Robert Callender

Probing protein dynamics using temperature jump relaxation spectroscopy

There have been recent advances in initiating and perturbing chemical reactions on very fast timescales, as short as picoseconds, thus making it feasible to study a vast range of chemical kinetics problems that heretofore could not be studied. One such approach is the rapid heating of water solutions using laser excitation. Laser-induced temperature jump relaxation spectroscopy can be used to determine the dynamics of protein motion, an area largely unstudied for want of suitable experimental and theoretical probes, despite the obvious importance of dynamics to protein function. Coupled with suitable spectroscopic probes of structure, relaxation spectroscopy can follow the motion of protein atoms over an enormous time range, from picoseconds to minutes (or longer), and with substantial structural specificity.

The Dynamical Nature of Enzymatic Catalysis

Conspectus As is well-known, enzymes are proteins designed to accelerate specific life essential chemical reactions by many orders of magnitude. A folded protein is a highly dynamical entity, best described as a hierarchy or ensemble of interconverting conformations on all time scales from femtoseconds to minutes. We are just beginning to learn what role these dynamics play in the mechanism of chemical catalysis by enzymes due to extraordinary difficulties in characterizing the conformational space, that is, the energy landscape, of a folded protein. It seems clear now that their role is crucially important. Here we discuss approaches, based on vibrational spectroscopies of various sorts, that can reveal the energy landscape of an enzyme–substrate (Michaelis) complex and decipher which part of the typically very complicated landscape is relevant to catalysis. Vibrational spectroscopy is quite sensitive to small changes in bond order and bond length, with a resolution of 0.01 Å or less. It is this sensitivity that is crucial to its ability to discern bond reactivity. Using isotope edited IR approaches, we have studied in detail the role of conformational heterogeneity and dynamics in the catalysis of hydride transfer by LDH (lactate dehydrogenase). Upon the binding of substrate, the LDH·substrate system undergoes a search through conformational space to find a range of reactive conformations over the microsecond to millisecond time scale. The ligand is shuttled to the active site via first forming a weakly bound enzyme·ligand complex, probably consisting of several heterogeneous structures. This complex undergoes numerous conformational changes spread throughout the protein that shuttle the enzyme·substrate complex to a range of conformations where the substrate is tightly bound. This ensemble of conformations all have a propensity toward chemistry, but some are much more facile for carrying out chemistry than others. The search for these tightly bound states is clearly directed by the forces that the protein can bring to bear, very much akin to the folding process to form native protein in the first place. In fact, the conformational subspace of reactive conformations of the Michaelis complex can be described as a “collapse” of reactive substates compared with that found in solution, toward a much smaller and much more reactive set. These studies reveal how dynamic disorder in the protein structure can modulate the on-enzyme reactivity. It is very difficult to account for how the dynamical nature of the ground state of the Michaelis complex modulates function by transition state concepts since dynamical disorder is not a starting feature of the theory. We find that dynamical disorder may well play a larger or similar sized role in the measured Gibbs free energy of a reaction compared with the actual energy barrier involved in the chemical event. Our findings are broadly compatible with qualitative concepts of evolutionary adaptation of function such as adaptation to varying thermal environments. Our work suggests a methodology to determine the important dynamics of the Michaelis complex.

Effect of osmolytes on protein dynamics in the lactate dehydrogenase-catalyzed reaction.

Laser-induced temperature jump relaxation spectroscopy was used to probe the effect of osmolytes on the microscopic rate constants of the lactate dehydrogenase-catalyzed reaction. NADH fluorescence and absorption relaxation kinetics were measured for the lactate dehydrogenase (LDH) reaction system in the presence of varying amounts of trimethylamine N-oxide (TMAO), a protein-stabilizing osmolyte, or urea, a protein-destabilizing osmolyte. Trimethylamine N-oxide (TMAO) at a concentration of 1 M strongly increases the rate of hydride transfer, nearly nullifies its activation energy, and also slightly increases the enthalpy of hydride transfer. In 1 M urea, the hydride transfer enthalpy is almost nullified, but the activation energy of the step is not affected significantly. TMAO increases the preference of the closed conformation of the active site loop in the LDH·NAD(+)·lactate complex; urea decreases it. The loop opening rate in the LDH·NADH·pyruvate complex changes its temperature dependence to inverse Arrhenius with TMAO. In this complex, urea accelerates the loop motion, without changing the loop opening enthalpy. A strong, non-Arrhenius decrease in the pyruvate binding rate in the presence of TMAO offers a decrease in the fraction of the open loop, pyruvate binding competent form at higher temperatures. The pyruvate off rate is not affected by urea but decreases with TMAO. Thus, the osmolytes strongly affect the rates and thermodynamics of specific events along the LDH-catalyzed reaction: binding of substrates, loop closure, and the chemical event. Qualitatively, these results can be understood as an osmolyte-induced change in the energy landscape of the protein complexes, shifting the conformational nature of functional substates within the protein ensemble.

