Dynamics of Hemoglobin Investigated by Mössbauer Spectroscopy

Abstract

Mössbauer Spectra of ⁵⁷Fe enriched horse hemoglobin and sperm whale myoglobin were measured in the temperature range from 80 K to 260 K. An analysis of the temperature dependence of the recoiless fraction (the Lamb-Mössbauer factor) shows it to be sensitive to conformational fluctuations which affect the mean square displacement of the iron. We have found that the protein conformation has a dramatic effect on these measurements. For hemoglobin greater conformational fluctuations at lower temperatures are observed for carbonmonoxyhemoglobin in the liganded conformation than for deoxyhemoglobin in the unliganded conformation. On the other hand, the Lamb-Mössbauer factor is insensitive to the binding of ligands to myoglobin and shows conformational fluctuations similar to deoxyhemoglobin even in the liganded state. It is also shown that a reversible complex with the distal histidine is formed in frozen deoxyhemoglobin solutions above 200 K where the Lamb-Mössbauer factor shows the excitation of new modes of conformational fluctuations. This complex is not formed with carbonmonoxyhemoglobin which already has a sixth ligand and with deoxymyoglobin which appears to undergo much more limited conformational fluctuations. A possible relationship between the formation of the distal histidine complex and the cooperative ligand binding reaction is suggested by results with partially liganded hemoglobin which indicate increased formation of the distal histidine complex.     

Protein dynamics: Mössbauer spectroscopy on deoxymyoglobin crystals

Mössbauer absorption experiments on ⁵⁷Fe of deoxygenated myoglobin crystals and on K₄⁵⁷Fe(CN)₆ dissolved in the water of metmyoglobin crystals were performed over a large temperature range. At low temperatures the mean square displacements, 〈x²〉, of the iron indicate solid-like behaviour of the whole system, whereas at higher temperatures protein-specific modes of motion contribute to 〈x²>. The protein dynamics are correlated with the mobility of the water within the protein crystals. A Brownian oscillator is used to model the protein-specific modes of motion measured at the ⁵⁷Fe nucleus. Three modes are necessary for understanding the Mössbauer spectrum. Two of them correspond to an extremely overdamped Brownian oscillator. The third mode can be understood as quasi-free diffusion. Whereas the protein molecule is frozen in conformational substates in the low temperature regime, it reaches transition states with a finite probability in the high temperature regime. The surface water mediates a possible trigger mechanism that switches on protein dynamics within a narrow temperature interval. Results from Mössbauer spectroscopy and from X-ray structure analysis are compared.     

Molecular dynamics simulation of carboxy-myoglobin embedded in a trehalose-water matrix

We report on a molecular dynamics (MD) simulation of carboxy-myoglobin (MbCO) embedded in a water-trehalose system. The mean square fluctuations of protein atoms, calculated at different temperatures in the 100-300 K range, are compared with those from a previous MD simulation on an H2O-solvated MbCO and with experimental data from M?ssbauer spectroscopy and incoherent elastic neutron scattering on trehalose-coated MbCO. The results show that, for almost all the atomic classes, the amplitude of the nonharmonic motions stemming from the interconversion among the protein's conformational substates is reduced with respect to the H2O-solvated system, and their onset is shifted toward higher temperature. Moreover, our simulation shows that, at 300 K, the heme performs confined diffusive motions as a whole, leaving the underlying harmonic vibrations unaltered.     

A Physical Picture of Protein Dynamics and Conformational Changes

A physical model is reviewed which explains different aspects of protein dynamics consistently. At low temperatures, the molecules are frozen in conformational substates. Their average energy is 3/2RT. Solid-state vibrations occur on a time scale of femtoseconds to nanoseconds. Above a characteristic temperature, often called the dynamical transition temperature, slow modes of motions can be observed occurring on a time scale between about 140 and 1 ns. These motions are overdamped, quasidiffusive, and involve collective motions of segments of the size of an α-helix. Molecules performing these types of motion are in the “flexible state”. This state is reached by thermal activation. It is shown that these motions are essential for conformational relaxation. Based on this picture, a new approach is proposed to understand conformational changes. It connects structural fluctuations and conformational transitions.     

