Role of CO2 in the formation of gold deposits.

Much of global gold production has come from deposits with uneconomic concentrations of base metals, such as copper, lead and zinc. These 'gold-only' deposits are thought to have formed from hot, aqueous fluids rich in carbon dioxide, but only minor significance has been attached to the role of the CO2 in the process of gold transport. This is because chemical bonding between gold ions and CO2 species is not strong, and so it is unlikely that CO2 has a direct role in gold transport. An alternative indirect role for CO2 as a weak acid that buffers pH has also appeared unlikely, because previously inferred pH values for such gold-bearing fluids are variable. Here we show that such calculated pH values are unlikely to record conditions of gold transport, and propose that CO2 may play a critical role during gold transport by buffering the fluid in a pH range where elevated gold concentration can be maintained by complexation with reduced sulphur. Our conclusions, which are supported by geochemical modelling, may provide a platform for new gold exploration methods.

Structure and dynamics of green fluorescent protein.

Many marine organisms are luminescent. The proteins that produce the light include a primary light producer (aequorin or luciferase) and often a secondary photoprotein that red shifts the light for better penetration in the ocean. Green fluorescent protein is one such secondary protein. It is remarkable in that it autocatalyzes the formation of its own fluorophore and thus can be expressed in a variety of organisms in its fluorescent form. The recent determination of its 3D structure and other physical characterizations are revealing its molecular mechanism of action.

The molecular structure of green fluorescent protein.

The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of beta-sheet with an alpha-helix inside and short helical segments on the ends of the cylinder. This motif, with beta-structure on the outside and alpha-helix on the inside, represents a new protein fold, which we have named the beta-can. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr66 with reduction of its C alpha-C beta bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests specific side chains that could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.

Motions of calmodulin characterized using both Bragg and diffuse X-ray scattering.

BACKGROUND: Calmodulin is a calcium-activated regulatory protein which can bind to many different targets. The protein resembles a highly flexible dumbbell, and bends in the middle as it binds. This and other motions must be understood to formulate a realistic model of calmodulin function. RESULTS: Using the Bragg reflections from X-ray crystallography, a multiple-conformer refinement of a calmodulin-peptide complex shows anisotropic displacements, with high variations of dihedral angles in several nonhelical domains: the flexible linker; three of the four calcium-binding sites (including both of the N-terminal sites); and a turn connecting the C-terminal EF-hand calcium-binding domains. Three-dimensional maps of the large scale diffuse X-ray scattering data show isotropic liquid-like motions with an unusually small correlation length. Three-dimensional maps of the small scale diffuse streaks show highly coupled, anisotropic motions along the head-to-tail molecular packing direction in the unit cell. There is also weak coupling perpendicular to the head-to-tail packing direction, particularly across a cavity occupied by the disordered linker domain of the molecule. CONCLUSIONS: Together, the Bragg and diffuse scattering present a self-consistent description of the motions in the flexible linker of calmodulin. The other mobile regions of the protein are also of great interest. In particular, the high variations in the calcium-binding sites are likely to influence how strongly they bind ions. This is especially important in the N-terminal sites, which regulate the activity of the molecule.

Characterizing global substates of myoglobin.

BACKGROUND: The massive amount of information generated from current molecular dynamics simulations makes the data difficult to analyze efficiently. Principal component analysis has been used for almost a century to detect and characterize data relationships and to reduce the dimensionality for problems in many fields. Here, we present an adaptation of principal component analysis using a partial singular value decomposition (SVD) for investigating both the localized and global motions of macromolecules. RESULTS: Configuration space projections from the SVD analysis of a variety of myoglobin simulations are used to characterize the dynamics of the protein. This technique reveals new dynamical motifs, which quantify proposed hierarchical structures of conformational substates for proteins and provide a means by which configuration space sampling efficiency may be probed. The SVD clearly shows that solvent effects facilitate transitions between global conformational substates for myoglobin molecular dynamics simulations. Lyapunov exponents calculated from the configuration space divergence of 15 trajectories agree with previous predictions for the chaotic behavior of complex protein systems. CONCLUSIONS: Configuration space projections provide invaluable information about protein motions that would be extremely difficult to obtain otherwise. While the configuration space for myoglobin is quite large, it does have structure. Our analysis of this structure shows that the protein hops between a number of distinct global conformational states, much like the local behavior observed for an individual residue.

