Myoglobin In Solution Research Articles

Expression of concern: Ligand discrimination of myoglobin in solution: an iron L-edge X-ray absorption study of the active centre.

Expression of concern for 'Ligand discrimination of myoglobin in solution: an iron L-edge X-ray absorption study of the active centre' by Kathrin M. Lange et al., Chem. Commun., 2013, 49, 4163-4165.

NanoArmoring of Enzymes by Polymer-Functionalized Iron Oxide Nanoparticles.

Polymer-armored enzymes loaded onto magnetic nanoparticles, as efficient nanobioreactors with enhanced properties, are described in this chapter. Polymers are useful macromolecules carrying a large number of surface charges and repeating units of desired chemical functional groups for linking enzymes onto them. Magnetic micro/nanoparticles have been widely used as enzyme carriers with the incorporation of suitable polymer layers. Synthesized iron oxide magnetic nanoparticles have been used to immobilize a peroxide-catalyzing enzyme-like heme protein: myoglobin using covalent and noncovalent strategies. The stability, scalability, and kinetics of the conjugate were studied in detail using spectroscopic and electrochemical analysis. Compared to the free myoglobin in solution, myoglobin conjugated to magnetic nanoparticles demonstrated high catalytic stability and easy recovery from the reaction medium for further use. Due to the large surface area offered by the magnetic nanoparticles, a large amount of myoglobin could be loaded with a small amount of magnetic nanoparticles. Selected examples of polymer-enzyme and polymer-magnetic nanoparticle-enzyme conjugates developed by us and others are presented in this chapter, and representative methods for making cost-effective scalable and reusable enzymatic reactors have been described.

Ligand discrimination of myoglobin in solution: an iron L-edge X-ray absorption study of the active centre

Iron L-edge X-ray absorption spectra of the active centre of myoglobin in the met-form, in the reduced form and upon ligation to O2, CO, NO and CN are presented. The strength of ligation with the iron centre is finger-printed through the variation of the L3 : L2 intensity ratio. Charge Transfer Multiplet calculations are performed and give qualitative information about oxidation states as well as charge transfer.

Revisiting the nitrosyl complex of myoglobin by high-field pulse EPR spectroscopy and quantum mechanical calculations

The binding of NO to reduced myoglobin in solution results in the formation of two paramagnetic nitrosyl myoglobin (MbNO) complexes: one with a rhombic g-factor and the other with an axial one, referred to as the R- and A-forms. In spite of past extensive studies of MbNO by crystallography, spectroscopy and quantum chemical calculations it is still not clear what factors determine the appearance of the two forms. In this work we applied a combination of state of the art quantum chemical calculations and high field pulsed EPR spectroscopy (W-band, 3.4 T/95 GHz) to further characterize the two forms. Specifically, we have used (1)H and (2)H electron-nuclear double resonance (ENDOR) spectroscopy to identify and characterize the H-bond to the NO, and hyperfine sub-level correlation (HYSCORE) spectroscopy to determine the hyperfine and quadrupole interactions of the Fe(ii) coordinated (14)N of the proximal histidine (14)N(His93). The calculations employed quantum mechanics (QM), particularly density functional theory (DFT) methods in combination with molecular mechanics (MM) force-field to model the protein environment. Through QM/MM calculations of the EPR parameters we have explored their dependence on several geometrical factors of the Fe-NO bond and found those that reproduce the best experimental results. The spread of the W-band EPR spectrum of MbNO due to the g-anisotropy is large and there is a significant part of the spectrum where the R-form is the sole contributor. This allowed us to resolve some new characteristics of the R-form: (i) a NO-H hydrogen bond has been detected and characterized and through QM/MM calculations has been unambiguously assigned to (epsilon2)H(His64). (ii) The complete hyperfine and quadrupole interactions of (14)N(His93) have been determined and correlated with structural parameters again using QM/MM calculations. The agreement between the experimental results and calculations varied between excellent and good, depending on the EPR parameter in question. As for the more elusive A-form, the results only suggest that it does have a (14)N(His93) ligand with a hyperfine comparable to that of the R-form and it has less hydrogen bonding interaction with His(64). The calculations also established the orientation of the principal g-values, finding that they are closely related to the orientation of the NO bond. This information is essential for deriving structural information from the experimental orientation selective HYSCORE and ENDOR data.

