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S G Boxer

Publications and source records attributed to S G Boxer.

At least 55 records · Page 3Linked to original sources
PubMed1994

Determination of the carbon monoxide binding constants of myoglobin mutants: comparison of kinetic and equilibrium methods.

The carbon monoxide (CO) binding constants of human myoglobin (Mb) and several single-site mutants have been determined using two different methods. In the kinetic method, which is commonly used for this ligand, the overall association (k(on)) and dissociation (k(off)) rates of CO were measured by flash photolysis and NO replacement, respectively, and the ratio k(on)/k(off) was calculated. In the equilibrium method, the binding constant Keq was measured directly using a thin-layer technique. These two measurements yield similar results for human wild-type Mb but differ significantly for some of the mutants. Possible reasons for these discrepancies are analyzed. A model assuming the presence of interconverting conformers with different association and dissociation rates is considered in light of infrared measurements on the CO stretching frequency in the MbCO forms of the same proteins [Balasubramanian et al. (1993a) Proc. Natl. Acad. Sci, U.S.A. 90, 4718]. It is suggested that in the case of some mutants which exhibit multiple conformations, this model may lead to nonequilibrium kinetics, which could produce the observed discrepancies between the kinetic and equilibrium determinations of the binding constant. These results suggest that both equilibrium and kinetic data should be obtained, even for a monomeric protein such as Mb, before the relative stabilities of mutants can be meaningfully compared.

Binding Sites↗
PubMed1994

Anatomy and dynamics of a ligand-binding pathway in myoglobin: the roles of residues 45, 60, 64, and 68.

In order for diatomic ligands to enter and exit myoglobin, there must be substantial displacements of amino acid side chains from their positions in the static X-ray structure. One pathway, involving Arg/Lys45, His64, and Val68, has been studied in greatest detail. In an earlier study (Lambright et al., 1989) we reported the surprising result that mutation of the surface residue Lys45 to arginine lowers the inner barrier to CO rebinding. Until then, it had been thought that this barrier primarily involves interior distal pocket residues such as His64 and Val68. In this report, we present a detailed study of the CO rebinding kinetics in aqueous solution of a series of single- and double-site mutants of human myoglobin at positions 64, 68, 45, and 60. On the basis of the observed kinetics, we propose that the effect of surface residue 45 on the inner barrier can be explained by a chain of interactions between surface and pocket residues. Very large, and in some cases unexpected, changes are observed in the kinetics of recombination and in the partitioning between geminate and bimolecular recombination.

Carbon Monoxide↗
PubMed1994

Ultrafast measurements of geminate recombination of NO with site-specific mutants of human myoglobin.

Flash photolysis studies of NO recombination to heme proteins offer a direct probe of protein structural changes on the tens of picoseconds timescale where they can be compared with molecular dynamics simulations. The geminate recombination of NO to site-specific mutants of human myoglobin (Mb) was studied following photodissociation of the MbNO form. Single amino acid changes were introduced at positions Val68, His64, Lys45 and Asp60 because motions of residues at these positions are generally regarded as important for the mechanism of ligand binding. In sharp contrast to the properties of simple porphyrin-NO complexes, the rebinding kinetics are found to be non-exponential for all mutants, even in aqueous solution at 298 K. The Val68 and His64 mutants substantially affect the NO rebinding rates but, surprisingly, so do changes on the protein surface that are further away from the iron. These changes in kinetics occur on a tens of picoseconds timescale, and therefore there is either a fast communication between protein residues over quite long distances or there are subtle differences in protein structure that exert great control over the reaction dynamics. Various models for the rebinding kinetics are evaluated. A model-free approach to data analysis using the maximum entropy method is found to be most useful. This analysis shows that the rate distributions are very different for the mutants, but are generally bimodal.

Asparagine↗
PubMed1994

Discovery of new ligand binding pathways in myoglobin by random mutagenesis.

A random library of single amino acid mutants of myoglobin was generated using a highly efficient, single-base-misincorporation random mutagenesis method to discover new ligand-binding pathways in myoglobin. A surprisingly large fraction of the library exhibits ligand-binding kinetics that are substantially different from the wild-type protein. In addition to residues 45, 64 and 68, which comprise the best studied ligand-binding pathway single mutations of several other clusters of residues far away from that pathway are discovered which profoundly affect the ligand-binding kinetics. These results provide a new approach to explore the relationship between the fluctuations in protein structure and function.

Amino Acid Sequence↗
PubMed1993

Dynamics of protein relaxation in site-specific mutants of human myoglobin.

