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.     

Disease-causing mutations in proteins: structural analysis of the CYP1B1 mutations causing primary congenital glaucoma in humans

In this communication, we report an in-depth structure-based analysis of the human CYP1b1 protein carrying disease-causing mutations that are discovered in patients suffering from primary congenital glaucoma (PCG). The "wild-type" and the PCG mutant structures of the human CYP1b1 protein obtained from comparative modeling were subjected to long molecular dynamics simulations with an intention of studying the possible impact of these mutations on the protein structure and hence its function. Analysis of time evolution as well as time averaged values of various structural properties--especially of those of the functionally important regions: the heme binding region, substrate binding region, and substrate access channel--gave some insights into the possible structural characteristics of the disease mutant and the wild-type forms of the protein. In a nutshell, compared to the wild-type the core regions in the mutant structures are associated with subtle but significant changes, and the functionally important regions seem to adopt such structures that are not conducive for the wild-type-like functionality.     

Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface

We have shown that the molecular conformation of a protein at an interface can be probed spatially using time-resolved evanescent wave-induced fluorescence spectroscopic (TREWIFS) techniques. Specifically, by varying the penetration depth of the evanescent field, variable-angle TREWIFS, coupled with variable-angle evanescent wave-induced time-resolved fluorescence anisotropy measurements, allow us to monitor how fluorescence intensity and fluorescence depolarization vary normal to an interface as a function of time after excitation. We have applied this technique to the study of bovine serum albumin (BSA) complexed noncovalently with the fluorophore 1-anilinonaphthalene-8-sulfonic acid. The fluorescence decay varies as a function of the penetration depth of the evanescent wave in a manner that indicates a gradient of hydrophobicity through the adsorbed protein, normal to the interface. Restriction of the fluorescent probes motion also occurs as a function of distance normal to the interface. The results are consistent with a model of partial protein denaturation: at the surface, an adsorbed BSA molecule unfolds, thus optimizing protein–silica interactions and the number of points of attachment to the surface. Further away, normal to the surface, the protein molecule maintains its coiled structure.Submitted as a record of the 2002 Australian Biophysical Society meeting     

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.     

The dynamical response of hen egg white lysozyme to the binding of a carbohydrate ligand

It has become clear that the binding of small and large ligands to proteins can invoke significant changes in side chain and main chain motion in the fast picosecond to nanosecond timescale. Recently, the use of a "dynamical proxy" has indicated that changes in these motions often reflect significant changes in conformational entropy. These entropic contributions are sometimes of the same order as the total entropy of binding. Thus, it is important to understand the connections amongst motion between the manifold of states accessible to the native state of proteins, the corresponding entropy, and how this impacts the overall energetics of protein function. The interaction of proteins with carbohydrate ligands is central to a range of biological functions. Here, we examine a classic carbohydrate interaction with an enzyme: the binding of wild-type hen egg white lysozyme (HEWL) to the natural, competitive inhibitor chitotriose. Using NMR relaxation experiments, backbone amide and side chain methyl axial order parameters were obtained across apo and chitotriose-bound HEWL. Upon binding, changes in the apparent amplitude of picosecond to nanosecond main chain and side chain motions are seen across the protein. Indeed, binding of chitotriose renders a large contiguous fraction of HEWL effectively completely rigid. Changes in methyl flexibility are most pronounced closest to the binding site, but average to only a small overall change in the dynamics across the protein. The corresponding change in conformational entropy is unfavorable and estimated to be a significant fraction of the total binding entropy.     