On the Pathway of Forming Enzymatically Productive Ligand-Protein Complexes in Lactate Dehydrogenase☆

We have carried out a series of studies on the binding of a substrate mimic to the enzyme lactate dehydrogenase (LDH) using advanced kinetic approaches, which begin to provide a molecular picture of the dynamics of ligand binding for this protein. Binding proceeds via a binding-competent subpopulation of the nonligated form of the protein (the LDH/NADH binary complex) to form a protein-ligand encounter complex. The work here describes the collapse of the encounter complex to form the catalytically competent Michaelis complex. Isotope-edited static Fourier transform infrared studies on the bound oxamate protein complex reveal two kinds of oxamate environments: 1), a major populated structure wherein all significant hydrogen-bonding patterns are formed at the active site between protein and bound ligand necessary for the catalytically productive Michaelis complex and 2), a minor structure in a configuration of the active site that is unfavorable to carry out catalyzed chemistry. This latter structure likely simulates a dead-end complex in the reaction mixture. Temperature jump isotope-edited transient infrared studies on the binding of oxamate with LDH/NADH suggest that the evolution of the encounter complex between LDH/NADH and oxamate collapses via a branched reaction pathway to form the major and minor bound species. The production of the catalytically competent protein-substrate complex has strong similarities to kinetic pathways found in two-state protein folding processes. Once the encounter complex is formed between LDH/NADH and substrate, the ternary protein-ligand complex appears to fold to form a compact productive complex in an all or nothing like fashion with all the important molecular interactions coming together at the same time.

Primary folding dynamics of sperm whale apomyoglobin: core formation.

The structure, thermodynamics, and kinetics of heat-induced unfolding of sperm whale apomyoglobin core formation have been studied. The most rudimentary core is formed at pH(*) 3.0 and up to 60 mM NaCl. Steady state for ultraviolet circular dichroism and fluorescence melting studies indicate that the core in this acid-destabilized state consists of a heterogeneous composition of structures of approximately 26 residues, two-thirds of the number involved for horse heart apomyoglobin under these conditions. Fluorescence temperature-jump relaxation studies show that there is only one process involved in Trp burial. This occurs in 20 micro s for a 7 degrees jump to 52 degrees C, which is close to the limits placed by diffusion on folding reactions. However, infrared temperature jump studies monitoring native helix burial are biexponential with times of 5 micro s and 56 micro s for a similar temperature jump. Both fluorescence and infrared fast phases are energetically favorable but the slow infrared absorbance phase is highly temperature-dependent, indicating a substantial enthalpic barrier for this process. The kinetics are best understood by a multiple-pathway kinetics model. The rapid phases likely represent direct burial of one or both of the Trp residues and parts of the G- and H-helices. We attribute the slow phase to burial and subsequent rearrangement of a misformed core or to a collapse having a high energy barrier wherein both Trps are solvent-exposed.

Vibrational study of phosphate modes in GDP and GTP and their interaction with magnesium in aqueous solution.

Raman and infrared spectra were examined for guanosine 5-diphosphate (GDP) and guanosine 5-triphosphate (GTP) in aqueous solution. The vibrational modes were assigned on the basis of isotopic frequency shifts and relative intensities in the Raman and infrared spectra. The observed frequency shifts on 18O isotope labeling made it possible to identify the bands from each phosphate group (alpha, beta, gamma). Frequency shifts were observed as Mg2+ complexes with GDP and GTP. The results suggested that Mg2+ binds to GDP in a bidentate manner to the alpha, beta P[symbol: see text]O bonds and in a tridentate manner to the alpha, beta and gamma P[symbol: see text]O bonds of Mg.GTP. The results indicate that structure of Mg2+ coordinated to GTP in aqueous solution differs somewhat to that found for Mg.ATP.