Backbone dynamics and energetics of a calmodulin domain mutant exchanging between closed and open conformations.

Previous studies have suggested that the Ca2+-saturated E140Q mutant of the C-terminal domain of calmodulin exhibits equilibrium exchange between "open" and "closed" conformations similar to those of the Ca2+-free and Ca2+-saturated states of wild-type calmodulin. The backbone dynamics of this mutant were studied using15N spin relaxation experiments at three different temperatures. Measurements at each temperature of the15N rate constants for longitudinal and transverse auto-relaxation, longitudinal and transverse cross-correlation relaxation, and the1H-15N cross-relaxation afforded unequivocal identification of conformational exchange processes on microsecond to millisecond time-scales, and characterization of fast fluctuations on picosecond to nanosecond time-scales using model-free approaches. The results show that essentially all residues of the protein are involved in conformational exchange. Generalized order parameters of the fast internal motions indicate that the conformational substates are well folded, and exclude the possibility that the exchange involves a significant population of unfolded or disordered species. The temperature dependence of the order parameters offers qualitative estimates of the contribution to the heat capacity from fast fluctuations of the protein backbone, revealing significant variation between the well-ordered secondary structure elements and the more flexible regions. The temperature dependence of the conformational exchange contributions to the transverse auto-relaxation rate constants directly demonstrates that the microscopic exchange rate constants are greater than 2.7x10(3)s-1at 291 K. The conformational exchange contributions correlate with the chemical shift differences between the Ca2+-free and Ca2+-saturated states of the wild-type protein, thereby substantiating that the conformational substates are similar to the open and closed states of wild-type calmodulin. Taking the wild-type chemical shifts to represent the conformational substates of the mutant and populations estimated previously, the microscopic exchange rate constants could be estimated as 2x10(4)to 3x10(4)s-1at 291 K for a subset of residues. The temperature depen dence of the exchange allows the characterization of apparent energy barriers of the conformational transition, with results suggesting a complex process that does not correspond to a single global transition between substates.     

ANCA: Anharmonic Conformational Analysis of Biomolecular Simulations

Anharmonicity in time-dependent conformational fluctuations is noted to be a key feature of functional dynamics of biomolecules. Although anharmonic events are rare, long-timescale (μs–ms and beyond) simulations facilitate probing of such events. We have previously developed quasi-anharmonic analysis to resolve higher-order spatial correlations and characterize anharmonicity in biomolecular simulations. In this article, we have extended this toolbox to resolve higher-order temporal correlations and built a scalable Python package called anharmonic conformational analysis (ANCA). ANCA has modules to: 1) measure anharmonicity in the form of higher-order statistics and its variation as a function of time, 2) output a storyboard representation of the simulations to identify key anharmonic conformational events, and 3) identify putative anharmonic conformational substates and visualization of transitions between these substates.     

Conformational transitions in protein-protein association: binding of fasciculin-2 to acetylcholinesterase

The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.     

Comparison of the dynamical structures of lipoamide dehydrogenase and glutathione reductase by time-resolved polarized flavin fluorescence.

Time-resolved polarized fluorescence spectroscopy has been applied to the bound FAD in the structurally related flavoproteins lipoamide dehydrogenase from Azotobacter vinelandii (LipDH-AV) and glutathione reductase (GR) from human erythrocytes. The fluorescence parameters as obtained from the maximum entropy analysis differ considerably in both enzymes, reflecting the unique properties of the flavin microenvironment. Three conformational substates are revealed in LipDH-AV and five in GR. Almost 90% of the population of GR molecules has a fluorescence lifetime in the order of 30 ps which originates from efficient exciplex formation with Tyr197. Equilibrium fluctuations between conformational substates are observed for LipDH-AV on a nanosecond time scale in the temperature range 277-313 K. Interconversion between conformational substates in GR is slow, indicating that large activation barriers exist between the states. In agreement with these results, a model is postulated which ascribes a role in catalysis to equilibrium fluctuations between conformational substates in GR and LipDH-AV. From time-resolved fluorescence anisotropy as a function of temperature, distinction can be made between flavin reorientational motion and interflavin energy transfer. In both proteins intersubunit energy transfer between the prosthetic groups is observed. Furthermore, it is revealed that only the flavin in glutathione reductase exhibits rapid restricted reorientational motion. Geometric information concerning the relative orientation and distance of the flavins can be extracted from the parameters describing the energy-transfer process. The obtained spatial arrangement of the flavins is in excellent agreement with crystallographic data.     