Kinetics of Carbon Monoxide Migration and Binding in Solvated Myoglobin as Revealed by Molecular Dynamics Simulations and Quantum Mechanical Calculations

By using multiple (independent) molecular dynamics (MD) trajectories (about 500 ns in total) of photolized carbon monoxide (CO) within solvated myoglobin, a quantitative description of CO migration and corresponding kinetics is obtained. MD results combined with previously reported quantum mechanical calculations on the CO-heme binding-unbinding reaction step in myoglobin allowed construction of a detailed quantitative model, shedding light on the kinetic mechanism and relevant steps of CO migration and geminate binding. Finally, the obtained (unbiased) theoretical-computational model is critically compared with the available computational and experimental data for myoglobin in solution.

Oxygen equilibrium properties of myoglobin locked in the liganded and unliganded conformations.

A comparison of the O(2) equilibrium curves of sperm-whale myoglobin locked in the liganded (CO-bound) and unliganded (deoxy) conformations by encapsulation in a wet porous sol-gel silica reveals a marked difference between them. The CO-bound state-locked myoglobin showed a nearly monophasic (hyperbolic) O(2) equilibrium curve with a dissociation constant of 0.2 Torr, which is smaller than that of myoglobin in solution (0.5 Torr). On the other hand, the deoxy state-locked myoglobin exhibited a multiphasic O(2) equilibrium curve that can be represented by a sum of three independent components with dissociation constants of 0.19, 0.90, and 44 Torr, respectively, indicating that deoxymyoglobin exists in multiple conformations. These results show that myoglobin can be frozen into ligand-dependent conformational populations at room temperature in the wet sol-gel and suggest that the overall O(2) equilibrium properties of myoglobin in solution are generated by a redistribution of protein conformational populations in response to ligand binding.

Evidence of nonspecific surface interactions between laser-polarized xenon and myoglobin in solution.

The high sensitivity of the magnetic resonance properties of xenon to its local chemical environment and the large (129)Xe NMR signals attainable through optical pumping have motivated the use of xenon as a probe of macromolecular structure and dynamics. In the present work, we report evidence for nonspecific interactions between xenon and the exterior of myoglobin in aqueous solution, in addition to a previously reported internal binding interaction. (129)Xe chemical shift measurements in denatured myoglobin solutions and under native conditions with varying xenon concentrations confirm the presence of nonspecific interactions. Titration data are modeled quantitatively with treatment of the nonspecific interactions as weak binding sites. Using laser-polarized xenon to measure (129)Xe spin-lattice relaxation times (T(1)), we observed a shorter T(1) in the presence of 1 mM denatured apomyoglobin in 6 M deuterated urea (T(1) = 59 +/- 1 s) compared with that in 6 M deuterated urea alone (T(1) = 291 +/- 2 s), suggesting that nonspecific xenon-protein interactions can enhance (129)Xe relaxation. An even shorter T(1) was measured in 1 mM apomyoglobin in D(2)O (T(1) = 15 +/- 0.3 s), compared with that in D(2)O alone (T(1) = 506 +/- 5 s). This difference in relaxation efficiency likely results from couplings between laser-polarized xenon and protons in the binding cavity of apomyoglobin that may permit the transfer of polarization between these nuclei via the nuclear Overhauser effect.

Continuous flow infrared matrix-assisted laser desorption/ionization with a solvent matrix

Continuous flow matrix-assisted laser desorption/ionization (MALDI) was demonstrated with infrared laser desorption and an ethanol matrix. A capillary was used to deliver an analyte solution dissolved in ethanol to a metal frit embedded in a sample stage. Typical flow rates were 1.7&mgr;L/min. An optical parametric oscillator tuned to 2.8&mgr;m was used for desorption and ionization, and mass analysis was achieved with a 1 m linear time-of-flight mass spectrometer. Flow injection studies were performed with low picomolar quantities of insulin and myoglobin in solutions containing 0.1 to 1.0% glycerol in ethanol. Copyright 2000 John Wiley & Sons, Ltd.

