NMR Analysis of Amide Hydrogen Exchange Rates in a Pentapeptide-Repeat Protein from A. thaliana

At2g44920 from Arabidopsis thaliana is a pentapeptide-repeat protein (PRP) composed of 25 repeats capped by N- and C-terminal α-helices. PRP structures are dominated by four-sided right-handed β-helices typically consisting of mixtures of type II and type IV β-turns. PRPs adopt repeated five-residue (Rfr) folds with an Rfr consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Unlike other PRPs, At2g44920 consists exclusively of type II β-turns. At2g44920 is predicted to be located in the thylakoid lumen although its biochemical function remains unknown. Given its unusual structure, we investigated the biophysical properties of At2g44920 as a representative of the β-helix family to determine if it had exceptional global stability, backbone dynamics, or amide hydrogen exchange rates. Circular dichroism measurements yielded a melting point of 62.8°C, indicating unexceptional global thermal stability. Nuclear spin relaxation measurements indicated that the Rfr-fold core was rigid with order parameters ranging from 0.7 to 0.9. At2g44920 exhibited a striking range of amide hydrogen exchange rates spanning 10 orders of magnitude, with lifetimes ranging from minutes to several months. A weak correlation was found among hydrogen exchange rates, hydrogen bonding energies, and amino acid solvent-accessible areas. Analysis of contributions from fast (approximately picosecond to nanosecond) backbone dynamics to amide hydrogen exchange rates revealed that the average order parameter of amides undergoing fast exchange was significantly smaller compared to those undergoing slow exchange. Importantly, the activation energies for amide hydrogen exchange were found to be generally higher for the slowest exchanging amides in the central Rfr coil and decreased toward the terminal coils. This could be explained by assuming that the concerted motions of two preceding or following coils required for hydrogen bond disruption and amide hydrogen exchange have a higher activation energy compared to that required for displacement of a single coil to facilitate amide hydrogen exchange in either the terminal or penultimate coils.