Protein dynamics and the diversity of an antibody response

The immune system is remarkable in its ability to produce antibodies (Abs) with virtually any specificity from a limited repertoire of germ line precursors. Although the contribution of sequence diversity to this molecular recognition has been studied for decades, recent models suggest that protein dynamics may also broaden the range of targets recognized. To characterize the contribution of protein dynamics to immunological molecular recognition, we report the sequence, thermodynamic, and time-resolved spectroscopic characterization of a panel of eight Abs elicited to the chromophoric antigen 8-methoxypyrene-1,3,6-trisulfonate (MPTS). Based on the sequence data, three of the Abs arose from unique germ line Abs, whereas the remaining five comprise two sets of siblings that arose by somatic mutation of a common precursor. The thermodynamic data indicate that the Abs recognize MPTS via a variety of mechanisms. Although the spectroscopic data reveal small differences in protein dynamics, the anti-MPTS Abs generally show similar levels of flexibility and conformational heterogeneity, possibly representing the convergent evolution of the dynamics necessary for function. However, one Ab is significantly more rigid and conformationally homogeneous than the others, including a sibling Ab from which it differs by only five somatic mutations. This example of divergent evolution demonstrates that point mutations are capable of fixing significant differences in protein dynamics. The results provide unique insight into how high affinity Abs may be produced that bind virtually any target and possibly, from a more general perspective, how new protein functions are evolved.     

Characterization of the internal motions of a chimeric protein by 13C NMR highlights the important dynamic consequences of the engineering on a millisecond time scale.

By transferring the central curaremimetic beta hairpin of the snake toxin alpha into the scaffold of the scorpion charybdotoxin, a chimeric protein was constructed that reproduced the three-dimensional structure and partially reproduced the function of the parent beta hairpin, without perturbing the three-dimensional structure of the scaffold [1]. Picosecond to hour time scale motions of charybdotoxin and the engineered protein were observed, in order to evaluate the dynamic consequences of the six deletions and eight mutations differentiating the two molecules. The chimeric protein dynamics were also compared to that of toxin alpha, in order to examine the beta hairpin motions in both structural contexts. Thus, 13C R1, R1rho and 1H-->13C nOe were measured for all the CalphaHalpha and threonine CbetaHbeta vectors. As the proteins were not labeled, accordion techniques combined to coherence selection by pulsed field gradients and preservation of magnetization following equivalent pathways were used to considerably reduce the spectrometer time needed. On one hand, we observed that the chimeric protein and charybdotoxin are subjected to similar picosecond to nanosecond time scale motions except around the modified beta sheet region. The chimeric protein also exhibits an additional millisecond time scale motion on its whole sequence, and its beta structure is less stable on a minute to hour time scale. On the other hand, when the beta hairpin dynamics is compared in two different structural contexts, i.e. in the chimeric protein and the curaremimetic toxin alpha, the picosecond to nanosecond time scale motions are fairly conserved. However, the microsecond to millisecond time scale motions are different on most of the beta hairpin sequence, and the beta sheet seems more stable in toxin alpha than in the chimera. The slower microsecond to hour time scale motions seem to be extremely sensitive to the structural context, and thus poorly transferred from one protein to another.     

Opportunities and challenges for time-resolved studies of protein structural dynamics at X-ray free-electron lasers

X-ray free-electron lasers (XFELs) are revolutionary X-ray sources. Their time structure, providing X-ray pulses of a few tens of femtoseconds in duration; and their extreme peak brilliance, delivering approximately 10¹² X-ray photons per pulse and facilitating sub-micrometre focusing, distinguish XFEL sources from synchrotron radiation. In this opinion piece, I argue that these properties of XFEL radiation will facilitate new discoveries in life science. I reason that time-resolved serial femtosecond crystallography and time-resolved wide angle X-ray scattering are promising areas of scientific investigation that will be advanced by XFEL capabilities, allowing new scientific questions to be addressed that are not accessible using established methods at storage ring facilities. These questions include visualizing ultrafast protein structural dynamics on the femtosecond to picosecond time-scale, as well as time-resolved diffraction studies of non-cyclic reactions. I argue that these emerging opportunities will stimulate a renaissance of interest in time-resolved structural biochemistry.     

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.     

