state. However, the phenomenon of fold switching, where protein sequences can exist at the interface between completely different folds, creates serious challenges to our understanding of how amino acid sequence encodes 3D structure. Additionally, it has many implications for understanding how proteins evolve, how mutation is related to disease, and how function is annotated to sequences of unknown structure. Here, the overall objective is to determine experimentally how amino acid sequences migrate through fold space. We will determine the generality of fold switching and define its common principles by designing, engineering and analyzing a number of strategic protein switches. Our proposed studies employ small proteins that are widely used in experimental and computational folding studies, connecting our future results to a large body of knowledge.
We aim to show that: 1) many folds can switch into other completely different topologies;2) such switches can be designed/evolved;3) structures and energetics of switches can be understood;4) understanding can lead to prediction of other switches. Previous examination of both natural and engineered fold switches has shown that three conditions are generally necessary for a fold switch: 1) low stability of both folds;2) compatibility of hydrophobic cores between folds;and 3) long range interactions in one fold which can override local interactions in the other. Methodical studies of fold switching require design and selection methods robust enough to create multiple examples of switches. To create different switches, we have chosen a series of origin folds that represent a range of common topologies (orthogonal bundle, 3-helix bundle, ?- grasp, SH3 barrel) that will be switched into different context-driven, destination folds (?/? plait, ?/? sandwich, Rossman-like). This approach allows us to satisfy the three general conditions of switching mentioned above. It also mimics evolutionary migration of sub-domains through fold space. Selection through the use of phage display methods will produce heteromorphic and bi-functional proteins that will be used for structural and energetic analysis. These proteins will be studied by a variety of physical methods including microcalorimetry, CD and NMR. Detailed structural and thermodynamic analysis will give important insights into the physicochemical basis for fold switching. The energetic and structural results will reveal how multiple folds are connected through short mutational pathways. These mutational connectivities in fold space will create networks of probable fold migrations. Our results will also enable computational biologists to use these data in folding simulations, fold network studies, and for further development and refinement of stability and structure prediction algorithms.

Public Health Relevance

The overall objective is to determine experimentally how amino acid sequences migrate through fold space. Our proposed studies have many implications for understanding how proteins evolve, how mutation is related to disease, and how function is annotated to sequences of unknown structure. Our results will also enable computational biologists to use these data in folding simulations, fold network studies, and for further development and refinement of stability and structure prediction algorithms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM062154-10
Application #
8438722
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Smith, Ward
Project Start
2002-03-01
Project End
2017-03-31
Budget Start
2013-06-01
Budget End
2014-03-31
Support Year
10
Fiscal Year
2013
Total Cost
$307,800
Indirect Cost
$105,300
Name
University of Maryland College Park
Department
Miscellaneous
Type
Other Domestic Higher Education
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742
He, Yanan; Chen, Yihong; Alexander, Patrick A et al. (2012) Mutational tipping points for switching protein folds and functions. Structure 20:283-91
Morrone, Angela; McCully, Michelle E; Bryan, Philip N et al. (2011) The denatured state dictates the topology of two proteins with almost identical sequence but different native structure and function. J Biol Chem 286:3863-72
Bryan, Philip N; Orban, John (2010) Proteins that switch folds. Curr Opin Struct Biol 20:482-8
Shen, Yang; Bryan, Philip N; He, Yanan et al. (2010) De novo structure generation using chemical shifts for proteins with high-sequence identity but different folds. Protein Sci 19:349-56
Alexander, Patrick A; He, Yanan; Chen, Yihong et al. (2009) A minimal sequence code for switching protein structure and function. Proc Natl Acad Sci U S A 106:21149-54
He, Yanan; Chen, Yihong; Alexander, Patrick et al. (2008) NMR structures of two designed proteins with high sequence identity but different fold and function. Proc Natl Acad Sci U S A 105:14412-7
He, Yanan; Chen, Yihong; Rozak, David A et al. (2007) An artificially evolved albumin binding module facilitates chemical shift epitope mapping of GA domain interactions with phylogenetically diverse albumins. Protein Sci 16:1490-4
Alexander, Patrick A; He, Yanan; Chen, Yihong et al. (2007) The design and characterization of two proteins with 88% sequence identity but different structure and function. Proc Natl Acad Sci U S A 104:11963-8
Rozak, David A; Alexander, Patrick A; He, Yanan et al. (2006) Using offset recombinant polymerase chain reaction to identify functional determinants in a common family of bacterial albumin binding domains. Biochemistry 45:3263-71
He, Yanan; Rozak, David A; Sari, Nese et al. (2006) Structure, dynamics, and stability variation in bacterial albumin binding modules: implications for species specificity. Biochemistry 45:10102-9

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