Quantifying the importance of protein conformation on ligand migration in myoglobin

Myoglobin (Mb) is a model system for ligand binding and migration. The energy barriers (ΔG) for ligand migration in Mb have been studied in the past by experiment and theory and significant differences between different approaches were found. From experiment, it is known that Mb can assume a large number of conformational substates. In this work, these substates are investigated as a possible source of the differences in migration barriers. We show that the initial structure significantly affects the calculated ΔG for a particular transition and that fluctuations in barrier heights δΔG are of similar magnitude as the free energy barriers themselves. The sensitivity of ΔG to the initial structure is compared to other sources of errors. Different protein structures can affect the calculated ΔG by up to 4 kcal/mol, whereas differences between simple point charge models and more elaborate multipolar charge models for the CO-ligand are smaller by a factor of two. Analysis of the structural changes underlying the large effect of the conformational substate reveals the importance of coupling between protein and ligand motion for migration.     

Dynamics of hemoglobin investigated by M?ssbauer spectroscopy

M?ssbauer Spectra of Fe enriched horse hemoglobin and sperm whale myoglobin were measured in the temperature range from 80 K to 260 K. An analysis of the temperature dependence of the recoiless fraction (the Lamb-M?ssbauer factor) shows it to be sensitive to conformational fluctuations which affect the mean square displacement of the iron. We have found that the protein conformation has a dramatic effect on these measurements. For hemoglobin greater conformational fluctuations at lower temperatures are observed for carbonmonoxyhemoglobin in the liganded conformation than for deoxyhemoglobin in the unliganded conformation. On the other hand, the Lamb-M?ssbauer factor is insensitive to the binding of ligands to myoglobin and shows conformational fluctuations similar to deoxyhemoglobin even in the liganded state. It is also shown that a reversible complex with the distal histidine is formed in frozen deoxyhemoglobin solution above 200 K where the Lamb-M?ssbauer factor shows the excitation of new modes of conformational fluctuations. This complex is not formed with carbonmonoxyhemoglobin which already has a sixth ligand and with deoxymyoglobin which appears to undergo much more limited conformational fluctuations. A possible relationship between the formation of the distal histidine complex and the cooperative ligand binding reaction is suggested by results with partially liganded hemoglobin which indicate increased formation of the distal histidine complex.     

Instantaneous Normal Modes as an Unforced Reaction Coordinate for Protein Conformational Transitions

We present a novel sampling approach to explore large protein conformational transitions by determining unique substates from instantaneous normal modes calculated from an elastic network model, and applied to a progression of atomistic molecular dynamics snapshots. This unbiased sampling scheme allows us to direct the path sampling between the conformational end states over simulation timescales that are greatly reduced relative to the known experimental timescales. We use adenylate kinase as a test system to show that instantaneous normal modes can be used to identify substates that drive the structural fluctuations of adenylate kinase from its closed to open conformations, in which we observe 16 complete transitions in 4 μs of simulation time, reducing the timescale over conventional simulation timescales by two orders of magnitude. Analysis shows that the unbiased determination of substates is consistent with known pathways determined experimentally.     

Comparison study of ferrous ions in deoxygenated Hb-A and its isolated subunits

The Mössbauer effect in ⁵⁷Fe was used to probe the electronic structure of the ferrous ions in deoxy Hb-A and its isolated subunits, which are known to have characteristically different oxygen affinities. Within experimental error, the Mössbauer parameters obtained over the temperature range of 77°K to 200°K show no differences between these three proteins. Thus the difference in oxygen affinity between the deoxy Hb-A and its isolated subunits must not be a consequence of difference in the iron electronic structure.     

