Microscopic modeling of ligand diffusion through the protein leghemoglobin: computer simulations and experiments (original) (raw)
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Ligand and interfacial dynamics in a homodimeric hemoglobin
Structural Dynamics, 2016
The structural dynamics of dimeric hemoglobin (HbI) from Scapharca inaequivalvis in different ligand-binding states is studied from atomistic simulations on the ls time scale. The intermediates are between the fully ligandbound (R) and ligand-free (T) states. Tertiary structural changes, such as rotation of the side chain of Phe97, breaking of the Lys96-heme salt bridge, and the Fe-Fe separation, are characterized and the water dynamics along the R-T transition is analyzed. All these properties for the intermediates are bracketed by those determined experimentally for the fully ligand-bound and ligand-free proteins, respectively. The dynamics of the two monomers is asymmetric on the 100 ns timescale. Several spontaneous rotations of the Phe97 side chain are observed which suggest a typical time scale of 50-100 ns for this process. Ligand migration pathways include regions between the B/G and C/G helices and, if observed, take place in the 100 ns time scale. V
Biochemistry, 2007
Model-free-based NMR dynamics studies have been undertaken for polypeptide backbone amide N-H bond vectors for both the deoxy and carbonmonoxy forms of chain-specific, isotopically ( 15 N and 2 H) labeled tetrameric hemoglobin (Hb) using 15 N-relaxation parameters [longitudinal relaxation rate (R 1 ), transverse relaxation rate (R 2 ), and heteronuclear nuclear Overhauser effect (NOE)] measured at two temperatures (29 and 34°C) and two magnetic field strengths (11.7 and 14.1 T). In both deoxy and carbonmonoxy forms of human normal adult hemoglobin (Hb A), the amide N-H bonds of most amino acid residues are rigid on the fast time scale (nanosecond to picosecond), except for the loop regions and certain helix-helix connections. Although rigid in deoxy-Hb A, 146His has been found to be free from restriction of its backbone motions in the CO form, presumably due to the rupture of its hydrogen bond/salt bridge network. We now have direct dynamics evidence for this structural transition of Hb in solution. While remarkably flexible in the deoxy state, R31Arg and 123Thr, neighbors in the intradimer (R 1 1 ) interface, exhibit stiffening upon CO binding. These findings imply a role for R31Arg and 123Thr in the intradimer communication but contradict the results from X-ray crystallography. We have also found that there is considerable flexibility in the intradimer (R 1 1 ) interface (i.e., B, G, and H helices and the GH corner) and possible involvement of several amino acid residues (e.g., R31Arg, 3Leu, 41Phe, 123Thr, and 146His) in the allosteric pathway. Several amino acid residues at the intradimer interfaces, such as 109Val, appear to be involved in possible conformational exchange processes. The dynamic picture derived from the present study provides new insights into the traditional description of the stereochemical mechanism for the cooperative oxygenation of Hb A based on X-ray crystallographic results.
Backbone Dynamics of Deoxy and Carbonmonoxy Hemoglobin by NMR/SRLS
The Journal of Physical Chemistry B, 2011
The slowly relaxing local structure (SRLS) approach, developed for NMR spin relaxation analysis in proteins, is applied herein to amide 15 N relaxation in deoxy and carbonmonoxy hemoglobin. Experimental data including 15 N T 1 , T 2 and 15 N-{ 1 H} NOE, acquired at 11.7 and 14.1 T, and 29 and 34°C, are analyzed. The restricted local motion of the N-H bond is described in terms of the principal value (S 0 2 ) and orientation ( D ) of an axial local ordering tensor, S, and the principal values (R || L andR ⊥ L ) and orientation ( O ) of an axial local diffusion tensor, R L . The parameters c 0 2 (the potential coefficient in terms of which S 0 2 is defined), R || L , D , and O are determined by data fitting; R ⊥ L is set equal to the global motional rate, R C , found previously to be (5.2-5.8) × 10 6 1/s in the temperature range investigated. The principal axis of S is (nearly) parallel to the C i-1 R -C i R axis; when the two axes are parallel, D ) -101.3°(in the frame used). The principal axis of R L is (nearly) parallel to the N-H bond; when the two axes are parallel, O ) -101.3°. For "rigid" N-H bonds located in secondary structure elements the best-fit parameters are S 0 2 ) 0.88-0.95 (corresponding to local potentials of 8.6-19.9 k B T), R || L ) 10 9 -10 10 1/s, D ) -101.3°( 2.0°, and O ) -101.3°( 4°. For flexible N-H bonds located in loops the best-fit values are S 0 2 ) 0.75-0.80 (corresponding to local potentials of 4.5-5.5 k B T), R || L ) (1.0-6.3) × 10 8 1/s, D ) -101.3°( 4.0°, and O ) -101.3°( 10°. These results are important in view of their physical clarity, inherent potential for further interpretation, consistency, and new qualitative insights provided (vide infra).
