Endogenous Cu(II) Labeling for Distance Measurements on Proteins by EPR.

ABSTRACT: In-cell measurements of the relationship between structure and dynamics to protein function is at the forefront of biophysics. Recently, developments in EPR methodology have demonstrated the sensitivity and power of this method to measure structural constraints in-cell. However, the need to spin label proteins ex-situ or use noncanonical amino acids to achieve endogenous labeling remains a bottleneck. In this work we expand the methodology to endogenously spin label proteins with Cu(II) spin labels and describe how to assess in-cell spin labeling. We quantify the amount of Cu(II)-NTA in cells, assess spin labeling, and account for orientational effects during distance measurements. We compare the efficacy of using heat-shock and hypotonic swelling to deliver spin label, showing that hypoto

SUBMITTER: Hunter HR 

PROVIDER: S-EPMC11665492 | biostudies-literature | 2024 Dec

REPOSITORIES: biostudies-literature