Metal-ion affinity and specificity in EF-hand proteins: coordination geometry and domain plasticity in parvalbumin.

BACKGROUND: The EF-hand family is a large set of Ca(2+)-binding proteins that contain characteristic helix-loop-helix binding motifs that are highly conserved in sequence. Members of this family include parvalbumin and many prominent regulatory proteins such as calmodulin and troponin C. EF-hand proteins are involved in a variety of physiological processes including cell-cycle regulation, second messenger production, muscle contraction, microtubule organization and vision. RESULTS: We have determined the structures of parvalbumin mutants designed to explore the role of the last coordinating residue of the Ca(2+)-binding loop. An E101D substitution has been made in the parvalbumin EF site. The substitution decreases the Ca(2+)-binding affinity 100-fold and increases the Mg(2+)-binding affinity 10-fold. Both the Ca(2+)- and Mg(2+)-bound structures have been determined, and a structural basis has been proposed for the metal-ion-binding properties. CONCLUSIONS: The E101D mutation does not affect the Mg(2+) coordination geometry of the binding loop, but it does pull the F helix 1.1 A towards the loop. The E101D-Ca(2+) structure reveals that this mutant cannot obtain the sevenfold coordination preferred by Ca(2+), presumably because of strain limits imposed by tertiary structure. Analysis of these results relative to previously reported structural information supports a model wherein the characteristics of the last coordinating residue and the plasticity of the Ca(2+)-binding loop delimit the allowable geometries for the coordinating sphere.

Crystal structure of a nonsymbiotic plant hemoglobin.

BACKGROUND: Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O(2) conditions. RESULTS: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 A resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b(5). The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit. CONCLUSIONS: The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O(2) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(2) affinity is ultra-high (K(D) approximately 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified.

Crystal structures of CO-, deoxy- and met-myoglobins at various pH values.

The distal histidine residue, His64(E7), and the proximal histidine residue, His93(F8), in myoglobin (Mb) are important for the function of the protein. For example, the increase in the association rate constant for CO binding at low pH has been suggested to be caused by the protonation of these histidine residues. In order to investigate the influence of protonation on the structure of myoglobin, we determined the crystal structures of sperm whale myoglobin to 2.0 A or better in different states of ligation (MbCO, deoxyMb and metMb) at pH values of 4, 5 and 6. The most dramatic change found at low pH is that His64 swings out of the distal pocket in the MbCO structure at pH 4, opening a direct channel from the solvent to the iron atom. This rotation seems to be facilitated by conformational changes in the CD corner. The benzyl side-chain of Phe46(CD4), which has been suggested to be a critical residue in controlling the rotation of His64, moves away from His64 at pH 4 in the deoxyMb structure, allowing more free rotation of His64. Arg45(CD3) is also important for the dynamics of myoglobin, since it influences the pK(a) of His64 and forms a hydrogen bond lattice that hinders the rotation of His64 at neutral pH. This hydrogen-bond lattice disappears at low pH. Although His64 rotates out of the distal pocket in the MbCO structure at pH 4, leaving more space for the CO ligand, the Fe-C-O angle refines to about 130 degrees, the same as those at pH 5 and 6. In the MbCO structure at pH 4, significant conformational changes appear in the EF corner. The peptide plane between Lys79(EF2) and Gly80(EF3) flips about 150 degrees. The occupancy of this conformation in the MbCO structures increases with decreases in pH. On the proximal side of the heme, the bond between the heme iron atom and N(epsilon) of His93 remains intact under the experimental conditions in the MbCO and deoxyMb structures, but appears elongated in the metMb structure at pH 4, representing either a weakened bond or the breakage of the bond in some fraction of the molecules in the crystal.

Tropomyosin crystal structure and muscle regulation.

