The structural dynamics of myoglobin

Conformational fluctuations in proteins were initially invoked to explain the observation that diffusion of small ligands through the matrix is a global phenomenon. Small globular proteins contain internal cavities that play a role not only in matrix dynamics but also in controlling function, tracing a pathway for the diffusion of the ligand to and from the active site. This is the main point addressed in this Review, which presents pertinent information obtained on myoglobin (Mb). Mb, a simple globular heme protein which binds reversibly oxygen and other ligands. The bond between the heme Fe(II) and gaseous ligands can be photodissociated by a laser pulse, generating a non-equilibrium population of protein structures that relaxes on a picosecond to millisecond time range. This process is associated with migration of the ligand to internal cavities of the protein, which are known to bind xenon. Some of the results obtained by laser photolysis, molecular dynamics simulations, and X-ray diffraction of intermediate states of wild-type and mutant myoglobins are summarized. The extended relaxation of the globin moiety directly observed by Laue crystallography reflects re-equilibration among conformational substates known to play an essential role in controlling protein function.     

Cavities and packing defects in the structural dynamics of myoglobin

Small globular proteins contain internal cavities and packing defects that reduce thermodynamic stability but seem to play a role in controlling function by defining pathways for the diffusion of the ligand/substrate to the active site. In the case of myoglobin (Mb), a prototype for structure–function relationship studies, the photosensitivity of the adduct of the reduced protein with CO, O₂ and NO allows events related to the migration of the ligand through the matrix to be followed. The crystal structures of intermediate states of wild-type (wt) and mutant Mbs show the photolysed CO to be located either in the distal heme pocket (primary docking site) or in one of two alternative cavities (secondary docking sites) corresponding to packing defects accessible to an atom of xenon. These results convey the general picture that pre-existing internal cavities are involved in controlling the dynamics and reactivity of the reactions of Mb with O₂ and other ligands, including NO.     

Molecular dynamics simulation of sperm whale myoglobin: effects of mutations and trapped CO on the structure and dynamics of cavities

The results of extended (80-ns) molecular dynamics simulations of wild-type and YQR triple mutant of sperm whale deoxy myoglobin in water are reported and compared with the results of the simulation of the intermediate(s) obtained by photodissociation of CO in the wild-type protein. The opening/closure of pathways between preexistent cavities is different in the three systems. For the photodissociated state, we previously reported a clear-cut correlation between the opening probability and the presence of the photolyzed CO in the proximity of the passage; here we show that in wild-type deoxy myoglobin, opening is almost random. In wild-type deoxy myoglobin, the passage between the distal pocket and the solvent is strictly correlated to the presence/absence of a water molecule that simultaneously interacts with the distal histidine side chain and the heme iron; conversely, in the photodissociated myoglobin, the connection with the bulk solvent is always open when CO is in the vicinity of the A pyrrole ring. In YQR deoxy myoglobin, the mutated Gln(E7)64 is stably H-bonded with the mutated Tyr(B10)29. The essential dynamics analysis unveils a different behavior for the three systems. The motion amplitude is progressively restricted in going from wild-type to YQR deoxy myoglobin and to wild-type myoglobin photoproduct. In all cases, the principal motions involve mainly the same regions, but their directions are different. Analysis of the dynamics of the preexisting cavities indicates large fluctuations and frequent connections with the solvent, in agreement with the earlier hypothesis that some of the ligand may escape from the protein through these pathways.     

Extended molecular dynamics simulation of the carbon monoxide migration in sperm whale myoglobin

We report the results of an extended molecular dynamics simulation on the migration of photodissociated carbon monoxide in wild-type sperm whale myoglobin. Our results allow following one possible ligand migration dynamics from the distal pocket to the Xe1 cavity via a path involving the other xenon binding cavities and momentarily two additional packing defects along the pathway. Comparison with recent time resolved structural data obtained by Laue crystallography with subnanosecond to millisecond resolution shows a more than satisfactory agreement. In fact, according to time resolved crystallography, CO, after photolysis, can occupy the Xe1 and Xe4 cavities. However, no information on the trajectory of the ligand from the distal pocket to the Xe1 is available. Our results clearly show one possible path within the protein. In addition, although our data refer to a single trajectory, the local dynamics of the ligand in each cavity is sufficiently equilibrated to obtain local structural and thermodynamic information not accessible to crystallography. In particular, we show that the CO motion and the protein fluctuations are strictly correlated: free energy calculations of the migration between adjacent cavities show that the migration is not a simple diffusion but is kinetically or thermodynamically driven by the collective motions of the protein; conversely, the protein fluctuations are influenced by the ligand in such a way that the opening/closure of the passage between adjacent cavities is strictly correlated to the presence of CO in its proximity. The compatibility between time resolved crystallographic experiments and molecular dynamics simulations paves the way to a deeper understanding of the role of internal dynamics and packing defects in the control of ligand binding in heme proteins.     