Reduction of protein volume and compressibility by macromolecular cosolvents: dependence on the cosolvent molecular weight

The partial specific volume ( V) and adiabatic compressibility ( β) of myoglobin have been shown to be reduced by small cosolvents such as glycerol (A. Priev, A. Almagor, S. Yedgar, B. Gavish, Biochemistry 35 (1996) 2061–2066). To elucidate the effect of the cosolvent size on these protein properties, in the present study we determined V and β of myoglobin in solutions containing a homologous cosolvent series from sucrose to dextran - 500 (M.W. 500 000). It was found that in addition to the expected effect of the cosolvent concentration, V and β decrease with increasing cosolvent M.W. This suggests that structural properties of the cosolvent contribute to its effect on the protein interior.

Temperature and solvent viscosity dependence of conformational dynamics of Zn-substituted myoglobin observed by nanosecond time-resolved transient hole-burning spectroscopy

Conformational fluctuations of a protein myoglobin in solution have been investigated by a time-resolved transient hole-burning experiment with a temporal resolution of ~ 10 ns over the temperature range of 180–300 K. The temperature dependence of the observed relaxation time τ r has been well fitted by the Vogel-Fulcher law. The solvent viscosity dependence of τ r has been also studied around room temperature.

Binding of CO to myoglobin from a heme pocket docking site to form nearly linear Fe-C-O.

The relative orientations of carbon monoxide (CO) bound to and photodissociated from myoglobin in solution have been determined with time-resolved infrared polarization spectroscopy. The bound CO is oriented < or = 7 degrees from the heme normal, corresponding to nearly linear FE-C-O. Upon dissociation from the Fe, CO becomes trapped in a docking site that orientationally constrains it to lie approximately in the plane of the heme. Because the bound and "docked" CO are oriented in nearly orthogonal directions CO binding from the docking site is suppressed. These solutions results help to establish how myoglobin discriminates against CO, a controversial issue dominated by the misconception that Fe-C-O is bent.

Structural heterogeneity in proteins at cryogenic temperatures. Cooling rate dependence

The influence of the cooling rate on the structural heterogeneity of sperm whale myoglobin in solution at cryogenic temperatures was studied. Samples were either cooled slowly (0.03 K/s) or rapidly by immersing in liquid propane at 77 K, which yielded a cooling rate of more than 100 K/s. FTIR spectra of the stretch bands of the heme-bound CO showed that the population of the A substates depends on the cooling rate. The structural heterogeneity within each A substate was assessed by temperature-derivative spectroscopy (TDS), which yields the distribution of enthalpy barriers for CO rebinding after photodissociation. No significant changes of the barrier distribution were found. It is concluded that, within the range explored, the cooling rate plays a negligible role in the structural heterogeneity of protein solutions at cryogenic temperatures

Relaxation dynamics of myoglobin in solution.

The geminate rebinding kinetics of MbCO in solution at high temperature (260--300 K) shows nonexponential behavior (stretched, \ensuremath{\beta}\ensuremath{\sim}1/2) that may be related to nonequilibrium relaxation of the distal pocket. A two-pulse photolysis experiment, which probes a kinetically selected subpopulation, demonstrates that the nonexponential behavior arises from a fluctuationally averaged system and also reveals slow interconversion times for large-scale protein motions. Measurements as a function of temperature lead to a direct determination of the Arrhenius barrier at the heme (18\ifmmode\pm\else\textpm\fi{}2 kJ/mole).

On the dielectric spectra of proteins in conducting media

The effect of conductivity on the dielectric measurements of proteins is studied. For that purpose the dielectric spectra (0.03–13 MHz) of serum albumin and myoglobin in solutions of varying conductivities were recorded. The results presented confirm that Maxwell's prediction of a threshold frequency in conducting materials also holds for protein solutions. The threshold frequency of a serum albumin solution is experimentally determined and the ionic screening of the electric field when performing dielectric spectra of these samples discussed. Three distinct frequency regions must be considered: a low frequency region where the sample behaves like a conductor; an intermediate region centered around the threshold frequency where the free charges partially screen the fixed charges; and a high frequency region where the sample behaves like a good dielectric. Dielectric measurements in the low frequency region defined above, are not possible.

