Myoglobin Crystals Research Articles

Infrared Crystallography: Structural Refinement through Spectroscopy

A method for determining the orientation of individual bonds within complex macromolecules from polarized IR measurements on oriented single crystals is described. At present, X-ray diffraction is the principal technique used to define the global structure and orientation of macromolecules in the crystalline state. However, resolution limitations and conformational disorder limit the accuracy of the resulting structural model. A quantitative understanding of protein function often requires a more precise description of structural features at a localized active site. Polarized IR measurements of internal stretching bands of N−3, CN−, OCN−, and SCN− bound at the ferric heme iron of single crystals of myoglobin are presented. A synthesis of such measurements on different crystal forms leads to an N−3 orientation consistent with IR measurements on both P21 and P212121 crystals, but significantly different from the orientation reported in X-ray crystal structures. Subtle structural changes between these two crystal forms of the CN− complex may account for small differences in the stretching frequency and orientation of the C–N bond.

Dielectric relaxation models applied of the dynamics of myoglobin as determined by Mössbauer spectroscopy

Abstract Protein specific modes of motions are found in myoglobin crystals above 180 K. In this contribution we show that this type of motions can be analyzed by a Davidson-Cole, a Cole-Cole or a Havriliak-Negami distribution in analogy to dielectric relaxation. However, the temperature dependence of the obtained parameters is quite unusual indicating a broadening of the distributions with temperature instead of motional narrowing. This can be understood from the picture of conformational substates if one assumes that more and more substates become accessible with increasing temperature. The result shows that the analogy between glass forming organic liquids and proteins should not be exaggerated.

Myoglobin solvent structure at different temperatures.

The structure of the solvent surrounding myoglobin crystals has been analyzed using neutron diffraction data, and the results indicate that the water around the protein is not disordered, but rather lies in well-defined hydration shells. We have analyzed the structure of the solvent surrounding the protein by collecting neutron diffraction data at four different temperatures, namely, 80, 130, 180, and 240K. Relative Wilson Statistics applied to low resolution data showed evidence of a phase transition in the region of 180K. A plot of the liquidity factor, Bsn, versus distance from the protein surface begins with a high plateau near the surface of the protein and drops to two minima at distances from the protein surface of about 2.35A and 3.85A. Two distinct hydration shells are observed. Both hydration shells are observed to expand as the temperature is increased.

Simultaneous Multiwavelength Anomalous Diffraction (SMAD): an Energy-Dispersive Synchrotron Diffraction Method for Macromolecules

A new synchrotron data acquisition method, simultaneous multiwavelength anomalous diffraction (SMAD), employing a variable bandwidth curved crystal monochromator (polychromator) coupled with an energy-selecting grid plate is described. The SMAD experiment demonstrates the ability to measure reflections at six different wavelengths and their Bijvoet pairs at the same time. On one diffraction image, both the dispersive and Bijvoet information is recorded from a myoglobin crystal for multiwavelength anomalous diffraction (MAD) phasing.

Thermal Behavior of Protein Crystals at Low Temperatures.

The low-temperature behavior of tetragonal lysozyme and monoclinic myoglobin crystals was investigated with an adiabatic microcalorimeter. Glass transitions were obseived in both crystals. From the water content dependence of the glass transition temperatures, it is concluded that the glass transitions are due to the immobilization of the cooperative motions among the protein molecules and the crystal water molecules.

Photolysis-induced structural changes in single crystals of carbonmonoxy myoglobin at 40 K

Myoglobin's reversible binding of oxygen is a model for studies of protein control of ligand binding and discrimination. Protein relaxation and geminate ligand rebinding subsequent to ligand photodissociation have been studied extensively by a variety of techniques. The ps to ns time scales for these processes are still much shorter than the ms time resolution of X-ray diffraction experiments, but it may be possible to trap these intermediates at low temperatures. We report here an X-ray diffraction investigation of structural changes induced by photolysis of carbonmonoxy myoglobin crystals at 40 K. Our results provide a structural basis for the interpretation of ambient and low temperature spectroscopic observations and molecular dynamics simulations of the ligand photodissociation and binding processes in haem proteins.