Apparent Tradeoff of Higher Activity in MMP-12 for Enhanced Stability and Flexibility in MMP-3

The greater activity of MMP-12 than MMP-3 toward substrates from protein fibrils has been quantified. Why is MMP-12 the more active protease? We looked for behaviors associated with the higher activity of MMP-12 than MMP-3, using nuclear magnetic resonance to monitor backbone dynamics and residue-specific stabilities of their catalytic domain. The proteolytic activities are likely to play important roles in inflammatory diseases of arteries, lungs, joints, and intestines. Nuclear magnetic resonance line broadening indicates that regions surrounding the active sites of both proteases sample conformational substates within milliseconds. The more extensive line broadening in MMP-3 suggests greater sampling of conformational substates, affecting the full length of helix B and β-strand IV forming the active site, and more remote sites. This could suggest more excursions to functionally incompetent substates. MMP-3 also has enhanced subnanosecond fluctuations in helix A, in the β-hairpin of strands IV and V, and before and including helix C. Hydrogen exchange protection in the EX2 regime suggests that MMP-3 possesses 2.8 kcal/mol higher folding stability than MMP-12(E219A). The β-sheet of MMP-3 appears to be stabilized still more. The higher stability of MMP-3 relative to MMP-12 coincides with the former''s considerably lower proteolytic activity. This relationship is consistent with the hypothesis that enzymes often trade stability for higher activity.     

Starting‐structure dependence of nanosecond timescale intersubstate transitions and reproducibility of MD‐derived order parameters

Factors affecting the accuracy of molecular dynamics (MD) simulations are investigated by comparing generalized order parameters for backbone NH vectors of the B3 immunoglobulin‐binding domain of streptococcal protein G (GB3) derived from simulations with values obtained from NMR spin relaxation (Yao L, Grishaev A, Cornilescu G, Bax A, J Am Chem Soc 2010;132:4295‐4309.). Choices for many parameters of the simulations, such as buffer volume, water model, or salt concentration, have only minor influences on the resulting order parameters. In contrast, seemingly minor conformational differences in starting structures, such as orientations of sidechain hydroxyl groups, resulting from applying different protonation algorithms to the same structure, have major effects on backbone dynamics. Some, but not all, of these effects are mitigated by increased sampling in simulations. Most discrepancies between simulated and experimental results occur for residues located at the ends of secondary structures and involve large amplitude nanosecond timescale transitions between distinct conformational substates. These transitions result in autocorrelation functions for bond vector reorientation that do not converge when calculated over individual simulation blocks, typically of length similar to the overall rotational diffusion time. A test for convergence before averaging the order parameters from different blocks results in better agreement between order parameters calculated from different sets of simulations and with NMR‐derived order parameters. Thus, MD‐derived order parameters are more strongly affected by transitions between conformational substates than by fluctuations within individual substates themselves, while conformational differences in the starting structures affect the frequency and scale of such transitions. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Ligand binding to heme proteins: II. Transitions in the heme pocket of myoglobin.

Phenomena occurring in the heme pocket after photolysis of carbonmonoxymyoglobin (MbCO) below about 100 K are investigated using temperature-derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substrates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experiments on MbCO solution samples at different pH and on orthorhombic crystals. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A substate. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilibrium populations of the B substates after photolysis below 25 K and determine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those between A substates have only been observed above about 160 K. The transitions between the B substates are nonexponential in time, providing evidence for a distribution of substates. The temperature dependence of the B substate transitions implies that they occur mainly by quantum-mechanical tunneling below 10 K. Taken together, the observations suggest that the transitions between the B substates within the same A substate reflect motions of the CO in the heme pocket and not conformational changes. Geminate rebinding of CO to Mb, monitored in the Soret band, depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a population shift among the substates and not from a change of the rebinding to an individual A substate.     

