Single molecule techniques have fueled key insights into the molecular basis of protein function by facilitating the ability to characterize individual conformational populations and dynamics. In recent years, there has been an explosion in the capabilities of instrumentation, and a vast number of biological questions have been identified that could be addressed using this technique, especially if multiple distances could be simultaneously measured in large protein molecules (requiring 3-6 site specific labels). However, methodologies for generating proteins with the requisite labels have lagged, limiting applications needing multi-site labeling. We propose to develop, adapt and combine a robust set of protein synthesis / semisynthesis methodologies centered around the mechanistically related chemistries of native chemical ligation and protein trans splicing to the challenge of developing novel tools for building multiply-labeled proteins. Through careful synthetic design, we have developed a strategy that will facilitate site-specific protein labeling at multiple positions in a protein using a single, commonly-practiced labeling reaction. This semisynthetic approach is compatible with any protein, regardless of length or sequence and has the potential to close the gap between experiments that can be imagined and those that can be performed in the laboratory.

Public Health Relevance

Structural complexity in proteins often underlies the critical roles they play in cellular function. We propose to develop new synthetic tools that will substantially facilitate our ability to make direct measurements of protein structure and dynamics using single-molecule techniques. The resulting methods are expected to result in new insight into a broad range of protein systems, with implications in biology and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21GM132787-02
Application #
9937779
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Mcguirl, Michele
Project Start
2019-06-01
Project End
2021-02-28
Budget Start
2020-03-01
Budget End
2021-02-28
Support Year
2
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037