Evolution of sequence specific molecular recognition in telomere end binding proteins from Sterkiella nova

Abstract

Specificity in molecular recognition is the binding of the correct substrate and exclusion of incorrect though very similar looking substrates. We have examined how Sterkiella nova telomere proteins distinguish cognate versus non‐cognate single stranded DNA sequences using approaches measuring both thermodynamic and kinetic performance. Features from X‐ray crystal structures that correlate strongly with high specificity include Watson‐Crick like H‐bonds between guanine bases and aspartate or glutamate amino acid residues. We further tested the role of these interactions by mutagenesis, and surprisingly, binding affinities measured for single amino acid substitution variants were very similar to those measured for wild type protein. In contrast, our study of kinetic on and off rates of different complexes shows that the rate with which a protein releases substrate DNA is more predictive of specificity performance. In this system, specificity appears to have evolved by optimization of kinetic off rates, and possibly on rates as well, not equilibrium binding constants. These findings likely reflect the reality that specificity operates in a dynamic and responsive (i.e. living) system. C.Y.D. (University of Utah Graduate Research Fellow), M.P.H. (NIH R01 GM067994).