Biomedical subjects
Publications and source records attributed to M Karavitis.
Quantum coherent vibrational relaxation of an impurity strongly coupled to its solid host is demonstrated through four-wave mixing measurements to infer sustained coherence in the bath, which is recognized as a superposition of macroscopically distinct states.
Pump-probe, four-wave, and six-wave mixing measurements of I2 isolated in solid argon are used to provide a clear experimental measure for the onset of vibrational quantum decoherence on the excited electronic state. The electronically resonant, six-wave mixing measurements bypass the rapid electronic dephasing, and measure the quantum cross-correlation between two packets launched on the B-state. The vibrational quantum coherence survives one period of motion, 400 fs, during which approximately 2000 cm(-1) of energy is transferred to the lattice. The decoherence occurs during the second cycle of motion, while classically coherent motion measured via pump-probe spectroscopy using the same electronic resonances continues for approximately 15 periods. This is contrasted with vibrational dephasing on the ground electronic surface, which lasts for 10(2) periods, as measured through time-resolved coherent anti-Stokes Raman scattering. The measurements and observables are discussed through time-circuit diagrams, and a mechanistic description of decoherence is derived through semiclassical analysis and simulations that reproduce the experiments.
Pump-probe measurements of I2 in solid Ar are reported and analyzed to extract a description of cage response to impulsive excitation, from the gentle kick, up to the breaking point. The most informative data are obtained through wavepacket motion on cage-bound, but otherwise dissociative, potentials where the chromophore acts as a transducer to drive the cage and to report on the local dynamics. This general class of dynamics is identified and analyzed as a function of energy in Ar, Kr, and Xe. The overdriven cage rebounds with a characteristic period of 1.2 ps that shows little dependence on excitation amplitude in all hosts. After rebound, the cage rings as a local resonant mode in Ar, with a period of 1 ps and dephasing time of 3 ps. This mode remains at the Debye edge in Kr and Xe, with periods of 630 and 800 fs, and dephasing times of 8 and 6 ps, respectively. In the bound B-state, the cage fluctuates toward its dilated equilibrium structure on a time scale of 3 ps, which is extracted from the down-chirp in the molecular vibrational frequency. When kicked with excess energy of 4 eV, the Ar cage breaks with 50% probability, and the molecule dissociates. The kinetics of polarization selective, multiphoton dissociation with Gaussian laser intensity profiles is delineated and the ballistics of cage breakout is described: The photodissociation proceeds by destruction of the local lattice, by creating interstitials and vacancies. During large amplitude motion on cage-bound potentials, sudden, nonadiabatic spin-flip transitions can be observed and quantified in space and time. The spin-flip occurs with unit probability in Ar when the I*-I bond is stretched beyond 6 A.
Time-resolved coherent anti-Stokes Raman scattering, with a resolution of 20 fs, is used to prepare a broadband vibrational superposition on the ground electronic state of I2 isolated in solid Kr. The coherent evolution of a packet consisting of nu=1-6 is monitored for as many as 1000 periods, allowing a precise analysis of the material response and radiation coherence. The molecular vibrations are characterized by omega(e)=211.330(2) cm(-1), omega(e)x(e)=0.6523(6) cm(-1), omega(e)y(e)=2.9(1) x 10(-3) cm(-1); the dephasing rates at 32 K range from 110 ps for nu=1 to 34 ps for nu=6, with nu dependence: gamma(nu)=8.5 x 10(-3)+4.9 x 10(-4)nu2+2.1 x 10(-6)nu4 ps(-1). The signal amplitude is also modulated at omega(q)=41.56(3) cm(-1); which can be interpreted as coupling between the molecule and a local mode. The surprising implication is that this resonant local mode is decoupled from the lattice phonons, a finding that cannot be rationalized based on a normal-mode analysis.