A quantum-chemical picture of hemoglobin affinity
Proceedings of The National Academy of Sciences, 2007
Understanding the molecular mechanism of hemoglobin cooperativity remains an enduring challenge. Protein forces that control ligand affinity are not directly accessible by experiment. We demonstrate that computational quantum mechanics/molecular mechanics methods can provide reasonable values of ligand binding energies in Hb, and of their dependence on allostery. About 40% of the binding energy differences between the relaxed state and tense state quaternary structures result from strain induced in the heme and its ligands, especially in one of the pyrrole rings. The proximal histidine also contributes significantly, in particular, in the ␣-chains. The remaining energy difference resides in protein contacts, involving residues responsible for locking the quaternary changes. In the ␣-chains, the most important contacts involve the FG corner, at the ''hinge'' region of the ␣12 quaternary interface. The energy differences are spread more evenly among the -chain residues, suggesting greater flexibility for the than for the ␣-chains along the quaternary transition. Despite this chain differentiation, the chains contribute equally to the relaxed substitute state energy difference. Thus, nature has evolved a symmetric response to the quaternary structure change, which is a requirement for maximum cooperativity, via different mechanisms for the two kinds of chains.
Structural factors controlling ligand binding to myoglobin: A kinetic hole-burning study
Proceedings of the National Academy of Sciences, 1998
Using temperature-derivative spectroscopy in the temperature range below 100 K, we have studied the dependence of the Soret band on the recombination barrier in sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation at 12 K. The spectra were separated into contributions from the photodissociated species, Mb*CO, and CObound myoglobin. The line shapes of the Soret bands of both photolyzed and liganded myoglobin were analyzed with a model that takes into account the homogeneous bandwidth, coupling of the electronic transition to vibrational modes, and static conformational heterogeneity. The analysis yields correlations between the activation enthalpy for rebinding and the model parameters that characterize the homogeneous subensembles within the conformationally heterogeneous ensemble. Such couplings between spectral and functional parameters arise when they both originate from a common structural coordinate. This effect is frequently denoted as ''kinetic hole burning.'' The study of these correlations gives direct insights into the structure-function relationship in proteins. On the basis of earlier work that assigned spectral parameters to geometric properties of the heme, the connections with the heme geometry are discussed. We show that two separate structural coordinates inf luence the Soret line shape, but only one of the two is coupled to the enthalpy barrier for rebinding. We give evidence that this coordinate, contrary to widespread belief, is not the iron displacement from the mean heme plane.
Biochemistry, 2001
W This paper contains enhanced objects available on the Internet at http://pubs.acs.org/biochemistry. ABSTRACT: A time-resolved Laue X-ray diffraction technique has been used to explore protein relaxation and ligand migration at room temperature following photolysis of a single crystal of carbon monoxymyoglobin. The CO ligand is photodissociated by a 7.5 ns laser pulse, and the subsequent structural changes are probed by 150 ps or 1 µs X-ray pulses at 14 laser/X-ray delay times, ranging from 1 ns to 1.9 ms. Very fast heme and protein relaxation involving the E and F helices is evident from the data at a 1 ns time delay. The photodissociated CO molecules are detected at two locations: at a distal pocket docking site and at the Xe 1 binding site in the proximal pocket. The population by CO of the primary, distal site peaks at a 1 ns time delay and decays to half the peak value in 70 ns. The secondary, proximal docking site reaches its highest occupancy of 20% at ∼100 ns and has a half-life of ∼10 µs. At ∼100 ns, all CO molecules are accounted for within the protein: in one of these two docking sites or bound to the heme. Thereafter, the CO molecules migrate to the solvent from which they rebind to deoxymyoglobin in a bimolecular process with a second-order rate coefficient of 4.5 × 10 5 M -1 s -1 . Our results also demonstrate that structural changes as small as 0.2 Å and populations of CO docking sites of 10% can be detected by time-resolved X-ray diffraction.
