George N. Phillips

Dynamics of proteins in crystals: comparison of experiment with simple models.

The dynamic behavior of proteins in crystals is examined by comparing theory and experiments. The Gaussian network model (GNM) and a simplified version of the crystallographic translation libration screw (TLS) model are used to calculate mean square fluctuations of C(alpha) atoms for a set of 113 proteins whose structures have been determined by x-ray crystallography. Correlation coefficients between the theoretical estimations and experiment are calculated and compared. The GNM method gives better correlation with experimental data than the rigid-body libration model and has the added benefit of being able to calculate correlations between the fluctuations of pairs of atoms. By incorporating the effect of neighboring molecules in the crystal the correlation is further improved.

Kinetic Pathways and Barriers for Ligand Binding to Myoglobin

Myoglobin is a small globular heme protein that increases the aerobic capacity of striated vertebrate muscle cells by taking up oxygen from blood during rest and delivering O2 to mitochondria during muscle contraction when blood flow through capillaries is restricted. The ferrous form of myoglobin can also react with CO and NO, which are produced in vivo as second messengers for regulating various physiological functions including blood pressure, platelet aggregation, and neurotransmission. Its tertiary structure consists of eight tightly packed helices, and the resulting “myoglobin fold” is very similar to that found for the a and b subunits of hemoglobin. Since Gibson’s minireview in 1989, a number of exciting new studies have led to detailed molecular mechanisms of myoglobin function. The rapid progress made in the past 7 years is primarily the result of ultrafast kinetic measurements, mutagenesis experiments, and theoretical molecular dynamics simulations. Significant contributions have been made individually by these approaches, but more progress has occurred when these endeavors have been combined (e.g. Gibson et al., 1992; Braunstein et al., 1993; Schlichting et al., 1994; Teng et al., 1994; Huang and Boxer, 1994; Petrich et al., 1994; Carlson et al., 1994; Quillin et al. 1995). This review focuses on NO, O2, and CO binding to myoglobin mutants under physiological conditions. The objectives are to summarize successes in correlating theoretical, structural, and kinetic results and to identify the major remaining questions in ligand binding dynamics.

Myoglobin discriminates between O2, NO, and CO by electrostatic interactions with the bound ligand

Abstract Most biological substrates have distinctive sizes, shapes, and charge distributions which can be recognized specifically by proteins. In contrast, myoglobin must discriminate between the diatomic gases O2, CO, and NO which are apolar and virtually the same size. Selectivity occurs at the level of the covalent Fe-ligand complexes, which exhibit markedly different bond strengths and electrostatic properties. By pulling a water molecule into the distal pocket, His64(E7)1 inhibits the binding of all three ligands by a factor of ∼10 compared to that observed for protoheme-imidazole complexes in organic solvents. In the case of O2 binding, this unfavorable effect is overcome by the formation of a strong hydrogen bond between His64(E7) and the highly polar FeO2 complex. This favorable electrostatic interaction stabilizes the bound O2 by a factor of ∼1000, and the net result is a 100-fold increase in overall affinity compared to model hemes or mutants with an apolar residue at position 64. Electrostatic interaction between FeCO and His64 is very weak, resulting in only a two- to three-fold stabilization of the bound state. In this case, the inhibitory effect of distal pocket water dominates, and a net fivefold reduction in KCO is observed for the wild-type protein compared to mutants with an apolar residue at position 64. Bound NO is stabilized ∼tenfold by hydrogen bonding to His64. This favorable interaction with FeNO exactly compensates for the tenfold inhibition due to the presence of distal pocket water, and the net result is little change in KNO when the distal histidine is replaced with apolar residues. Thus, it is the polarity of His64 which allows discrimination between the diatomic gases. Direct steric hindrance by this residue plays a minor role as judged by: (1) the independence of KO2, KCO, and KNO on the size of apolar residues inserted at position 64, and (2) the observation of small decreases, not increases, in CO affinity when the mobility of the His64 side chain is increased. Val68(E11) does appear to hinder selectively the binding of CO. However, the extent is no more than a factor of 2–5, and much smaller than electrostatic stabilization of bound O2 by the distal histidine.