Adsorption of the protein antigen myoglobin affects the binding of conformation-specific monoclonal antibodies

Five monoclonal antibodies against sperm whale myoglobin have been used to investigate the physical state of the antigen adsorbed onto a polydimethylsiloxane surface. The binding of each antibody is sensitive to the antigen's conformation in solution while the locations of the antigenic sites on the myoglobin molecule for three of the antibodies have been determined (Berzofsky, J.A., G.K. Buckenmeyer, G. Hicks, F.R.N. Gurd, R.J. Feldmann, and J. Minna. 1982. J. Biol. Chem. 257:3189-3198). The binding of the fluorescein isothiocyanate-labeled IgG and Fab antibodies to previously adsorbed myoglobin has been observed using total internal reflection fluorescence. Three of the antibodies bind specifically to surface-adsorbed myoglobin with affinities at least 50% relative to myoglobin in solution whereas two of the antibodies show affinities for the surface-adsorbed myoglobin diminished by at least two orders of magnitude relative to myoglobin in solution. The specific loss of certain antigenic determinants on the adsorbed myoglobin, coupled with the retention of others, indicates a nonrandom adsorption of the myoglobin molecules.

Heme orientational disorder in reconstituted and native sperm whale myoglobin: Proton nuclear magnetic resonance characterizations by heme methyl deuterium labeling in the met-cyano protein

The solution proton nuclear magnetic resonance spectrum of the Met-cyano form of sperm whale myoglobin reveals the presence of two sets of comparably intense resonances immediately after reacting the apoprotein with hemin, only one of which corresponds to that of the accepted native protein. Isotope labeling of individual methyl groups of hemin reveals that the methyl assignments differ characteristically in that similar resonance positions for the two components arise from the methyl groups related by a 180 degrees rotation about the alpha-gamma-meso axis. This phenomenon, observed earlier only for myoglobin with modified hemin, dictates that the second protein component in solution immediately after reconstitution must have the heme rotated by 180 degrees about the alpha-gamma-meso axis as compared to that found in the single crystal. The two components in the reconstituted protein equilibrate to yield the spectrum of the native Met-cyanomyoglobin for which there still exists approximately 8% of the minor component. Thus native myoglobin in solution is structurally heterogeneous in the heme pocket. Proton nuclear magnetic resonance spectra of deoxymyoglobin produced from both native and freshly reconstituted protein shown that the heterogeneity is also a property of the physiologically relevant reduced protein forms. It is suggested that, contrary to available X-ray data, heme orientational heterogeneity may be the rule rather than the exception in b-type hemoproteins, and that such disorder must be carefully considered in detailed correlations between structure and function even in native hemoproteins.

The reaction of carbon monoxide with myoglobin in solution, in an amorphous state, and in crystals.

The stopped flow and flash photolysis methods were applied for the kinetic study of the reaction of carbon monoxide with myoglobin in solution, in an amorphous state, and in crystals. From the flash photolysis data, the reactivity of myoglobin was concluded to be essentially the same in all three states. When the reaction was started with a stopped flow apparatus, the rate became slower as the state of myoglobin was changed from solution to amorphous precipitate, and to crystals. The bigger the crystals, the slower the reaction became. Therefore, the change of the rate could be explained in terms of a diffusion layer formed on the crystals. In the reaction of crystalline myoglobin, the effective concentration of CO was increased locally by about 20 microM after flash photolysis and approached the bulk concentration during the reaction. In contrast, in the reaction of amorphous precipitate of myoglobin, such an increase in the CO concentration was apparently dispersed homogeneously just after flash photolysis.

EXAFS study of myoglobin in solution and in crystalline form

EXAFS study of myoglobin in solution and in crystalline form

Electron paramagnetic resonance of Cu2+:Mb single crystal. Conformational changes

Copper introduced into met-myoglobin crystals occupies various sites as indicated by electron paramagnetic resonance parameters. Cu2+ (A) is probably liganded to histidine A10, lysine A14, and asparagine GH4 (Banaszak et al., 1965) and shows superhyperfine interaction with a single (imidazole) nitrogen. Cu2+ (B) and Cu2+ (C) correspond to other anisotropic sites described in less detail. Cu2+ (A) exhibits a transition to an isotropic form with a transition temperature of 40.5 degrees C. This transition indicates a conformational change in myoglobin and could correspond to a motion of A helix away from the GH section. The transition temperature is 7 degrees C higher than the one previously reported (Atanasov, 1971) for myoglobin in solution.

Comparison of the three basic scattering functions of myoglobin in solution with those from the known structure in crystalline state.

Abstract X-ray small-angle scattering of myoglobin in aqueous solutions enhances the probability that the structure of myoglobin is conserved on passing to the dissolved state. The agreement of measured and calculated results could be extended up to k = 1.2 A −1 which roughly corresponds to 5 A resolution.