Elasticity of globular proteins. The relation between mechanics, thermodynamics and mobility.

An analysis of elasticity of lysozyme and myoglobin crystals in terms of thermodynamics has revealed a direct relation between entropy and enthalpy of deformation and delta S* and delta H* terms in the standard free energy change in proteins, delta G(o), (K.P. Murphy, P.L. Privalov, S.J. Gill (1990) Science 247, 559-561), so that at any temperature (between the glass-transition and denaturation temperatures) free energy of deformation is proportional to the hydration independent part of delta G(o). Both energies are characterized with large enthalpy-entropy compensation and tend to zero at the same temperature, Tm = (delta H*/delta S*) = 353 +/- 20 K. Large positive entropy contribution to deformation energy causes large linear decrease in protein elasticity, and increase in thermal mobility of protein atoms with temperature. Being plotted in inverse coordinates, temperature dependence of the mean-square amplitudes, obtained in neutron and mossbauer experiments as well as in molecular dynamic simulations, gives the same 353 +/- 10 K for the temperature, where the amplitudes tend to infinity. Mechanism explaining large positive entropy contribution in deformation energy of native protein molecules presumably involves emergence of more room for motion of protein side-chain groups squeezed between alpha-helices and other rigid skeleton elements, when precise packing of atoms in native protein molecule is distorted as a result of deformation.

Glass transition of myoglobin crystal

Heat capacities of monoclinic horse myoglobin crystals containing different amounts of water have been measured in the temperature range between 8 and 300 K. The samples containing 48.9 and 19.0 mass% of water underwent glass transitions at T g=188 and 216 K, respectively. The heat capacity increased from the rigid lattice value for T < T g to the supercooled liquid value for T > T g with concomitant spontaneous exothermic and endothermic enthalpy relaxation. The glass transition at 188 K shifted to 172 K when the sample was annealed at 230 K. The temperature drift rate due to the relaxation was analyzed to derive relaxation times as a function of the temperature and water content. The Arrhenius parameters of the relaxation time are discussed in relation to the molecular mechanism of the glass transition.

Conformational interconversion in protein crystals

We present evidence that the structure of carbonmonoxy myoglobin crystals can be altered by lowering the pH. This structural change is monitored by the characteristic FeCO Raman modes at 508 and 491 cm −1 and is thought to involve a localized distal pocket transition from a “closed” conformation at pH 7 to a more “open” conformation at pH 4. These changes take place in the crystal without loss of intensity of a conformationally sensitive Raman mode at 252 cm −1 that signals a partial unfolding of the globin structure in solution. Quantitative studies, which monitor the open and closed populations as a function of laser photolysis, demonstrate that the interconversion rates ( k ±) in solution at 298 K are fast compared to the photolysis and CO entry rates (i.e. k ± ⪢ 10 3 s −1), while in frozen samples the interconversion is much slower than the experimental time scale (minutes). Since the open conformation is a minority species at pH 7, rapid exchange in aqueous solution is a necessary condition for this species to play a functional role. In the crystal, the interconversion rates are slowed compared to solution and begin to approach the photolysis rate (i.e. k ± ~ 10 3 to 10 4 s −1). This indicates that the barriers for conformational exchange are increased in the crystal environment, compared to the solution, apparently due to the packing forces of the surrounding molecules. X-ray and neutron diffraction studies of MbCO crystals at high and low pH are needed to characterize the details of the structural changes and to test the hypothesis that closed and open distal pocket structures are associated with the 508 and 491 cm −1 FeCO modes.

Repulsive restraints for hydrogen bonding in least-squares refinement of protein crystals. A neutron diffraction study of myoglobin crystals

The purpose of this article is to describe stereochemical restraints on hydrogen bonding within proteins and their associated solvent which can be included in the refinement (PROLSQ) of X-ray or neutron structures of protein crystals. The parameters which define the geometry of hydrogen bonding, i.e. the correlation between distances and angles, are based on the results of an analysis of hydrogen bonding in crystal structures of myoglobin derivatives analyzed by neutron diffraction.