Profiling of dynamics in protein–lipid–water systems: a time-resolved fluorescence study of a model membrane protein with the label BADAN at specific membrane depths

Profiles of lipid-water bilayer dynamics were determined from picosecond time-resolved fluorescence spectra of membrane-embedded BADAN-labeled M13 coat protein. For this purpose, the protein was labeled at seven key positions. This places the label at well-defined locations from the water phase to the center of the hydrophobic acyl chain region of a phospholipid model membrane, providing us with a nanoscale ruler to map membranes. Analysis of the time-resolved fluorescence spectroscopic data provides the characteristic time constant for the twisting motion of the BADAN label, which is sensitive to the local flexibility of the protein–lipid environment. In addition, we obtain information about the mobility of water molecules at the membrane–water interface. The results provide an unprecedented nanoscale profiling of the dynamics and distribution of water in membrane systems. This information gives clear evidence that the actual barrier of membranes for ions and aqueous solvents is located at the region of carbonyl groups of the acyl chains.     

A recombinant glutamine-binding protein from Escherichia coli: effect of ligand-binding on protein conformational dynamics

We have investigated the effect of the binding of glutamine on the conformational dynamics of the recombinant glutamine binding protein (GlnBP) from Escherichia coli by steady-state and time-resolved fluorescence techniques. The structural stability of the protein was also studied by far-UV circular dichroism spectroscopy in the range of temperature between 25 and 80 degrees C. The results showed that the interaction of the protein with the ligand resulted in a marked change of the structural and conformational dynamics features of the protein. In particular, the fluorescence and circular dichroism data showed that the presence of glutamine resulted in a dramatic increase of the protein thermal stability of about 10 degrees C. In addition, the fluorescence time-resolved data pointed out that both in the absence and in the presence of glutamine the protein structure was highly rigid with small amplitude of segmental motion up to 65 degrees C and a low accessibility of the protein tryptophan residues to acrylamide. The obtained results on the structural properties of the recombinant glutamine-binding protein in the absence and in the presence of glutamine can contribute to a better understanding of the transport-related functions of the protein and structurally similar periplasmic transport proteins, as well as to the design and development of new biotechnological applications of this class of proteins.     

Rapid structural fluctuations of the free HIV protease flaps in solution: Relationship to crystal structures and comparison with predictions of dynamics calculations

Crystal structures have shown that the HIV-1 protease flaps, domains that control access to the active site, are closed when the active site is occupied by a ligand. Although flap structures ranging from closed to semi-open are observed in the free protease, crystal structures reveal that even the semi-open flaps block access to the active site, indicating that the flaps are mobile in solution. The goals of this paper are to characterize the secondary structure and fast (sub-ns) dynamics of the flaps of the free protease in solution, to relate these results to X-ray structures and to compare them with predictions of dynamics calculations. To this end we have obtained nearly complete backbone and many sidechain signal assignments of a fully active free-protease construct that is stabilized against autoproteolysis by three point mutations. The secondary structure of this protein was characterized using the chemical shift index, measurements of (3h)J(NC') couplings across hydrogen bonds, and NOESY connectivities. Analysis of these measurements indicates that the protease secondary structure becomes irregular near the flap tips, residues 49-53. Model-free analysis of (15)N relaxation parameters, T(1), T(2) (T(1rho)) and (15)N-[(1)H] NOE, shows that residues in the flap tips are flexible on the sub-ns time scale, in contrast with previous observations on the inhibitor-bound protease. These results are compared with theoretical predictions of flap dynamics and the possible biological significance of the sub-ns time scale dynamics of the flap tips is discussed.     

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.     

Conservation of Flexible Residue Clusters among Structural and Functional Enzyme Homologues

Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins.     

Accessibility to internal cavities and ligand binding sites monitored by protein crystallographic thermal factors

Protein structures are flexible both in solution and in the solid state. X-ray crystallographically determined thermal factors monitor the flexibility of protein atoms. A method utilizing such factors is proposed to delineate protein regions through which a ligand can exchange between binding site and bulk solvent. It is based on the assumption that thermally excited protein regions are excellent candidates for opening a ligand channel. Computationally simple and inexpensive, the method analyzes directions from which thermal factors can propagate within the protein, resulting in thermal motion paths (TMPs). Applications to engineered T4 lysozymes, where an artificial internal cavity can host hydrophobic molecules, and to sperm whale myoglobins, where the active site is completely buried, yielded results in agreement with other independent structural observations and with previous hypotheses. Further new features could also be suggested. The proposed TMP analysis could aid molecular dynamics simulation studies as well as time-resolved and site-directed mutagenesis experimental studies, especially given its modest computational expense and its direct roots in experimental results based on thermal factors determined in high-resolution crystallographic studies. Proteins 31:201–213,1998. © 1998 Wiley-Liss, Inc.     