Protein Collective Motions Coupled to Ligand Migration in Myoglobin

Ligand migration processes inside myoglobin and protein dynamics coupled to the migration were theoretically investigated with molecular dynamics simulations. Based on a linear response theory, we identified protein motions coupled to the transient migration of ligand, carbon monoxide (CO), through channels. The result indicates that the coupled protein motions involve collective motions extended over the entire protein correlated with local gating motions at the channels. Protein motions, coupled to opening of a channel from the distal pocket to a neighboring xenon site, were found to share the collective motion with experimentally observed protein motions coupled to a doming motion of the heme Fe atom upon photodissociation of the ligand. Analysis based on generalized Langevin dynamics elucidated slow and diffusive features of the protein response motions. Remarkably small transmission coefficients for rates of the CO migrations through myoglobin were found, suggesting that the CO migration dynamics are characterized as motions governed by the protein dynamics involving the collective motions, rather than as thermally activated transitions across energy barriers of well-structured channels.     

Structural dynamics of ligand diffusion in the protein matrix: A study on a new myoglobin mutant Y(B10) Q(E7) R(E10)

A triple mutant of sperm whale myoglobin (Mb) [Leu(B10) --> Tyr, His(E7) --> Gln, and Thr(E10) --> 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, O2, 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 O2 makes two H-bonds with Tyr(B10)29 and Gln(E7)64; on deoxygenation, these two residues move toward the space occupied by O2. The bimolecular rate constant for NO binding is the same as for wild-type, but those for CO and O2 binding are reduced 10-fold. While there is no geminate recombination with O2 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 O2 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 O2 delivery to tissue at a rate compatible with physiology.     

Locally enhanced sampling molecular dynamics study of the dioxygen transport in human cytoglobin

Cytoglobin (Cyg)--a new member of the vertebrate heme globin family--is expressed in many tissues of the human body but its physiological role is still unclear. It may deliver oxygen under hypoxia, serve as a scavenger of reactive species or be involved in collagen synthesis. This protein is usually six-coordinated and binds oxygen by a displacement of the distal HisE7 imidazole. In this paper, the results of 60 ns molecular dynamics (MD) simulations of dioxygen diffusion inside Cyg matrix are discussed. In addition to a classical MD trajectory, an approximate Locally Enhanced Sampling (LES) method has been employed. Classical diffusion paths were carefully analyzed, five cavities in dynamical structures were determined and at least four distinct ligand exit paths were identified. The most probable exit/entry path is connected with a large tunnel present in Cyg. Several residues that are perhaps critical for kinetics of small gaseous diffusion were discovered. A comparison of gaseous ligand transport in Cyg and in the most studied heme protein myoglobin is presented. Implications of efficient oxygen transport found in Cyg to its possible physiological role are discussed.     

Infrared Study of Carbon Monoxide Migration among Internal Cavities of Myoglobin Mutant L29W

Myoglobin, a small globular heme protein that binds gaseous ligands such asO₂, CO and NO reversibly at the heme iron, provides an excellent modelsystem for studying structural and dynamic aspects of protein reactions. Flashphotolysis experiments, performed over wide ranges in time and temperature, reveal a complex ligand binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein. Our recent studies of carbonmonoxy-myoglobin (MbCO) mutant L29W, using time-resolved infrared spectroscopy in combination with x-ray crystallography, have correlated kinetic intermediates with photoproduct structures that are characterized by the CO residing in different internal protein cavities, so-called xenon holes. Here we have used Fourier transform infrared temperature derivative spectroscopy (FTIR-TDS) to further examine the role of internal cavities in the dynamics. Different cavities can be accessed by the CO ligands at different temperatures, and characteristic infrared absorption spectra have been obtained for the different locations of the CO ligand within the protein, enabling us to monitor ligand migration through the protein as well as conformational changes of the protein.     

Molecular dynamics simulation of deoxy and carboxy murine neuroglobin in water

Globins are respiratory proteins that reversibly bind dioxygen and other small ligands at the iron of a heme prosthetic group. Hemoglobin and myoglobin are the most prominent members of this protein family. Unexpectedly a few years ago a new member was discovered and called neuroglobin (Ngb), being predominantly expressed in the brain. Ngb is a single polypeptide of 151 amino acids and despite the small sequence similarity with other globins, it displays the typical globin fold. Oxygen, nitric oxide, or carbon monoxide can displace the distal histidine which, in ferrous Ngb as well as in ferric Ngb, is bound to the iron, yielding a reversible adduct. Recent crystallographic data on carboxy Ngb show that binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities; in particular, the huge internal tunnel that connects the bulk with the active site, peculiar to Ngb, is heavily reorganized. We report the results of extended (90 ns) molecular dynamics simulations in water of ferrous deoxy and carboxy murine neuroglobin, which are both coordinated on the distal site, in the latter case by CO and in the former one by the distal His(64)(E7). The long timescale of the simulations allowed us to characterize the equilibrated protein dynamics and to compare protein structure and dynamical behavior coupled to the binding of an exogenous ligand. We have characterized the heme sliding motion, the topological reorganization of the internal cavities, the dynamics of the distal histidine, and particularly the conformational change of the CD loop, whose flexibility depends ligand binding.     

