Ligand binding and protein relaxation in heme proteins: a room temperature analysis of NO geminate recombination

Ultrafast absorption spectroscopy is used to study heme-NO recombination at room temperature in aqueous buffer on time scales where the ligand cannot leave its cage environment. While a single barrier is observed for the cage recombination of NO with heme in the absence of globin, recombination in hemoglobin and myoglobin is nonexponential. Examination of hemoglobin with and without inositol hexaphosphate points to proximal constraints as important determinants of the geminate rebinding kinetics. Molecular dynamics simulations of myoglobin and heme-imidazole subsequent to ligand dissociation were used to investigate the transient behavior of the Fe-proximal histidine coordinate and its possible involvement in geminate recombination. The calculations, in the context of the absorption measurements, are used to formulate a distinction between nonexponential rebinding that results from multiple protein conformations (substates) present at equilibrium or from nonequilibrium relaxation of the protein triggered by a perturbation such as ligand dissociation. The importance of these two processes is expected to depend on the time scale of rebinding relative to equilibrium fluctuations and nonequilibrium relaxation. Since NO rebinding occurs on the picosecond time scale of the calculated myoglobin relaxation, a time-dependent barrier is likely to be an important factor in the observed nonexponential kinetics. The general implications of the present results for ligand binding in heme proteins and its time and temperature dependence are discussed. It appears likely that, at low temperatures, inhomogeneous protein populations play an important role and that as the temperature is raised, relaxation effects become significant as well.     

Ligand binding and protein dynamics in lactate dehydrogenase

Recent experimental studies suggest that lactate dehydrogenase (LDH) binds its substrate via the formation of a LDH/NADH.substrate encounter complex through a select-fit mechanism, whereby only a minority population of LDH/NADH is binding-competent. In this study, we perform molecular dynamics calculations to explore the variations in structure accessible to the binary complex with a focus on identifying structures that seem likely to be binding-competent and which are in accord with the known experimental characterization of forming binding-competent species. We find that LDH/NADH samples quite a range of protein conformations within our 2.148 ns calculations, some of which yield quite facile access of solvent to the active site. The results suggest that the mobile loop of LDH is perhaps just partially open in these conformations and that multiple open conformations, yielding multiple binding pathways, are likely. These open conformations do not require large-scale unfolding/melting of the binary complex. Rather, open versus closed conformations are due to subtle protein and water rearrangements. Nevertheless, the large heat capacity change observed between binding-competent and binding-incompetent can be explained by changes in solvation and an internal rearrangement of hydrogen bonds. We speculate that such a strategy for binding may be necessary to get a ligand efficiently to a binding pocket that is located fairly deep within the protein's interior.     

The approach to the Michaelis complex in lactate dehydrogenase: the substrate binding pathway

We examine here the dynamics of forming the Michaelis complex of the enzyme lactate dehydrogenase by characterizing the binding kinetics and thermodynamics of oxamate (a substrate mimic) to the binary lactate dehydrogenase/NADH complex over multiple timescales, from nanoseconds to tens of milliseconds. To access such a wide time range, we employ standard stopped-flow kinetic approaches (slower than 1 ms) and laser-induced temperature-jump relaxation spectroscopy (10 ns-10 ms). The emission from the nicotinamide ring of NADH is used as a marker of structural transformations. The results are well explained by a kinetic model that has binding taking place via a sequence of steps: the formation of an encounter complex in a bimolecular step followed by two unimolecular transformations on the microsecond/millisecond timescales. All steps are well described by single exponential kinetics. It appears that the various key components of the catalytically competent architecture are brought together as separate events, with the formation of strong hydrogen bonding between active site His(195) and substrate early in binding and the closure of the catalytically necessary protein surface loop over the bound substrate as the final event of the binding process. This loop remains closed during the entire period that chemistry takes place for native substrates; however, motions of other key molecular groups bringing the complex in and out of catalytic competence appear to occur on faster timescales. The on-enzyme K(d) values (the ratios of the microscopic rate constants for each unimolecular step) are not far from one. Either substantial, approximately 10-15%, transient melting of the protein or rearrangements of hydrogen bonding and solvent interactions of a number of water molecules or both appear to take place to permit substrate access to the protein binding site. The nature of activating the various steps in the binding process seems to be one overall involving substantial entropic changes.     

Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape

An acid-destabilized form of apomyoglobin, the so-called E state, consists of a set of heterogeneous structures that are all characterized by a stable hydrophobic core composed of 30-40 residues at the intersection of the A, G, and H helices of the protein, with little other secondary structure and no other tertiary structure. Relaxation kinetics studies were carried out to characterize the dynamics of core melting and formation in this protein. The unfolding and/or refolding response is induced by a laser-induced temperature jump between the folded and unfolded forms of E, and structural changes are monitored using the infrared amide I' absorbance at 1648-1651 cm(-1) that reports on the formation of solvent-protected, native-like helix in the core and by fluorescence emission changes from apomyoglobin's Trp14, a measure of burial of the indole group of this residue. The fluorescence kinetics data are monoexponential with a relaxation time of 14 micros. However, infrared kinetics data are best fit to a biexponential function with relaxation times of 14 and 59 micros. These relaxation times are very fast, close to the limits placed on folding reactions by diffusion. The 14 micros relaxation time is weakly temperature dependent and thus represents a pathway that is energetically downhill. The appearance of this relaxation time in both the fluorescence and infrared measurements indicates that this folding event proceeds by a concomitant formation of compact secondary and tertiary structures. The 59 micros relaxation time is much more strongly temperature dependent and has no fluorescence counterpart, indicating an activated process with a large energy barrier wherein nonspecific hydrophobic interactions between helix A and the G and H helices cause some helix burial but Trp14 remains solvent exposed. These results are best fit by a multiple-pathway kinetic model when U collapses to form the various folded core structures of E. Thus, the results suggest very robust dynamics for core formation involving multiple folding pathways and provide significant insight into the primary processes of protein folding.     

Toward an understanding of the role of dynamics on enzymatic catalysis in lactate dehydrogenase

The motions of key residues at the substrate binding site of lactate dehydrogenase (LDH) were probed on the 10 ns to 10 ms time scale using laser-induced temperature-jump relaxation spectroscopy employing both UV fluorescence and isotope-edited IR absorption spectroscopy as structural probes. The dynamics of the mobile loop, which closes over the active site and is important for catalysis and binding, were characterized by studies of the inhibitor oxamate binding to the LDH/NADH binary complex monitoring the changes in emission of bound NADH. The bound NAD-pyruvate adduct, whose pyruvate moiety likely interacts with the same residues that interact with pyruvate in its ternary complex with LDH, served as a probe for any relative motions of active site residues against the substrate. The frequencies of its C=O stretch and -COO(-) antisymmetric stretch shift substantially should any relative motion of the polar moieties at the active site (His-195, Asp-168, Arg-109, and Arg-171) occur. The dynamics associated with loop closure are observed to involve several steps with motions from 1 to 300 microms. Apart from the "melting" of a few residues on the protein's surface, no kinetics were observed on any time scale in experiments of the bound NAD-pyr adduct although the measurements were made with a high degree of accuracy, even for final temperatures close to the unfolding transition of the protein. This is contrary to simple physical considerations and models. These results show that, once a productive protein/substrate complex is formed, the binding pocket is very rigid with very little, if any, motion apart from the mobile loop. The results also show that loop opening involves concomitant movement of the substrate out of the binding pocket.     

35-Cl nuclear magnetic resonance studies of anion-binding sites in proteins: lactate dehydrogenase.

³⁵Cl nmr relaxation rate measurements have been used to study anion-binding sites in pig heart lactate dehydrogenase. These studies reveal two types of sites, one is intimately associated with the active site, the other is not. The nonactive site has been ascribed to a subunit site in analogy with crystallographic results from the dogfish M₄ enzyme. The binding of either the reduced or the oxidized form of NAD results in an increase in the ³⁵Cl nmr relaxation rate by a factor of 1.8–2. The enhanced nmr relaxation rate of the binary lactate dehydrogenase-NAD complex is reduced on binding of the substrate inhibitor molecules oxamate or oxalate to a value less than that exhibited by lactate dehydrogenase alone. The enhancement of the nmr relaxation rate is attributed to a decrease in the dissociation constant of Cl for the enzyme. The K_p values for Cl binding to the active center site of lactate dehydrogenase is 0.85 m and for lactate dehydrogenase-NADH is 0.25 m. The ratio of these constants, 3.4, agrees well with the measured enhancement value 3.7. The effect of coenzyme analogs on the ³⁵Cl nmr relaxation rate has been examined. 3-Acetylpyridine NAD produces an enhancement of 4.3, thionicotinamide NAD of 2.3, whereas 3-pyridinealdehyde, adenosinediphosphoribose, and adenosine diphosphate do not affect the nmr relaxation state of Cl bound to lactate dehydrogenase.     

Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding

Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions.     

C-terminal domain of insulin-like growth factor (IGF) binding protein 6: conformational exchange and its correlation with IGF-II binding

Insulin-like growth factor binding proteins (IGFBPs) function as carriers and regulators of the insulin-like growth factors (IGF-I and -II). Within the family of six binding proteins, IGFBP-6 is unique in having a 20-100-fold higher affinity for IGF-II over IGF-I and appears to act primarily as an inhibitor of IGF-II actions. We have recently determined the solution structure of the C-terminal domain of IGFBP-6 (C-BP-6), which shows the presence of substantial flexible regions, including three loop regions. In this paper, we report results from (15)N relaxation measurements carried out in both the laboratory and rotating frames. Analysis of conventional (15)N relaxation data (R(1), R(2), and steady-state (15)N-[(1)H] nuclear Overhauser effect) indicated that there was a considerable number of residues involved in conformational/chemical exchange. Measurements of off-resonance (15)N R(1)(rho) in the rotating frame and (15)N relaxation dispersion using an in- and antiphase coherence-averaged Carr-Purcell-Meiboom-Gill sequence were thus carried out to gain further insight into the solution dynamics of C-BP-6. Although the off-resonance (15)N relaxation data showed no clear evidence for residues undergoing microsecond motion, the (15)N relaxation dispersion data allowed us to identify 15 residues that clearly exhibit submilli- to millisecond motion. A good correlation was observed between residues exhibiting motion at submilli- to millisecond time scales and those affected by IGF-II binding, as identified through the perturbation of nuclear magnetic resonance (NMR) spectra of C-BP-6 following IGF-II addition. A complete NMR relaxation study of C-BP-6 dynamics in complex with IGF-II was hampered by peak broadening and disappearance of C-BP-6 in the presence of IGF-II. Nonetheless, current results strongly suggest possible conformation switching or population shifting between pre-existing conformations in C-BP-6 upon binding to IGF-II.     

NMR dynamic studies suggest that allosteric activation regulates ligand binding in chicken liver bile acid-binding protein

Apo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via (15)N relaxation and steady state heteronuclear (15)N((1)H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the beta-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His(98), located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His(98) to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His(98) protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids.     

Properties of metabolic networks: structure versus function

The dynamical nature of the binding of a substrate surrogate to lactate dehydrogenase is examined on the nanoseconds to milliseconds timescale by laser-induced temperature-jump relaxation spectroscopy. Fluorescence emission of the nicotinamide group of bound NADH is used to define the pathway and kinetics of substrate binding. Assignment of specific kinetic states and elucidation of their structures are accomplished using isotope edited infrared absorption spectroscopy. Such studies are poised to yield a detailed picture of the coupling of protein dynamics to function.     

Structural Transformations in the Dynamics of Michaelis Complex Formation in Lactate Dehydrogenase

The dynamical nature of the binding of a substrate surrogate to lactate dehydrogenase is examined on the nanoseconds to milliseconds timescale by laser-induced temperature-jump relaxation spectroscopy. Fluorescence emission of the nicotinamide group of bound NADH is used to define the pathway and kinetics of substrate binding. Assignment of specific kinetic states and elucidation of their structures are accomplished using isotope edited infrared absorption spectroscopy. Such studies are poised to yield a detailed picture of the coupling of protein dynamics to function.     