The crystal structure of tropomyosin filaments has been solved to 15 A resolution by refinement of models against the diffraction data and heavy atom labeling of cysteine residues. These results confirm and extend earlier findings. The improved maps reveal the pitch of the coiled coil, the location of the cysteine residues, and the location and features of the overlapping molecular ends in the filaments. A correlation can now be made between regions of the amino acid sequence and key features of the molecule, such as contact sites in the lattice and departures from regularity along the coiled coil. The crystal shows remarkable dynamic features and the relative flexibility of different parts of the molecule as well as its anisotropic character have been determined. The structure and motions of tropomyosin in the crystal provide information on the structure of tropomyosin in muscle and its possible role in regulation. An atomic model of the molecule has been constructed, based on the low resolution X-ray results, together with the stereochemistry of alpha-helical coiled coils. In contrast to previous views, the molecule appears to display but one set of seven alpha-sites that permit weak linkages of the flexible tropomyosin filament to the actin helix. Correspondingly, we picture that in the "off" state of ATPase activity, the alpha-sites are not occupied; in the "on" state, they are only partly occupied; and in the "potentiated" state, they are more completely saturated. Control of contraction is therefore seen as a statistical mechanism requiring at least three distinct average conformations for the tropomyosin molecule on the actin helix.

A new crystal form of tropomyosin. Preliminary X-ray diffraction analysis.

A new crystalline form of tropomyosin has been produced that diffracts to about 4 A resolution. The crystals are grown at room temperature by slowly lowering the concentration of spermine. This polyamine apparently neutralizes the acidic amino acid side-chains of tropomyosin and allows close side-by-side packing of molecules. The space group is C2, with unit cell dimensions a = 259.7 A, b = 55.3 A, c = 135.6 A, and beta = 97.2 degrees. The tropomyosin molecules appear to be bonded head-to-tail to form straight filaments that run along the crystallographic (332) direction in an arrangement closely related to thin crystalline sheets previously described.

Structure of myoglobin-ethyl isocyanide. Histidine as a swinging door for ligand entry.

The structure of myoglobin(Fe II)-ethyl isocyanide has been solved at 1.68 A resolution by X-ray crystallography. The isocyano group of the ligand is distorted from the linear conformation observed in solution and in model compounds. Local changes in the protein conformation are also seen. The side-chain of Arg-CD3 moves out into the solvent, and the side-chain of His-E7 swings up and away from the ligand. Both of these side-chains show disorder indicative of dynamic behavior. These outward movements of His-E7 and Arg-CD3 side-chains clear a path from the solvent to the heme iron, suggesting a mechanism for ligand entry.

Coiled-coil packing in spermine-induced tropomyosin crystals. A comparative study of three forms.

Electron microscope images of highly ordered spermine-induced microcrystals of tropomyosin have been analyzed to determine the packing of the molecular filaments. Negatively stained microcrystals terminate in a distinctive "double fringe", which reveals the location of the molecular ends. This information, together with the symmetry of the structure in projection, shows that the microcrystals can be accounted for by a packing scheme of four layers of molecules in the unit cell. Knowing the position of the symmetry elements relating the layers then allows the three-dimensional space group of the microcrystals to be established as C222(1). Using cryo-electron microscopy and simulation studies, the run of the filaments and their packing in the C222(1) form have been shown to be related to those in the spermine-induced C2 crystal of tropomyosin whose structure has been solved to 9 A by X-ray crystallography. This result allows us to infer the location of the molecular ends in the C2 crystal as well, and this inference has been confirmed by analysis of thin sections of the C2 crystal. The C222(1) microcrystal has also been shown to be closely related to the classical divalent cation tropomyosin paracrystal. Based on knowledge of the molecular packing in the divalent cation paracrystal, the polarity of the molecules has been deduced in the other two crystal forms. The tropomyosin filament packing in all these forms may be accounted for by coiled-coil close packing and specific cationic bridging of negatively charged zones on the molecule. Taken together the results reveal a hierarchy of interactions in these close-packed crystalline forms, whose principles may apply to the packing in other fibrous proteins. This study also shows the usefulness of co-ordinating results from cryo-electron microscopy with negative staining in the structure analysis of such ordered arrays, and how these findings can complement the results of low resolution X-ray crystallographic studies.

High-resolution crystal structures of distal histidine mutants of sperm whale myoglobin.