Dynamics of ligand rebinding to unfolded MbCO by guanidine HCl

Femtosecond vibrational spectroscopy was used to probe a functionally important dynamics and residual structure of myoglobin unfolded by 4 M guanidine HCl. The spectra of the dissociated CO indicated that the residual structure of unfolded myoglobin (Mb) forms a few hydrophobic cavities that could accommodate the dissociated ligand. Geminate rebinding (GR) of CO to the unfolded Mb is three-orders-of-magnitude faster and more efficient than the native Mb but similar to a model heme in a viscous solvent, suggesting that the GR of CO to heme is accelerated by the longer retention of the dissociated ligand near the Fe atom by the poorly-structured protein matrix of the unfolded Mb or viscous solvent. The inefficient GR of CO in native Mb, while dissociated CO is trapped in the primary heme pocket located near the active binding site, indicates that the tertiary structure of the pocket in native Mb plays a functionally significant role.     

Structural dynamics of myoglobin: effect of internal cavities on ligand migration and binding

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation. Photoproduct intermediates, characterized by CO in different locations, were selectively enhanced by laser illumination at specific temperatures. Measurements were performed on the wild-type protein and a series of mutants (L104W, I107W, I28F, and I28W) in which bulky amino acid side chains were introduced to block passageways between cavities or to fill these sites. Binding of xenon was also employed as an alternative means of filling cavities. In all samples, photolyzed CO ligands were observed to initially bind at primary docking site B in the vicinity of the heme iron, from where they migrate to the secondary docking sites, the Xe4 and/or Xe1 cavities. To examine the relevance of these internal docking sites for physiological ligand binding, we have performed room-temperature flash photolysis on the entire set of proteins in the CO- and O(2)-bound form. Together with the cryospectroscopic results, these data provide a clear picture of the role of the internal sites for ligand escape from and binding to myoglobin.     

Geminate rebinding in trehalose-glass embedded myoglobins reveals residue-specific control of intramolecular trajectories.

It is becoming increasingly apparent that hydrophobic cavities (also referred to as xenon cavities) within proteins have significant functional implications. The potential functional role of these cavities in modulating the internal dynamics of carbon monoxide in myoglobin (Mb) is explored in the present study by using glassy matrices derived from trehalose to limit protein dynamics and to eliminate ligand exchange between the solvent and the protein. By varying the temperature (-15 to 65 degrees C) and humidity for samples of carbonmonoxy myoglobin embedded in trehalose-glass, it is possible to observe a hierarchy of distinct geminate recombination phases that extend from nanosecond to almost seconds that can be directly associated with rebinding from specific hydrophobic cavities. The use of mutant forms of Mb reveals the role of key residues in modulating ligand access between these cavities and the distal hemepocket.     

Protein-ligand dynamics. A 96 picosecond simulation of a myoglobin-xenon complex

A 96 picosecond dynamics trajectory of myoglobin with five xenon-probe ligands in internal cavities is examined to study the effect of protein motions on ligand motion and internal cavity fluctuations. Average structural and energetic properties indicate that the simulation is well behaved. The average protein volume is similar to the volume of the X-ray model and the main-chain atom root-mean-square deviation between the X-ray model and the average dynamical structure is 1.25 A. The protein volume oscillates 3 to 4% around the volume of the X-ray structure. These fluctuations lead to changes in the internal free volume and in the size, shape and location of atom-sized cavity features. Transient cavities produced in the simulation have a crucial role in the movement of two of the ligands. One of the ligands escapes to the protein surface, whilst a second ligand travels through the protein interior. Complex gating processes involving several protein residues are responsible for producing the necessary pores through which the ligand passes between transient cavities or packing defects.     