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.

Structures and Analysis of Highly Homologous Psychrophilic, Mesophilic, and Thermophilic Adenylate Kinases*

The crystal structures of adenylate kinases from the psychrophile Bacillus globisporus and the mesophile Bacillus subtilis have been solved and compared with that from the thermophile Bacillus stearothermophilus. This is the first example we know of where a trio of protein structures has been solved that have the same number of amino acids and a high level of identity (66–74%) and yet come from organisms with different operating temperatures. The enzymes were characterized for their own thermal denaturation and inactivation, and they exhibited the same temperature preferences as their source organisms. The structures of the three highly homologous, dynamic proteins with different temperature-activity profiles provide an opportunity to explore a molecular mechanism of cold and heat adaptation. Their analysis suggests that the maintenance of the balance between stability and flexibility is crucial for proteins to function at their environmental temperatures, and it is achieved by the modification of intramolecular interactions in the process of temperature adaptation.

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.

Crystal structure of tropomyosin at 7 Angstroms resolution.

Tropomyosin is a 400Å‐long coiled coil that polymerizes to form a continuous filament that associates with actin in muscle and numerous non‐muscle cells. Tropomyosin and troponin together form a calcium‐sensitive switch that is responsible for thin‐filament regulation of striated muscle. Subtle structural features of the molecule, including non‐canonical aspects of its coiled‐coil motif, undoubtedly influence its association with f‐actin and its role in thin filament regulation. Previously, careful inspection of native diffraction intensities was sufficient to construct a model of tropomyosin at 9Å resolution in a spermine‐induced crystal form that diffracts anisotropically to 4Å resolution. Single isomorphous replacement (SIR) phasing has now provided an empirical determination of the structure at 7Å resolution. A novel method of heavy‐atom analysis was used to overcome difficulties in interpretation of extremely anisotropic diffraction. The packing arrangement of the molecules in the crystal, and important aspects of the tropomyosin geometry such as non‐uniformities of the pitch and variable bending and radius of the coiled coil are evident. Proteins 2000;38:49–59. ©2000 Wiley‐Liss, Inc.

Probing substates in sperm whale myoglobin using high-pressure crystallography.

Pressures in the 100 MPa range are known to have an enormous number of effects on the action of proteins, but straightforward means for determining the structural basis of these effects have been lacking. Here, crystallography has been used to probe effects of pressure on sperm whale myoglobin structure. A comparison of pressure effects with those seen at low pH suggests that structural changes under pressure are interpretable as a shift in the populations of conformational substates. Furthermore, a novel high-pressure protein crystal-cooling method has been used to show low-temperature metastability, providing an alternative to room temperature, beryllium pressure cell-based techniques. The change in protein structure due to pressure is not purely compressive and involves conformational changes important to protein activity. Correlation with low-pH structures suggests observed structural changes are associated with global conformational substates. Methods developed here open up a direct avenue for exploration of the effects of pressure on proteins.

Optimization and evaluation of a coarse-grained model of protein motion using x-ray crystal data.

Simple coarse-grained models, such as the Gaussian network model, have been shown to capture some of the features of equilibrium protein dynamics. We extend this model by using atomic contacts to define residue interactions and introducing more than one interaction parameter between residues. We use B-factors from 98 ultra-high resolution (<or=1.0 A) x-ray crystal structures to optimize the interaction parameters. The average correlation between Gaussian network-model fluctuation predictions and the B-factors is 0.64 for the data set, consistent with a previous large-scale study. By separating residue interactions into covalent and noncovalent, we achieve an average correlation of 0.74, and addition of ligands and cofactors further improves the correlation to 0.75. However, further separating the noncovalent interactions into nonpolar, polar, and mixed yields no significant improvement. The addition of simple chemical information results in better prediction quality without increasing the size of the coarse-grained model.

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.