Glass-like behaviour of proteins as seen by Mössbauer spectroscopy

Mössbauer spectroscopy reveals a strong analogy in the dynamic properties of 57Fe in glycerol and in myoglobin crystals. Above about 200 K, the shape and the area of the spectra can be explained by a jump diffusion model with a jump time distribution according to Cole-Davidson. In the high-viscosity region well below 200 K, the myoglobin molecules have a similar but not an identical structure indicating a highly degenerated ground state energy of the molecules.

Hemoglobin dynamics in rat erythrocytes investigated by M�ssbauer spectroscopy

Rats have been enriched in 57Fe and erythrocytes were isolated from the blood. Mössbauer absorption spectroscopy on the hemoglobin of these erythrocytes has shown rather similar dynamics as found earlier in crystals of myoglobin, in frozen solutions of human hemoglobin and in a number of other proteins. The results strongly indicate that the motion of the heme and presumably some part of the F-helix is mainly influenced by the average viscosity of the sample determined by a network of hydrogen bridges and other weak interactions. Extrapolations of Mössbauer results from protein crystals to proteins in their physiological surroundings seem to be suitable for heme proteins.

X-ray crystal structure of a recombinant human myoglobin mutant at 2·8 Å resolution

We have grown crystals in trigonal space group P3(2)21 of a mutant human myoglobin, aquomet form, in which lysine at position 45 has been replaced by arginine and cysteine at position 110 has been replaced by alanine. Suitable crystals of native recombinant human myoglobin have not been obtained. We have used the molecular replacement method to determine the X-ray crystal structure of the mutant at 2.8 A resolution. At the present stage of refinement, the crystallographic R-value for the model, with tightly restrained stereochemistry, is 0.158 for 5.0 to 2.8 A data. As expected, the overall structure is quite similar to the sperm whale myoglobin structure. Arginine 45 adopts a well-ordered conformation similar to that found in aquomet sperm whale myoglobin.

The similarity in the dynamics of myoglobin and glycerol as seen from Mössbauer spectroscopy on 57Fe

Abstract Mossbauer spectra of 57 Fe dissolved in glycerol are compared with spectra of 57 Fe in myoglobin crystals. Above 200 K the strong increase of the 2 >-values obtained from the Lamb-Mossbauer factor is similar for both materials indicating similar dynamics with characteristics times faster 100 ns. Moreover, in this temperature region both spectra are best fitted by a narrow and a broad Lorentzian which indicates the presence of diffusive motions limited in space. “Protein specific motions” become visible in the Mossbauer spectrum above a glass transition. They are characteristics for a hydrogen-bridged network of molecules or molecular segments.

Charge motion in MbCO crystals after flash photolysis

Charge motion accompanying the dissociation and recombination of carbon monoxide to oriented myoglobin crystals has been observed. The magnitude of the electrical signals detected after photodissociation by electrodes on either side of MbCO crystals of type A is consistent with the x-ray data showing that the doubly charged iron ion lies in the mean heme plane when a ligand is bound and moves out of the plane when deligated. Beyond 10 ms, the time development of the electrical signal is consistent with the kinetics observed optically after flash photolysis on the same crystals.

Protein crystal dynamics studied by time-resolved analysis of X-ray diffuse scattering

IT has been known for many years that information about the dynamics of molecules in crystals can be obtained from diffusely scattered radiation1–3. Intermolecular motions coupled on a large scale generate diffuse spots centred on the Bragg peaks, whereas locally coupled intramolecular motions produce a diffuse background. Although information about protein dynamics has recently been derived from diffusely scattered radiation4–6, molecular motions could not be distinguished from static disorder in these experiments, because of a lack of time resolution. Provided the energy of the radiation is well defined, however, time resolution can be obtained from an energy analysis of the scattered radiation. We report here the use of Mossbauer radiation, the energy of which is characteristically well defined, to probe the molecular dynamics of a myoglobin crystal. Information about molecular motions is extracted from the ratio of elastically to inelastically scattered photons, which by using Mossbauer radiation can be precisely determined7. At room temperature we obtain a rpot-mean-squared amplitude for intermolecular motions of 0.13 A.