Three variants of tetrameric human hemoglobin, with changes at the alpha1beta2/alpha2beta1-interface, at the alpha1beta1/alpha2beta2-interface, and at both interfaces, have been constructed. At alpha1beta2/alpha2beta1-interface the beta93 cysteine was replaced by alanine (betaC93A), and at the alpha1beta1/alpha2beta2-interface the beta112 cysteine was replaced by glycine (betaC112G). The alpha1beta2 interface variant, betaC93A, and the alpha1beta1/alpha1beta2 double mutant, beta(C93A+C112G), were crystallized in the T-state, and the structures determined at 2. 0 and 1.8 A resolution, respectively. A comparison of the structures with that of natural hemoglobin A shows the absence of detectable changes in the tertiary folding of the protein or in the T-state quaternary assembly. At the beta112 site, the void left by the removal of the cysteine side chain is filled by a water molecule, and the functional characteristics of betaC112G are essentially those of human hemoglobin A. At the beta93 site, water molecules do not replace the cysteine side chain, and the alanine substitution increases the conformational freedom of beta146His, weakening the important interaction of this residue with beta94Asp. As a result, when Cl- is present in the solution, at a concentration 100 mM, the Bohr effect of the two mutants carrying the beta93Cys-->Ala substitution, betaC93A and beta(C93A+C112G), is significantly modified being practically absent below pH 7.4. Based on the crystallographic data, we attribute these effects to the competition between beta94Asp and Cl- in the salt link with beta146His in T-state hemoglobin. These results point to an interplay between the betaHis146-betaAsp94 salt bridge and the Cl- in solution regulated by the Cys present at position beta93, indicating yet another role of beta93 Cys in the regulation of hemoglobin function.
The spectroscopic, conformational, and functional properties of mutant carbonmonoxy hemoglobins in which either the beta-globin Val67(E11) or the alpha-globin Val62(E11) is replaced by threonine have been investigated. The thermal evolution of the Soret absorption band and the stretching frequency of the bound CO were used to probe the stereodynamic properties of the heme pocket. The functional properties were investigated by kinetic measurements. The spectroscopic and functional data were related to the conformational properties through molecular analysis. The effects of this nonpolar-to-polar isosteric mutation are: (i) increase of heme pocket anharmonic motions, (ii) stabilization of the A0 conformer in the IR spectrum, (iii) increased CO dissociation rates. The spectroscopic data indicate that for the carbonmonoxy derivatives, the Val --> Thr mutation has a larger conformational effect on the beta-subunits than on the alpha-subunits. This is at variance with the deoxy derivatives where the conformational modification was larger in the heme pocket of the alpha-subunit (Cupane, A., Leone, M., Militello, V., Friedman, R. K., Koley, A. P., Vasquez, G. P., Brinigar, W. S., Karavitis, M., and Fronticelli, C. (1997) J. Biol. Chem. 272, 26271-26278). These effects are attributed to a different electrostatic interaction between Ogamma of Thr(E11) and the bound CO molecule. Molecular analysis indicates a more favorable interaction of the bound CO with Thr Ogamma in the beta-subunit heme pocket.
In the crystal structure of human T-state hemoglobin with a sebacyl residue cross-linking the two beta-subunit Lys82,s (DecHb), the Fe atoms of the alpha-subunit hemes are found to be oxidized with a water molecule bound. The three-dimensional structure and heme geometries were compared to those of deoxyhemoglobin and other partially and fully oxidized hemoglobins [R. Liddington, Z. Derewenda, E. Dodson, R. Hubbard, G. Dodson, J. Mol. Biol. 228 (1992) 551]. The heme geometries of the alpha-subunits are consistent with those observed in oxidized structures. The proximal histidines of the alpha-subunits move toward the heme plane shifting the F-helix and FG-corner in a manner observed for partially oxidized human hemoglobin. This supports the hypothesis that these perturbations may precede the T- to R-state transition. Circular dichroism studies comparing DecHb and natural human hemoglobin in the deoxy and CO ligated forms confirm that the conformations of the deoxy forms are identical, but the ligated forms have slight differences in the solution structures. DecHb is found to be more resistant to autoxidation than natural hemoglobin. The time course of autoxidation of DecHb shows that it is virtually absent for the first 1500 min followed by a rapid increase. Thus, the discovery of the oxidation of the alpha-subunits in the deoxy-crystals is quite unexpected. The data confirm that ligation of the alpha-subunits precedes that of the beta-subunits. This may suggest a low ligand affinity of the alpha-diligated form of hemoglobin.
