Protein kinetics: Structures of intermediates and reaction mechanism from time-resolved x-ray data

Abstract

We determine the number of authentic reaction intermediates in the later stages of the photocycle of photoactive yellow protein at room temperature, their atomic structures, and a consistent set of chemical kinetic mechanisms, by analysis of a set of time-dependent difference electron density maps spanning the time range from 5 s to 100 ms. The successful fit of exponentials to right singular vectors derived from a singular value decomposition of the difference maps demonstrates that a chemical kinetic mechanism holds and that structurally distinct intermediates exist. We identify two time-independent difference maps, from which we refine the structures of the corresponding intermediates. We thus demonstrate how structures associated with intermediate states can be extracted from the experimental, time-dependent crystallographic data. Stoichiometric and structural constraints allow the exclusion of one kinetic mechanism proposed for the photocycle but retain other plausible candidate kinetic mechanisms.chemical, kinetic mechanism ͉ time-resolved crystallography T he protein structures associated with short-lived intermediate states (ISs) that form and decay along a reaction pathway must be determined if the structural basis of function is to be fully understood. Models for such states are normally determined by physical or chemical trapping techniques designed both to greatly increase the lifetime of a particular state and to trap a structurally homogeneous species (1-4). However, the trapping techniques themselves may perturb the mechanism and the observed species may still not be structurally homogeneous (5). If homogeneous structures along the reaction path could be determined directly by using unmodified protein and reactants at near-physiological temperatures, the difficulties associated with trapping could be overcome.The reaction pathway of a protein is typically described by a chemical kinetic mechanism populated by a reactant state, a set of ISs, and a product state. For such a mechanism to hold, the transit time of an individual molecule from each state to its neighboring state(s) must be short, compared with the lifetimes of the states themselves. That is, equilibration across all other degrees of freedom must be fast with respect to progress along the reaction pathway. The time scales of structural transitions in proteins range from picoseconds for side-chain relaxations (6) to several nanoseconds for protein backbone relaxations (7). Such protein-specific motions are thought to enable both the transitions between protein conformations or substates within a single state (8,9), and those between ISs. If the lifetime of an IS is short compared with the time scales of protein-specific motions, a simple chemical kinetic mechanism (10) is unlikely to hold. If the converse is true, those distinct ISs that accumulate to sufficiently high concentration will be observable. If first-order reactions are considered, the rise and fall of the concentration of each state will be describable by a sum of exponentials...

Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

“…Although static structures of alternate states are available for a number of allosteric proteins, information about the kinetic pathway between such functionally important states is limited. Time-resolved crystallographic analysis (1)(2)(3)(4)(5)(6)(7)(8)(9) provides the unique opportunity to obtain direct, time-dependent structural information at high resolution on the entire protein molecule as it undergoes structural change.…”

Five-dimensional crystallography

“…Equation (2) is the result of integrating the coupled differential equations that describe the mechanism involving three intermediates assuming exponential kinetics. Equation (2) constitutes the mathematical base of simple chemical kinetics described in textbooks such as Steinfeld et al (1989) Schmidt et al (2003Schmidt et al ( , 2004 for post-SVD analysis of timeresolved data. Details are given also by Schmidt et al (2005b) and Schmidt (2008).…”

“…Time-resolved crystallography has been used to investigate reactions in heme-containing proteins such as myoglobin (Srajer et al, 1996Schmidt et al, 2005a;Bourgeois et al, 2003Bourgeois et al, , 2006Schotte et al, 2003Schotte et al, , 2004, FixL (Key et al, 2007) and hemoglobin (Knapp et al, 2006). In addition, the photocycle of the photoactive yellow protein was investigated extensively (Perman et al, 1998;Ren et al, 2001;Schmidt et al, 2004;Rajagopal et al, 2005;Ihee et al, 2005) to determine the structures of the intermediates of its photocycle that form and decay conjointly with the chemical kinetic mechanism that connects them.…”

X-ray free-electron lasers

“…This approach has been successfully applied in a variety of time-resolved crystallography measurements [98][99][100][101][102][103][104] and will certainly be a useful approach for experiments at XFELs. Crystallography does not, however, fully describe the influence of photoexcitation.…”