The Journal of Physical Chemistry B, 2010
The free-energy profile and the classical kinetics of the heme carbomonoxide binding-unbinding reaction have been derived by means of a theoretical method for the separated chains of human (HbA) and Trematomus newnesi major component (HbTn) hemoglobin. The results reveal that the Rand -chains of HbA have similar values of kinetic constants for the dissociation of the Fe-CO state, in agreement with experimental data. Comparisons of the present findings with the data obtained for the Rand -chains of HbTn and with theoretical and experimental results previously collected on myoglobin provide a detailed picture of this important biochemical reaction in globins. The sequence and structural differences among the globins are not reflected in meaningful variations in the rate of CO dissociation. These data support the conclusion that the differences observed for the reaction with CO of globins, if any, involve the rate of ligand migration to the solvent, rather than the Fe-CO complex formation/rupture. Furthermore, our results agree with the recent discovery that globin family proteins exhibit common dynamics, thus confirming the observation that the dynamic properties of proteins are strongly related to their overall architecture.
Biophysical Journal, 1999
A triple mutant of sperm whale myoglobin (Mb) [Leu(B10) 3 Tyr, His(E7) 3 Gln, and Thr(E10) 3 Arg, called Mb-YQR], investigated by stopped-flow, laser photolysis, crystallography, and molecular dynamics (MD) simulations, proved to be quite unusual. Rebinding of photodissociated NO, O 2 , and CO from within the protein (in a "geminate" mode) allows us to reach general conclusions about dynamics and cavities in proteins. The 3D structure of oxy Mb-YQR shows that bound O 2 makes two H-bonds with Tyr(B10)29 and Gln(E7)64; on deoxygenation, these two residues move toward the space occupied by O 2. The bimolecular rate constant for NO binding is the same as for wild-type, but those for CO and O 2 binding are reduced 10-fold. While there is no geminate recombination with O 2 and CO, geminate rebinding of NO displays an unusually large and very slow component, which is pretty much abolished in the presence of xenon. These results and MD simulations suggest that the ligand migrates in the protein matrix to a major "secondary site," located beneath Tyr(B10)29 and accessible via the motion of Ile(G8)107; this site is different from the "primary site" identified by others who investigated the photolyzed state of wild-type Mb by crystallography. Our hypothesis may rationalize the O 2 binding properties of Mb-YQR, and more generally to propose a mechanism of control of ligand binding and dissociation in hemeproteins based on the dynamics of side chains that may (or may not) allow access to and direct temporary sequestration of the dissociated ligand in a docking site within the protein. This interpretation suggests that very fast (picosecond) fluctuations of amino acid side chains may play a crucial role in controlling O 2 delivery to tissue at a rate compatible with physiology.
Small ligand–globin interactions: Reviewing lessons derived from computer simulation
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2013
In this work we review the application of classical and quantum-mechanical atomistic computer simulation tools to the investigation of small ligand interaction with globins. In the first part, studies of ligand migration, with its connection to kinetic association rate constants (k on ), are presented. In the second part, we review studies for a variety of ligands such as O 2 , NO, CO, HS − , F − , and NO 2 − showing how the heme structure, proximal effects, and the interactions with the distal amino acids can modulate protein\ligand binding. The review presents mainly results derived from our previous works on the subject, in the context of other theoretical and experimental studies performed by others. The variety and extent of the presented data yield a clear example of how computer simulation tools have, in the last decade, contributed to our deeper understanding of small ligand interactions with globins. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.