The highly conserved distal histidine residue (His64) of sperm whale myoglobin modulates the affinity of ligands. In an effort to fully characterize the effects of mutating residue 64, we have determined the high-resolution crystal structures of the Gly64, Val64, Leu64, Thr64 and Gln64 mutants in several liganded forms. Metmyoglobins with hydrophobic substitutions at residue 64 (Val64 and Leu64) lack a water molecule at the sixth coordination position, while those with polar amino acid residues at this position (wild-type and Gln64) retain a covalently bound water molecule. In the Thr64 mutant, the bound water position is only partially occupied. In contrast, mutating the distal histidine residue to glycine does not cause loss of the coordinated water molecule, because the hydrogen bond from the imidazole side-chain is replaced by one from a well-ordered solvent water molecule. Differences in water structure around the distal pocket are apparent also in the structures of deoxymyoglobin mutants. The water molecule that is hydrogen-bonded to the N epsilon atom of histidine 64 in wild-type deoxymyoglobin is not found in any of the position 64 mutant structures that were determined. Comparison of the carbonmonoxy structures of wild-type, Gly64, Leu64 and Gln64 myoglobins in the P6 crystal form shows that the conformation of the Fe-C-O complex is nearly linear and is independent of the identity of the amino acid residue at position 64. However, the effect of CO binding on the conformation of residue 64 is striking. Superposition of deoxy and carbonmonoxy structures reveals significant displacements of the residue 64 side-chain in the wild-type and Gln64 myoglobins, but no displacement in the Leu64 mutant. These detailed structural studies provide key insights into the mechanisms of ligand binding and discrimination in myoglobin.

Structure of tropomyosin at 9 angstroms resolution.

We have used molecular replacement followed by a highly parameterized refinement to determine the structure of tropomyosin crystals to a resolution to 9 A. The shape, coiled-coil structure and interactions of the molecules in the crystals have been determined. These crystals have C2 symmetry with a = 259.7 A, b = 55.3 A, c = 135.6 A and beta = 97.2 degrees. Because of the unusual distribution of intensity in X-ray diffraction patterns from these crystals, it was possible to solve the rotation problem by inspection of qualitative aspects of the diffraction data and to define unequivocally the general alignment of the molecules along the (332) and (3-32) directions of the unit cell. The translation function was then solved by a direct search procedure, while electron microscopy of a related crystal form indicated the probable location of molecular ends in the asymmetric unit, as well as the anti-parallel arrangement. The structural model we have obtained is much clearer than that obtained previously with crystals of extraordinarily high solvent content and shows the two alpha-helices of the coiled coil over most of the length of the molecules and establishes the coiled-coil pitch at 140(+/- 10) A. Moreover, the precise value of the coiled-coil pitch varies along the molecule, probably in response to local variations in the amino acid sequence, which we have determined by sequencing the appropriate cDNA. The crystals are constructed from layers of tropomyosin filaments. There are two molecules in the crystallographic asymmetric unit and the molecules within a layer are bent into an approximately sinusoidal profile. Molecules in consecutive layers in the crystal lie at an angle relative to one another as found in crystalline arrays of actin and myosin rod. There are three classes of interactions between tropomyosin molecules in the spermine-induced crystals and these give some insights into the molecular interactions between coiled-coil molecules that may have implications for assemblies such as muscle thick filaments and intermediate filaments. In interactions within a layer, the geometry of coiled-coil contacts is retained, whereas in contacts between molecules in adjacent layers the coiled-coil geometry varies and these interactions instead appear to be dominated by the repeating pattern of charged zones along the molecule.

Structural and functional effects of apolar mutations of the distal valine in myoglobin.