31 P nuclear magnetic resonance studies of the interaction of pyridine nucleotide coenzymes with dehydrogenases.

31P nuclear magnetic resonance spectra of the pyrophosphate group in NAD+ and NADH were recorded in the presence of beef heart lactate dehydrogenase and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. At high lactate dehydrogenase concentrations (60 mg/ml), two NADH resonances are observed: a slowly exchanging peak which is shifted to 1.9 ppm downfield (relative to free NADH) and a rapidly exchanging peak with a downfield shift of 0.5-0.6 ppm. At lover concentrations (15 mg/ml) only the rapidly exchanging peak is observed thus indicating that the peak observed at-1.9 ppm is due to coenzyme bound to an aggregated enzyme species. With NAD+, rapid exchange and downfield shifts are observed at both enzyme and concentrations, with shifts of about 1.5 ppm and 0.6 ppm at 60 and 15 mg/ml, respectively. In the presence of glyceraldehydephosphate dehydrogenase, the results are independent of enzyme concentration, and slow exchange and upfield shifts of 0.4-0.6 ppm occur with each coenzyme. These data indicate that the environment of the pyrophosphate group of oxidized and reduced coenzyme is the same for a given dehydrogenase, but is different in one enzyme from the other. The resonances observed with glyceraldehydephosphate dehydrogenase are broader than those observed with lactate dehydrogenase. This is indicative of either shorter relaxation times with the former enzyme, or the presence of multiple, unresolved resonances.     

The role of backbone motions in ligand binding to the c-Src SH3 domain.

The Src homology 3 (SH3) domain of pp60(c-src) (Src) plays dual roles in signal transduction, through stabilizing the repressed form of the Src kinase and through mediating the formation of activated signaling complexes. Transition of the Src SH3 domain between a variety of binding partners during progression through the cell cycle requires adjustment of a delicate free energy balance. Although numerous structural and functional studies of SH3 have provided an in-depth understanding of structural determinants for binding, the origins of binding energy in SH3-ligand interactions are not fully understood. Considering only the protein-ligand interface, the observed favorable change in standard enthalpy (DeltaH=-9.1 kcal/mol) and unfavorable change in standard entropy (TDeltaS=-2.7 kcal/mol) upon binding the proline-rich ligand RLP2 (RALPPLPRY) are inconsistent with the predominantly hydrophobic interaction surface. To investigate possible origins of ligand binding energy, backbone dynamics of free and RLP2-bound SH3 were performed via (15)N NMR relaxation and hydrogen-deuterium (H/(2)H) exchange measurements. On the ps-ns time scale, assuming uncorrelated motions, ligand binding results in a significant reduction in backbone entropy (-1.5(+/-0.6) kcal/mol). Binding also suppresses motions on the micros-ms time scale, which may additionally contribute to an unfavorable change in entropy. A large increase in protection from H/(2)H exchange is observed upon ligand binding, providing evidence for entropy loss due to motions on longer time scales, and supporting the notion that stabilization of pre-existing conformations within a native state ensemble is a fundamental paradigm for ligand binding. Observed changes in motion on all three time scales occur at locations both near and remote from the protein-ligand interface. The propagation of ligand binding interactions across the SH3 domain has potential consequences in target selection through altering both free energy and geometry in intact Src, and suggests that looking beyond the protein-ligand interface is essential in understanding ligand binding energetics.     

A possible role for melatonin in regulation of chick liver xanthine dehydrogenase activity.

³⁵Cl nuclear magnetic resonance longitudinal and transverse relaxation times were employed to study anion binding to rabbit muscle lactate dehydrogenase. The correlation time, obtained from a comparison of the two relaxation times, shows that coenzyme has a marked retarding effect on the anion mobility at the binding site. The quadrupole coupling constant is estimated from the magnitude of the relaxation rate change on oxamate addition.     