The dynamic and functional properties of mutant deoxyhemoglobins in which either the beta-globin Val67(E11) or the alpha-globin Val62(E11) is replaced by threonine have been investigated through the thermal evolution of the Soret absorption band in the temperature range 300 to 20 K and through the kinetics of CO rebinding after flash photolysis at room temperature. The conformational properties of the modified alpha chain and beta chain distal heme pockets were also studied through x-ray crystallography and molecular modeling. The data obtained with the various techniques consistently indicate that the polar isosteric mutation in the distal side of the alpha chain heme pocket has a larger effect on the investigated properties than the analogous mutation on the beta chain. We attribute the observed differences to the presence of a water molecule in the distal heme pocket of the modified alpha chains, interacting with the hydroxyl of the threonine side chain. This is indicated by molecular modeling which showed that the water molecule present in the alpha chain distal heme pocket can bridge by H bonding between Thr62(E11) and His58(E7) without introducing any unfavorable steric interactions. Consistent with the dynamic and functional data, the presence of a water molecule in the distal heme pocket of the modified beta chains is not observed by x-ray crystallography.
The alpha-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native beta-chains. The functional and structural properties of the expressed alpha-chains were assessed in the isolated state and after assembly into a functional hemoglobin tetramer. The recombinant and native hemoglobins were essentially identical on the basis of sensitivity to effectors (Cl- and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the alpha1beta1 and alpha1beta2 interfaces. However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed alpha-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relative to native HbA. These results indicate that subtle conformational changes in the heme pocket can alter hemoglobin cooperativity in the absence of modifications of quaternary interface contacts or protein dynamics. In addition to incorporation into a HbA tetramer, the alpha-globin refolds and incorporates heme in the absence of the partner beta-chain. Although the CO binding kinetics of recombinant alpha-chains were the same as that of native alpha-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics revealed an additional faster component. These results show that recombinant alpha-chain assumes alternating conformations in the absence of beta-chain and indicate that the isolated alpha-chain exhibits a higher degree of conformational flexibility than the alpha-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility of the expressed alpha-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships.
The crystal structure of the mutant deoxyhemoglobin in which the beta-globin Val67(E11) has been replaced with threonine [Fronticelli et al. (1993) Biochemistry 32, 1235-1242] has been determined at 2.2 A resolution. Prior to the crystal structure determination, molecular modeling indicated that the Thr67(E11) side chain hydroxyl group in the distal beta-heme pocket forms a hydrogen bond with the backbone carbonyl of His63(E7) and is within hydrogen-bonding distance of the N delta of His63(E7). The mutant crystal structure indicates only small changes in conformation in the vicinity of the E11 mutation confirming the molecular modeling predictions. Comparison of the structures of the mutant beta-subunits and recombinant porcine myoglobin with the identical mutation [Cameron et al. (1993) Biochemistry 32, 13061-13070] indicates similar conformations of residues in the distal heme pocket, but there is no water molecule associated with either of the threonines of the beta-subunits. The introduction of threonine into the distal heme pocket, despite having only small perturbations in the local structure, has a marked affect on the interaction with ligands. In the oxy derivative there is a 2-fold decrease in O2 affinity [Fronticelli et al. (1993) Biochemistry 32, 1235-1242], and the rate of autoxidation is increased by 2 orders of magnitude. In the CO derivative the IR spectrum shows modifications with respect to that of normal human hemoglobin, suggesting the presence of multiple CO conformers. In the nitrosyl derivative an interaction with the O gamma atom of Thr67(E11) is probably responsible for the 10-fold increase in the rate of NO release from the beta-subunits. In the aquomet derivative there is a 6-fold decrease in the rate of hemin dissociation suggesting an interaction of the Fe-coordinated water with the O gamma of Thr67(E11).
Bovine erythrocytes do not contain 2,3-diphosphoglycerate, the principal allosteric effector of human hemoglobin. Bovine hemoglobin has a lower oxygen affinity than human hemoglobin and is regulated by physiological concentrations of chloride (Fronticelli, C., Bucci, E., and Razynska, A. (1988) J. Mol. Biol. 202, 343-348). It has been proposed that the chloride regulation in bovine hemoglobin is introduced by particular amino acid residues located in the amino-terminal region of the A helix and in the E helix of the beta subunits (Fronticelli, C. (1990) Biophys. Chem. 37, 141-146). In accordance with this proposal we have constructed two mutant human hemoglobins, beta(V1M+H2deleted+T4I+P5A) and beta(V1M+H2deleted+T4I+P5A+A76K). These are the residues present at the proposed locations in bovine hemoglobin except for isoleucine at position 4. Oxygen binding studies demonstrate that these mutations have introduced into human hemoglobin the low oxygen affinity and chloride sensitivity of bovine hemoglobin and reveal the presence of a previously unrecognized allosteric mechanism of oxygen affinity regulation where all the interactions responsible for the lowered affinity and chloride binding appear to be confined to individual beta subunits.