Protein design offers a promising strategy to alter and optimize proteins for biotechnology, industry, and medicine. Though there are many examples of successful protein designs, reliable design of stable proteins with high biological activity remains challenging. Protein consensus sequence design (using multiple sequence alignment of homologous proteins to identify the sequence of maximal conservation) may provide proteins with high activity as well as high stability, as functionally important residues are likely to be conserved. Several studies have provided anecdotal accounts of stability enhancement using consensus sequence information, although other studies describe mixed results. In this proposal, I aim to generalize the viability of the consensus sequence design strategy to create protein design targets with high stability and biological activity. Motivated by my preliminary results demonstrating stability enhancement and maintenance of biological activity for a small set of consensus protein constructs, I plan to create a larger set of consensus proteins and characterize structure, stability, and function. I have selected ten proteins families for consensus design. I will determine the structures of these target proteins using a combination of NMR and x-ray crystallography, will, determine equilibrium thermodynamic stabilities, and will measure biological activities. As part of the functional characterization, I will examine thermodynamic and kinetic parameters of molecular recognition and enzyme catalysis, quantify consensus protein conformational dynamics, and determine the effects of consensus proteins on cellular fitness. This project will comprehensively evaluate consensus design as an effective protein design strategy, testing the generality to which it may produce proteins of high stability and biological activity. Moreover, the proposed project will answer longstanding unresolved questions on the interrelated nature of protein evolution, stability, dynamics, and function.

Public Health Relevance

The proposed research will evaluate consensus sequence design as a strategy to produce proteins of high stability and activity. This will be achieved by creating a large set of consensus proteins with different folds and activities, and characterizing them for structure, stability, and function. These results will help to develop guidelines for how consensus design may be used to create proteins with attractive properties for medicine, industry, and biotechnology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31GM128295-01
Application #
9541271
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Brown, Anissa F
Project Start
2018-04-01
Project End
2020-03-31
Budget Start
2018-04-01
Budget End
2019-03-31
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21205