Probing the Role of Dynamics in Hydride Transfer Catalyzed by Lactate Dehydrogenase

The dynamic nature of the interconversion of pyruvate to lactate as catalyzed by lactate dehydrogenase (LDH) is characterized by laser-induced temperature jump relaxation spectroscopy with a resolution of 20 ns. An equilibrium system of LDH.NADH plus pyruvate and LDH.NAD+ plus lactate is perturbed by a sudden T-jump, and the relaxation of the system is monitored by NADH emission and absorption changes. The substrate binding pathway is observed to be similar, although not identical, to previous work on substrate mimics: an encounter complex is formed between LDH.NADH and pyruvate, which collapses to the active Michaelis complex. The previously unresolved hydride transfer event is characterized and separated from other unimolecular isomerizations of the protein important for the catalytic mechanism, such as loop closure, a slower step, and faster events on the nanosecond-microsecond timescales whose structural basis is not understood. The results of this study show that this approach can be applied quite generally to enzyme systems and report on the dynamic nature of proteins over a very wide time range.

Direct Evidence of Catalytic Heterogeneity in Lactate Dehydrogenase by Temperature Jump Infrared Spectroscopy

Protein conformational heterogeneity and dynamics are known to play an important role in enzyme catalysis, but their influence has been difficult to observe directly. We have studied the effects of heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase using isotope edited infrared spectroscopy, laser-induced temperature jump relaxation, and kinetic modeling. The isotope edited infrared spectrum reveals the presence of multiple reactive conformations of pyruvate bound to the enzyme, with three major reactive populations having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm–1, respectively. The temperature jump relaxation measurements and kinetic modeling indicate that these substates form a heterogeneous branched reaction pathway, and each substate catalyzes the conversion of pyruvate to lactate with a different rate. Furthermore, the rate of hydride transfer is inversely correlated with the frequency of the C2 carbonyl stretch (the rate increases as the frequency decreases), consistent with the relationship between the frequency of this mode and the polarization of the bond, which determines its reactivity toward hydride transfer. The enzyme does not appear to be optimized to use the fastest pathway preferentially but rather accesses multiple pathways in a search process that often selects slower ones. These results provide further support for a dynamic view of enzyme catalysis where the role of the enzyme is not just to bring reactants together but also to guide the conformational search for chemically competent interactions.

Energy landscape of the Michaelis complex of lactate dehydrogenase: relationship to catalytic mechanism.

Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicotinamide adenine dinucleotide (NAD) as a cofactor. Using isotope-edited difference Fourier transform infrared spectroscopy on the “live” reaction mixture (LDH·NADH·pyruvate ⇌ LDH·NAD+·lactate) for the wild-type protein and a mutant with an impaired catalytic efficiency, a set of interconverting conformational substates within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these substates include (1) electronic orbital overlap between pyruvate’s C2=O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties, and (2) a characteristic hydrogen bond distance between the pyruvate C2=O group and active site residues, as shown by the observation of at least four C2=O stretch bands indicating varying degrees of C2=O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the substate population ratios in the Michaelis complex provides a unique opportunity to probe the structure–activity relationship in LDH catalysis. The various substates have a strong variance in their propensity toward on enzyme chemistry. Our results suggest a physical mechanism for understanding the LDH-catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational substates as compared to the same chemistry in solution.

Conformational heterogeneity within the Michaelis complex of lactate dehydrogenase

A series of isotope edited IR measurements, both static as well as temperature jump relaxation spectroscopy, are performed on lactate dehydrogenase (LDH) to determine the ensemble of structures available to its Michaelis complex. There clearly has been a substantial reduction in the number of states available to the pyruvate substrate (as modeled by the substrate mimic, oxamate) and NADH when bound to protein compared to dissolved in solution, as determined by the bandwidths and positions of the critical C(2)═O band of the bound substrate mimic and the C(4)-H stretch of the NADH reduced nicotinamide group. Moreover, it is found that a strong ionic bond (characterized by a signature IR band discovered in this study) is formed between the carboxyl group of bound pyruvate with (presumably) Arg171, forming a strong anchor within the protein matrix. However, conformational heterogeneity within the Michaelis complex is found that has an impact on both catalytic efficiency and thermodynamics of the enzyme.