Angular dependent rayleigh scattering of Mössbauer radiation on proteins

RSMR experiments with57Fe radiation were performed on myoglobin. An areasensitive detector was employed for simultaneous angular dependent collection of the scattered quanta up to a maximum angle 2θ of 17‡. Experimental data of polycrystalline and lyophilized myoglobin are compared with computer calculations of the scattering which are based on the atomic coordinates determined by X-ray structure analysis. Special attention has been paid to the influence of coherence effects from collectively moving parts of the protein. A simple model is introduced in order to take into account these segmental motions. Our first results indicate that the sizes of collectively moving segments are comparable with spheres of about 6 a in diameter in dry myoglobin. In myoglobin crystals, where the molecules are surrounded by large hydration shells, the movements appear to be correlated in segments with sizes comparable to helices.

Energy distributions at the high-spin ferric sites in myoglobin crystals

The orientation and temperature dependence (4.2-2.5 K) of electron paramagnetic resonance (EPR) power saturation and spin-lattice relaxation rate, and the orientation dependence of signal linewidth, were measured in single crystals of the aquo complex of ferric sperm whale skeletal muscle myoglobin. The spin-packet linewidth was found to be temperature independent and to vary by a factor of seven within the heme plane. An analysis is presented which enables one to arrive at (a) hyperfine component line-widths and, from the in-plane angular variation of the latter, at (b) the widths of distributions in energy differences between low-lying electronic levels and (c) the angular spread in the in-plane principal g-directions. The values of the energy level distributions in crystals obtained from the measurements and analysis reported here are compared with those obtained by a different method for the same protein complex in frozen solution. The spread in the rhombic energy splitting is significantly greater in solution than in the crystal.

Solvent effect in protein crystals: A neutron diffraction analysis of solvent and ion density

In protein crystallography, it has been customary to omit the low-order data in refinement procedures. These data contain, however, important information about the gross features of the unit cell content and particularly the scattering density of the solvent, i.e. solvent structure. In order to use the low-order Bragg reflections, a solvent evaluation procedure has been developed that permits the description of the low-order structure factors ( F) as a combination of solvent and protein terms. This permits the use of all observed F values in a least-squares refinement, results in better refinement (lower R factor) and permits easier placement of water and ion locations. Coupled with the measurement of the crystal density by a density-gradient technique, the evaluation of the solvent scattering makes it possible to determine the amount of salt present in the solvent space. For myoglobin crystals grown from solutions containing close to 40% ( w w ) ammonium sulfate only 13% ( w w ) of salt is present in the solvent space. This is equivalent to seven (ionized) ammonium sulfate molecules, which is larger than the two to three sulfate ions observed in the crystal solvent space by X-ray diffraction.

Apomyoglobin as a molecular recognition surface: expression, reconstitution and crystallization of recombinant porcine myoglobin in Escherichia coli.

Recombinant porcine myoglobin has been produced in Escherichia coli using the lambda cII fusion expression system of Nagai and Thøgersen [Nature, 309, 810-812 (1984)]. After processing and reconstitution with haem, the protein is gel-electrophoretically and spectrophotometrically indistinguishable from native pig myoglobin. Large crystals of both native and recombinant porcine myoglobin were grown from 50 mM sodium phosphate, pH 7.1, 80% ammonium sulphate. The crystals belong to space group C2 (a = 156.9 A, b = 42.0 A, c = 92.2 A, beta = 127.9 degrees) and diffract to a nominal 2.5 A resolution. We plan to explore apomyoglobin as a binding surface in studies combining site-directed mutagenesis and X-ray analysis. These experiments will be extended by studying the binding of haem analogues to the mutant apoproteins.