Structural dynamics of myoglobin: spectroscopic and structural characterization of ligand docking sites in myoglobin mutant L29W

We have studied CO binding to the heme and CO migration among protein internal cavities after photodissociation in sperm whale carbonmonoxy myoglobin (MbCO) mutant L29W using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) and kinetic experiments at cryogenic temperatures. Photoproduct intermediates, characterized by CO at particular locations in the protein, were selectively enhanced by applying special laser illumination protocols. These studies were performed on the L29W mutant protein and a series of double mutants constructed so that bulky amino acid side chains block passageways between cavities or fill these sites. Binding of xenon was also employed as an alternative means of occluding cavities. All mutants exhibit two conformations, A(I) and A(II), with distinctly different photoproduct states and ligand binding properties. These differences arise mainly from different positions of the W29 and H64 side chains in the distal heme pocket [Ostermann, A., et al. (2000) Nature 404, 205-208]. The detailed knowledge of the interplay between protein structure, protein dynamics, and ligand migration at cryogenic temperatures allowed us to develop a dynamic model that explains the slow CO and O(2) bimolecular association observed after flash photolysis at ambient temperature.     

Structural dynamics of distal histidine replaced mutants of myoglobin accompanied with the photodissociation reaction of the ligand

Protein dynamics observed by the transient grating (TG) method are studied for some site-directed mutants at the distal histidine of myoglobin (H64L, H64Q, H64V). The time profiles of the TG signals are very sensitive to the amino acid residue of the 64 position. It was found that the sensitivity is mostly caused by the different rates of the ligand escape from the protein to solvent and the magnitude of the molecular volume change. Several molecular origins of the volume difference between MbCO and Mb, such as the electrostatic interaction in the distal pocket, movement of helices, and distal water, are proposed. Interestingly, the volume difference between the CO-trapped Mb inside the protein interior and Mb is similar to that of the partial molar volume of CO in organic solvent. The effect of mutation on the nature of the CO trapped site is discussed.     

Mapping the pathways for O2 entry into and exit from myoglobin

The effects of mutagenesis on geminate and bimolecular O2 rebinding to 90 mutants at 27 different positions were used to map pathways for ligand movement into and out of sperm whale myoglobin. By analogy to a baseball glove, the protein "catches" and then "holds" incoming ligand molecules long enough to allow bond formation with the iron atom. Opening of the glove occurs by outward movements of the distal histidine (His(64)), and the ligands are trapped in the interior "webbing" of the distal pocket, in the space surrounded by Ile(28), Leu(29), Leu(32), Val(68), and Ile(107). The size of this pocket is a major determinant of the rate of ligand entry into the protein. Immediately after photo- or thermal dissociation, O2 moves away from the iron into this interior pocket. The majority of the dissociated ligands return to the active site and either rebind to the iron atom or escape through the His(64) gate. A fraction of the ligands migrate further away from the heme group into cavities that have been defined as Xe binding sites 4 and 1; however, most of these ligands also return to the distal pocket, and net escape through the interior of wild-type myoglobin is <20-25%.     

Ligand migration and binding in the dimeric hemoglobin of Scapharca inaequivalvis

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of the dimeric hemoglobin of Scapharca inaequivalvis (HbI) after photodissociation. By combining these studies with X-ray crystallography, three transient ligand docking sites were identified: a primary docking site B in close vicinity to the heme iron, and two secondary docking sites C and D corresponding to the Xe4 and Xe2 cavities of myoglobin. To assess the relevance of these findings for physiological binding, we also performed flash photolysis experiments on HbICO at room temperature and equilibrium binding studies with dioxygen. Our results show that the Xe4 and Xe2 cavities serve as transient docking sites for unbound ligands in the protein, but not as way stations on the entry/exit pathway. For HbI, the so-called histidine gate mechanism proposed for other globins appears as a plausible entry/exit route as well.     

Quenching of the zinc-protoporphyrin triplet state as a measure of small-molecule diffusion through the structure of myoglobin

The diffusion of small molecules through the myoglobin structure was studied. It has been shown that the fluorescent Zn-protoporphyrin substitutes easily for the native nonfluorescent Fe-protoporphyrin in myoglobin. The quenching rate of the E-type delayed fluorescence of Zn-protoporphyrin in a substituted myoglobin by the quenchers oxygen and anthraquinonesulfonate was used to measure their diffusion from the ambient solution through the protein to the ligand binding site. The quenching rate constant (at 21 degrees C) for oxygen is kq = (9.6 +/- 0.9) X 10(7) M-1 S-1, only 1 order of magnitude less than that for Zn-hematoporphyrin quenching in aqueous solution. The activation energy in the range between 2 and 40 degrees C is Ea = 6.0 +/- 0.6 kcal/mol. The corresponding data for anthraquinonesulfonate are kq = (2.1 +/- 0.3) X 10(8) M-1 S-1 and Ea = 5.8 +/- 0.6 kcal/mol. Taking into account the statistical factor involved in the oxygen quenching of the Zn-porphyrin triplet, the quenching rates are very similar. The data are discussed in terms of the "gated reaction" theory of Northrup and McCammon. The similar rate constants and activation energies indicate that the diffusion rate in the protein is determined by the frequency of the conformational changes that open "gates" for the passage of the quencher through the protein.