Lactate and malate dehydrogenase binding to the microsomal fraction from chicken liver

Chicken liver microsomal fractions show lactate and malate dehydrogenase activities which behave differently with respect to successive extractions by sonication in 0.15 M NaCl, 0.2% Triton X-100 and 0.15 M NaCl, respectively. The Triton X-100-treated pellet did not show malate dehydrogenase activity but exhibited a 10-fold increase in lactate dehydrogenase activity with respect to the sonicated pellet. Total extracted lactate and malate dehydrogenase activities were, respectively, 7.5 and 1.7 times higher than that in the initial pellet. Different isoenzyme compositions were observed for cytosoluble and microsomal extracted lactate and malate dehydrogenases. When the ionic strength (0-500 mM) or the pH values (6.1-8.7) of the media were increased, an efficient release of lactate dehydrogenase was found at NaCl 30-70 mM and pH 6.6-7.3. Malate dehydrogenase solubilization under the same conditions was very small, even at NaCl 500 mM, but it attained a maximum in the 7.3-8.7 pH range. Cytosoluble lactate dehydrogenase bound in vitro to 0.15 M NaCl-treated (M2) and sonicated (M3) microsomal fractions but not to the crude microsomal fraction (M1). Particle saturation by lactate dehydrogenase occurred with M2 and M3, which contained binding sites with different affinities. Cytosoluble malate dehydrogenase did not bind to M1, M2 and M3 fractions, however, a little binding was found when purified basic malate dehydrogenase was incubated with M2 or M3 fractions.     

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.     

Interesting structural and dynamical behaviors exhibited by the AF-6 PDZ domain upon Bcr peptide binding

PDZ (postsynaptic density-95, disks large, zonula occludens-1) domains are small, protein-protein interaction modules that have multiple binding surfaces for the docking of diverse molecules. These domains can propagate signals from ligand-binding site to distal regions of the structure through allosteric communication. Recent works have revealed that picosecond to nanosecond time scale dynamics play a potential role in propagating long-range signals within a protein. Comparison of AF-6 PDZ domain structures in free and complex forms shows a conformation rearrangement of distal surface 2, which is far from the peptide binding groove. The relaxation dispersion experiments detected that the free AF-6 PDZ domain was sampling multiple conformations; millisecond dynamics mapped a network for allostery signal transmission throughout the AF-6 PDZ domain in the weak saturation state, and intramolecular motions were observed in distal surface 1 when the protein was saturated. These results provide evidence that the allosteric process in the AF-6 PDZ domain is not two-state; instead, the millisecond dynamic network provides a mechanism for the transmission of allosteric signals throughout a protein. Interestingly, the two distal surfaces of the AF-6 PDZ domain respond differently to peptide binding; distal surface 1 changes in millisecond dynamics, whereas distal surface 2 undergoes structural rearrangement. The significance of the different response patterns in the signaling pathway and its relevance to the function of the AF-6 PDZ domain should be studied further.     

Lactate dehydrogenase activity in the mitochondrial fraction of chicken liver: enzyme binding and kinetic behavior of soluble and bound enzyme

Chicken liver crude mitochondrial fraction showed lactate dehydrogenase activity (6.5% of cytoplasmic enzyme). Most of the mitochondrial lactate dehydrogenase was solubilized by sonication of the mitochondrial fraction in 0.15 M NaCl, pH 6. Total extracted lactate deshydrogenase activity was 3-fold higher than the initial pellet activity. Different isoenzymatic compositions were observed for cytosoluble and mitochondrial extracted lactate dehydrogenase. The pI, values of the 5 lactate dehydrogenase isoenzymes were found to be independent of their origin. The cytosoluble lactate dehydrogenase and the separated H4,H3M and H2M2 isoenzymes were able to bind to the chicken liver mitochondrial fraction in 5 mM sodium phosphate buffered medium, and could be solubilized afterwards with 0.15 M NaCl, pH 6. The enzyme bound to the mitochondrial fraction was less active than the soluble one. Particle saturation by the bound enzyme occurred with all mitochondrial fractions assayed. According to the Langmuir isotherm, the non-sonicated mitochondrial fractions contain a single type of binding sites for lactate dehydrogenase; in contrast, the sonicated mitochondrial fraction should contain different binding sites. Chicken liver crude or sonicated active mitochondrial fractions showed a hyperbolic behavior with respect to NADH and a non-hyperbolic one with respect to pyruvate. This mechanism is different from the bi-bi compulsory order mechanism of the soluble enzyme. With hydroxypyruvate as the substrate, the active mitochondrial fraction fit a sequential mechanism but lost the rapid-equilibrium characteristics of the soluble enzyme.