Protein dynamics of a β-sheet protein

Rhodnius prolixus Nitrophorin 4 (abbreviated NP4) is an almost pure β-sheet heme protein. Its dynamics is investigated by X-ray structure determination at eight different temperatures from 122 to 304 K and by means of Mössbauer spectroscopy. A comparison of this β-sheet protein with the pure α-helical protein myoglobin (abbreviated Mbmet) is performed. The mean square displacement derived from the Mössbauer spectra increases linearly with temperature below a characteristic temperature T _c. It is about 10 K larger than that of myoglobin. Above T _c the mean square displacements increase dramatically. The Mössbauer spectra are analyzed by a two state model. The increased mean square displacements are caused by very slow motions occurring on a time scale faster than 140 ns. With respect to these motions NP4 shows the same protein specific modes as Mbmet. There is, however, a difference in the fast vibration regime. The B values found in the X-ray structures vary linearly over the entire temperature range. The mean square displacements in NP4 increase with slopes which are 60% larger than those observed for Mbmet. This indicates that nitrophorin has a larger structural distribution which makes it more flexible than myoglobin.     

Small- and large-scale conformational changes of adenylate kinase: a molecular dynamics study of the subdomain motion and mechanics

Adenylate kinase, an enzyme that catalyzes the phosphoryl transfer between ATP and AMP, can interconvert between the open and catalytically potent (closed) forms even without binding ligands. Several aspects of the enzyme elasticity and internal dynamics are analyzed here by atomistic molecular dynamics simulations covering a total time span of 100 ns. This duration is sufficiently long to reveal a partial conversion of the enzyme that proceeds through jumps between structurally different substates. The intra- and intersubstates contributions to the enzyme's structural fluctuations are analyzed and compared both in magnitude and directionality. It is found that, despite the structural heterogeneity of the visited conformers, the generalized directions accounting for conformational fluctuations within and across the substates are mutually consistent and can be described by a limited set of collective modes. The functional-oriented nature of the consensus modes is suggested by their good overlap with the deformation vector bridging the open and closed crystal structures. The consistency of adenylate kinase's internal dynamics over timescales wide enough to capture intra- and intersubstates fluctuations adds elements in favor of the recent proposal that the free (apo) enzyme possesses an innate ability to sustain the open/close conformational changes.     

Energy Landscape and Dynamics of Biomolecules Extended Abstract

Proteins are not isolated homogeneous systems. Each protein can exist in a very large number of conformations (conformational substates) that are characterized by an energy landscape. The main conformational motions, similar to the α and β fluctuations in glasses, are linked to fluctuations in the bulk solvent and the hydration shell.     

Myoglobin-CO conformational substate dynamics: 2D vibrational echoes and MD simulations

Two-dimensional (2D) infrared vibrational echoes were performed on horse heart carbonmonoxymyoglobin (MbCO) in water over a range of temperatures. The A(1) and A(3) conformational substates of MbCO are found to have different dephasing rates with different temperature dependences. A frequency-frequency correlation function derived from molecular dynamics simulations on MbCO at 298 K is used to calculate the vibrational echo decay. The calculated decay shows substantial agreement with the experimentally measured decays. The 2D vibrational echo probes protein dynamics and provides an observable that can be used to test structural assignments for the MbCO conformational substates.     

Nature of iron deposits on the cardiac walls in β-thalassemia by Mössbauer spectroscopy

An identification of the nature and an estimation of the particle size distribution of the iron deposits on thalassemic heart tissue is carried out by variable temperature Mössbauer spectroscopy. Comparison of Mössbauer spectra obtained for the thalassemic heart tissue (I) with those of normal heart tissue (II) and of horse spleen ferritin (III) identifies the iron deposits to be small, superparamagnetic particles of ferritin and/or hemosiderin, two closely related iron storage proteins containing an iron core of (FeOOH)₈(FeO · OPO₃H₂). The dependence of the superparamagnetic relaxation time, τ₂, of magnetically ordered fine particles on their volume V via the magnetic anisotropy constant K of the material and the condition τ > τ_L, the Larmor precession time of the nuclear magnetic moment of ⁵⁷Fe about an effective magnetic field, for observation of hyperfine structure are used in analyzing the Mössbauer data to yield the particle size distribution. Particle diameters are estimated to be $\text{74 ± 12}\text{A}\text{?}$.