High-resolution structures of the aquomet, deoxy, and CO forms of Ala68, Ile68, Leu68, and Phe68 sperm whale myoglobins have been determined by X-ray crystallography. These 12 new structures, plus those of wild-type myoglobin, have been used to interpret the effects of mutations at position 68 and the effects of cobalt substitution on the kinetics of O2, CO, and NO binding. Molecular dynamics simulations based on crystal structures have provided information about the time-dependent behavior of photolyzed ligands for comparison with picosecond geminate recombination studies. The Val68-->Ala mutation has little effect on the structure and function of myoglobin. In Ala68 deoxymyoglobin, as in the wild-type protein, a water molecule hydrogen-bonded to the N epsilon atom of the distal histidine restricts ligand binding and appears to be more important in regulating the function of myoglobin than direct steric interactions between the ligand and the C gamma atoms of the native valine side-chain. This distal pocket water molecule is displaced by the larger side-chains at position 68 in the crystal structures of Leu68 and Ile68 deoxymyoglobins. The Leu68 side-chain can rotate about its C alpha-C beta and C beta-C gamma bonds to better accommodate bound ligands, resulting in net increases in overall association rate constants and affinities due to the absence of the distal pocket water molecule. However, the flexibility of Leu68 makes simulation of picosecond NO recombination difficult since multiple starting conformations are possible. In the case of Ile68, rotation of the substituted side-chain is restricted due to branching at the beta carbon, and as a result, the delta methyl group is located close to the iron atom in both the deoxy and liganded structures. The favorable effect of displacing the distal pocket water molecule is offset by direct steric hindrance between the bound ligand and the terminal carbon atom of the isoleucine side-chain, resulting in net decreases in affinity for all three ligands and inhibition of geminate recombination which is reproduced in the molecular dynamics simulations. In Phe68 myoglobin, the benzyl side-chain is pointed away from the ligand binding site, occupying a region in the back of the distal pocket. As in wild-type and Ala68 myoglobins, a well-defined water molecule is found hydrogen bonded to the distal histidine in Phe68 deoxymyoglobin. This water molecule, in combination with the large size of the benzyl side-chain, markedly reduces the speed and extent of ligand movement into the distal pocket. (ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants.

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.

Structure and dynamics of the water around myoglobin.

The interplay between simulations at various levels of hydration and experimental observables has led to a picture of the role of solvent in thermodynamics and dynamics of protein systems. One of the most studied protein-solvent systems is myoglobin, which serves as a paradigm for the development of structure-function relationships in many biophysical studies. We review here some aspects of the solvation of myoglobin and the resulting implications. In particular, recent theoretical and simulation studies unify much of the diverse set of experimental results on water near proteins.

Protein structure determination using a database of interatomic distance probabilities.

The accelerated pace of genomic sequencing has increased the demand for structural models of gene products. Improved quantitative methods are needed to study the many systems (e.g., macromolecular assemblies) for which data are scarce. Here, we describe a new molecular dynamics method for protein structure determination and molecular modeling. An energy function, or database potential, is derived from distributions of interatomic distances obtained from a database of known structures. X-ray crystal structures are refined by molecular dynamics with the new energy function replacing the Van der Waals potential. Compared to standard methods, this method improved the atomic positions, interatomic distances, and side-chain dihedral angles of structures randomized to mimic the early stages of refinement. The greatest enhancement in side-chain placement was observed for groups that are characteristically buried. More accurate calculated model phases will follow from improved interatomic distances. Details usually seen only in high-resolution refinements were improved, as is shown by an R-factor analysis. The improvements were greatest when refinements were carried out using X-ray data truncated at 3.5 A. The database potential should therefore be a valuable tool for determining X-ray structures, especially when only low-resolution data are available.

Stabilization of myoglobin by multiple alanine substitutions in helical positions.

We have carried out a series of multiple Xaa-->Ala changes at nonadjacent surface positions in the sequence of sperm whale myoglobin. Although the corresponding single substitutions do not increase the thermal stability of the protein, multiple substitutions enhance the stability of the resulting myoglobins. The effect observed is an increase in the observed Tm (midpoint unfolding temperature) relative to that predicted from assuming additivity of the free energy changes corresponding to single mutations. The stabilization occurs in the presence of urea, as measured by the dependence of the unfolding temperature on urea concentration. The sites that have been altered occur in different helices and are not close in sequence or in the native structure of myoglobin. The observed effect is consistent with a role of multiple alanines in residual interactions in the unfolded state of the mutant proteins.

The structure of fibrinogen and fibrin: II. Architecture of the fibrin clot.

Our present low resolution model for fibrinogen based on electron microscopy and x-ray diffraction data has been described by Cohen et al. A unique aspect of the structural analysis of fibrous proteins is that the molecular packing in ordered arrays reflects biologically significant intermolecular interactions. We have shown that the orthogonal sheet microcrystals, which are closely related to fibrin, are made up of a highly regular arrangement of two-stranded protofibrils, and we have visualized aspects of both the substructure of the protofibrils as well as their packing to form the fibrin clot. By correlation of structural data with biochemical studies we have begun to identify certain functional regions of the fibrinogen model related to fibrin. Many aspects of fibrinogen's physiological activity remain to be related to its structure. As our present model is improved by higher resolution studies, we will see with increasing clarity molecular features critical for clot formation and fibrinolysis.