Adarsh D Pandit | PubMed Health

PubMed2007

Characterizing protein folding transition States using Psi-analysis.

We discuss the implementation of Psi-analysis for the structural characterization of protein folding transition states. In Psi-analysis, engineered bi-histidine metal ion binding sites are introduced at surface positions to stabilize secondary and tertiary structures. The addition of metal ions stabilizes the interaction between the two known histidines in a continuous fashion. Measuring the ratio of transition state stabilization to that of the native state provides information about the presence of the metal binding site in the transition state. Psi-Analysis uses noninvasive surface mutations and does not require specialized equipment, so it can be readily applied to characterize the folding of many proteins. As a result, this method can provide a wealth of high-resolution quantitative data for comparison with theoretical folding simulations. Additionally, investigations of other biological processes also may utilize metal binding sites and Psi-analysis to detect conformational events during catalysis, assembly, and function.

Binding Sites↗

PubMed2006

Small proteins fold through transition states with native-like topologies.

The folding pathway of common-type acyl phosphatase (ctAcP) is characterized using psi-analysis, which identifies specific chain-chain contacts using bi-histidine (biHis) metal-ion binding sites. In the transition state ensemble (TSE), the majority of the protein is structured with a near-native topology, only lacking one beta-strand and an alpha-helix. psi-Values are zero or unity for all sites except one at the amino terminus of helix H2. This fractional psi-value remains unchanged when three metal ions of differing coordination geometries are used, indicating this end of the helix experiences microscopic heterogeneity through fraying in the TSE. Ubiquitin, the other globular protein characterized using psi-analysis, also exhibits a single consensus TSE structure. Hence, the TSE of both proteins have converged to a single configuration, albeit one that contains some fraying at the periphery. Models of the TSE of both proteins are created using all-atom Langevin dynamics simulations using distance constraints derived from the experimental psi-values. For both proteins, the relative contact order of the TS models is approximately 80% of the native value. This shared value viewed in the context of the known correlation between contact order and folding rates, suggests that other proteins will have a similarly high fraction of the native contact order. This constraint greatly limits the range of possible configurations at the rate-limiting step.

Computer Simulation↗