Conformational Sub-states and Populations in Enzyme Catalysis

Abstract

Enzyme function involves substrate and cofactor binding, precise positioning of reactants in the active site, chemical turnover, and release of products. In addition to formation of crucial structural interactions between enzyme and substrate(s), coordinated motions within the enzyme-substrate complex allows reaction to proceed at a much faster rate, compared to the reaction in solution and in the absence of enzyme. An increasing number of enzyme systems show the presence of conserved protein motions that are important for function. A wide variety of motions are naturally sampled (over femtosecond to millisecond time-scales) as the enzyme complex moves along the energetic landscape, driven by temperature and dynamical events from the surrounding environment. Areas of low energy along the landscape form conformational substates, which show higher conformational populations than surrounding areas. A small number of these protein conformational sub-states contain functionally important structural and dynamical features, which assist the enzyme mechanism along the catalytic cycle. Identification and characterization of these higher-energy (also called excited) sub-states and the associated populations is challenging, as these sub-states are very short-lived and therefore rarely populated. Specialized techniques based on computer simulations, theoretical modeling and nuclear magnetic resonance (NMR) have been developed for quantitative characterization of these substates and populations. This chapter discusses these techniques and provides examples of their applications to enzyme systems.

Modulating hinge flexibility in the APP transmembrane domain alters γ-secretase cleavage

“…Although these results confirm previous studies (43,44,52) (96). Here, large-scale shape fluctuations might be selected to enable recognition, while lower-amplitude, more localized motions help to optimize and stabilize the enzyme-bound intermediate states (94)(95)(96)(97)99). With regard to C99, the essential property enabling the TMD to switch between different shapes of functional importance is the organization of rigidity/flexibility along the helix backbone where residues enjoying higher flexibility can coordinate the motions of more rigid flanking segments.…”

Applications of NMR and computational methodologies to study protein dynamics

“…The various force-constants associated with inter-atomic interactions are collectively referred to as a force-field. MD trajectories, which correspond to a collection of conformational snapshots sampled during the simulation, are analyzed using a variety of approaches to identify conformational states that are potentially important for function 53 . While conformational motions in proteins are observed over a wide range of time scales — from the fast femtosecond-picosecond (fs-ps) to the slower millisecond-second (ms-s) — the range of time scales accessible by MD is currently limited due to the speed of central/graphical processing units and related computer hardware.…”

“…The popular Carr-Purcell-Meiboom-Gill (CPMG) and R 1ρ rotating frame relaxation dispersion experiments have been collectively employed to investigate conformational exchange rates ( k ex ) for residues experiencing conformational exchange in proteins over time frames that roughly span ~100 to ~50,000 events per second (s −1 ), overlapping the time scale of relevant biological events. B) Energetic representation of the two-site exchange between ground state A (higher population, p A ), and excited state B (lower population p B , often invisible on fast and intermediate NMR time scales) 23,53,100 . NMR relaxation dispersion experiments can provide rates of exchange ( k ex ), populations ( p A p B ), and chemical shifts between interconverting species (Δω).…”

High-Pressure NMR Reveals Volume and Compressibility Differences Between Two Stably Folded Protein Conformations

“…Taken to an extreme, one arrives at the emerging concept of 'metamorphic' proteins, where one sequence can reversibly adopt different stable folds in different environmental conditions [8][9][10] . However, identifying and characterizing high energy states of a protein can be challenging, as these are sparsely populated by definition [11][12] . Several techniques are currently available for quantitative characterization of conformational sub-states, predominately through modeling, molecular dynamics simulations, and advanced nuclear magnetic resonance (NMR) techniques such as relaxation dispersion experiments [11][12][13][14] .…”