Integrating protein structural dynamics and evolutionary analysis with Bio3D

Background

Popular bioinformatics approaches for studying protein functional dynamics include comparisons of crystallographic structures, molecular dynamics simulations and normal mode analysis. However, determining how observed displacements and predicted motions from these traditionally separate analyses relate to each other, as well as to the evolution of sequence, structure and function within large protein families, remains a considerable challenge. This is in part due to the general lack of tools that integrate information of molecular structure, dynamics and evolution.

Results

Here, we describe the integration of new methodologies for evolutionary sequence, structure and simulation analysis into the Bio3D package. This major update includes unique high-throughput normal mode analysis for examining and contrasting the dynamics of related proteins with non-identical sequences and structures, as well as new methods for quantifying dynamical couplings and their residue-wise dissection from correlation network analysis. These new methodologies are integrated with major biomolecular databases as well as established methods for evolutionary sequence and comparative structural analysis. New functionality for directly comparing results derived from normal modes, molecular dynamics and principal component analysis of heterogeneous experimental structure distributions is also included. We demonstrate these integrated capabilities with example applications to dihydrofolate reductase and heterotrimeric G-protein families along with a discussion of the mechanistic insight provided in each case.

Conclusions

The integration of structural dynamics and evolutionary analysis in Bio3D enables researchers to go beyond a prediction of single protein dynamics to investigate dynamical features across large protein families. The Bio3D package is distributed with full source code and extensive documentation as a platform independent R package under a GPL2 license from http://thegrantlab.org/bio3d/.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0399-6) contains supplementary material, which is available to authorized users.     

Use of structural phylogenetic networks for classification of the ferritin-like superfamily

In the postgenomic era, bioinformatic analysis of sequence similarity is an immensely powerful tool to gain insight into evolution and protein function. Over long evolutionary distances, however, sequence-based methods fail as the similarities become too low for phylogenetic analysis. Macromolecular structure generally appears better conserved than sequence, but clear models for how structure evolves over time are lacking. The exponential growth of three-dimensional structural information may allow novel structure-based methods to drastically extend the evolutionary time scales amenable to phylogenetics and functional classification of proteins. To this end, we analyzed 80 structures from the functionally diverse ferritin-like superfamily. Using evolutionary networks, we demonstrate that structural comparisons can delineate and discover groups of proteins beyond the "twilight zone" where sequence similarity does not allow evolutionary analysis, suggesting that considerable and useful evolutionary signal is preserved in three-dimensional structures.     

Time-resolved resonance Raman study on ultrafast structural relaxation and vibrational cooling of photodissociated carbonmonoxy myoglobin

A localized small structural change is converted to a higher order conformational change of protein and extends to a mesoscopic scale to induce a physiological function. To understand such features of protein, ultrafast dynamics of myoglobin (Mb) following photolysis of carbon monoxide were investigated. Recent results are summarized here with a stress on structural and vibrational energy relaxation. The core expansion of heme takes place within 2 ps but the out of plane displacement of the heme iron and the accompanying protein conformational change occur in 10 and 100 s of the picosecond regimes, respectively. Unexpectedly, it was found from UV resonance Raman spectra that Trp7 in the N-terminal region and Tyr151 in the C-terminal region undergo appreciable structural changes upon ligand binding-dissociation while Tyr104, Tyr146, and Trp14 do not. Because of the communication between the movements of these surface residues and the heme iron, the rate of spectral change of the iron-histidine (Fe- His) stretching band after CO photodissociation is influenced by the viscosity of solvent. Temporal changes of the anti-Stokes Raman intensity demonstrated immediate generation of vibrationally excited heme upon photodissociation and its decay with a time constant of 1-2 ps.