Proline 54 trans‐cis isomerization is responsible for the kinetic partitioning at the last‐step photocycle of photoactive yellow protein

• Published in: Protein science Vol. 17; no. 12; pp. 2101 - 2110
• Main Authors: Lee, Byoung‐Chul, Hoff, Wouter D.
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.12.2008
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Crystallography, X-Ray; kinetic partitioning; Kinetics; Light; Models, Molecular; photoactive yellow protein; photocycle; Photoreceptors, Microbial - chemistry; Photoreceptors, Microbial - genetics; Photoreceptors, Microbial - metabolism; Proline - chemistry; Proline - metabolism; proline trans‐cis isomerization; Protein Conformation; Protein Folding; slow phase; Solvents; Stereoisomerism
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.037655.108

Photoactive yellow protein (PYP), a blue‐light photoreceptor for Ectothiorhodospira halophila, has provided a unique system for studying protein folding that is coupled with a photocycle. Upon receptor activation by blue light, PYP proceeds through a photocycle that includes a partially folded signaling state. The last‐step photocycle is a thermal recovery reaction from the signaling state to the native state. Bi‐exponential kinetics had been observed for the last‐step photocycle; however, the slow phase of the bi‐exponential kinetics has not been extensively studied. Here we analyzed both fast and slow phases of the last‐step photocycle in PYP. From the analysis of the denaturant dependence of the fast and slow phases, we found that the last‐step photocycle proceeds through parallel channels of the folding pathway. The burial of the solvent‐accessible area was responsible for the transition state of the fast phase, while structural rearrangement from the compact state to the native state was responsible for the transition state of the slow phase. The photocycle of PYP was linked to the thermodynamic cycle that includes both unfolding and refolding of the fast‐ and slow‐phase intermediates. In order to test the hypothesis of proline‐limited folding for the slow phase, we constructed two proline mutants: P54A and P68A. We found that only a single phase of the last‐step photocycle was observed in P54A. This suggests that there is a low energy barrier between trans to cis conformation in P54 in the light‐induced state of PYP, and the resulting cis conformation of P54 generates a slow‐phase kinetic trap during the photocycle‐coupled folding pathway of PYP.