We have recently reported spectroscopic evidence for structural relaxation of myoglobin (Mb) following photodissociation of MbCO [Lambright, D. G., Balasubramanian, S., & Boxer, S. G. (1991) Chem. Phys. 158, 249-260]. In this paper we report measurements for a series of single amino acid mutants of human myoglobin on the distal side of the heme pocket (positions 45, 64, and 68) in order to examine specific structural determinants involved in this conformational relaxation and to determine the nature of the coupling between relaxation and the functional process of ligand binding. The kinetics of ligand binding and conformational relaxation were monitored by transient absorption spectroscopy in the Soret spectral region, and the results are analyzed using a four-state ligand binding model. Two principal results emerge: (1) amino acid substitutions in the distal heme pocket affect the kinetics of the nonequilibrium conformational relaxation and (2) the rate of ligand escape from the protein matrix is not significantly perturbed by the distal heme pocket mutations.

Alanine↗
PubMed1993

Perturbations of the distal heme pocket in human myoglobin mutants probed by infrared spectroscopy of bound CO: correlation with ligand binding kinetics.

The infrared spectra of CO bound to human myoglobin and myoglobin mutants at positions His-64, Val-68, Asp-60, and Lys-45 on the distal side have been measured between 100 and 300 K. Large differences are observed with mutations at His-64 and Val-68 as well as with temperature and pH. Although distal His-64 is found to affect CO bonding, Val-68 also plays a major role. The variations are analyzed qualitatively in terms of a simple model involving steric interaction between the bound CO and the distal residues. A strong correlation is found between the final barrier height to CO recombination and the CO stretch frequency: as compared to wild type, the barrier is smaller in those mutants that have a higher CO stretch frequency (vCO) and vice versa. Possible reasons for this correlation are discussed. It is emphasized that the temperature and pH dependence of both the kinetics and the infrared spectra must be measured to obtain a consistent picture.

Carbon Monoxide↗
PubMed1993

CO recombination to human myoglobin mutants in glycerol-water solutions.

The kinetics of CO recombination to site-specific mutants of human myoglobin have been studied by flash photolysis in the temperature range 250-320 K on the nanosecond to second time scale in 75% glycerol at pH 7. The mutants were constructed to examine specific proposals concerning the roles of Lys 45, Asp 60, and Val 68 in the ligand binding process. It is found that ligand recombination is nonexponential for all the mutants and that both the geminate amplitude and rate show large variations. The results are interpreted in terms of specific models connecting the dynamics and structure. It is shown that removal of the charged group at position 45 does not substantially affect the barrier height for escape or entry of the ligand; therefore the breakage of the salt bridge linking Lys 45, Asp 60, and a heme propionate is ruled out as the rate-determining barrier for this process. On the other hand, it is found that the escape barrier decreases roughly as size of the residue at position 68 increases, in the order Ala > Val > Asn > Leu. The residue at position 68 is also a major contributor to the final barrier to rebinding, but the barrier height shows no correlation with residue size and is more dependent on the stereochemistry of the residue. A molecular mechanism for ligand binding that is consistent with the results is discussed, and supporting evidence for this mechanism is examined.

Carbon Monoxide↗
PubMed1992

Biochemical characterization and electron-transfer reactions of sym1, a Rhodobacter capsulatus reaction center symmetry mutant which affects the initial electron donor.

A 51 bp section of the Rhodobacter capsulatus photosynthetic reaction center M subunit gene (nucleotides M562-M612 of the pufM structural sequence) encoding amino acids M187-M203 was replaced by the homologous region of the L subunit gene. This resulted in the symmetrization of much of the amino acid environment of the reaction center initial electron donor, P. This is the first in a series of large-scale symmetry mutations and is referred to as sym1. The sym1 mutant was able to grow photosynthetically, indicating that reaction center function was largely intact. Isolated reaction centers showed an approximately 10-nm blue shift in the QY band of P. The standard free energy change between P* and P+BphA- determined from analysis of the long-lived fluorescence from quinone-reduced reaction centers decreased from about -120 meV in the wild-type to about -75 meV in the sym1 mutant. A 65-70% quantum yield of electron transfer from P* to P+QA- was observed, most of the yield loss occurring between P* and P+BphA-. The decay of the stimulated emission from P* was about 3-fold slower in this mutant than in the wild-type. Time-resolved spectral analysis of the charge-separated intermediates formed in sym1 reaction centers indicated that the major product was P+BphA-. A model-dependent analysis of the observed rates and electron-transfer yields gave the following microscopic rate constants for sym1 reaction centers (wild-type values under the same conditions are given in parentheses): [formula: see text] Analysis of the sym1 mutant, mutants near P made by other groups, and interspecies variation of amino acids in the vicinity of P suggests that the protein asymmetry in the environment of the initial electron donor is important for optimizing the rate and yield of electron transfer, but is not strictly required for overall reaction center function.

Amino Acid Sequence↗
PubMed1992

Spectroscopic and redox properties of sym1 and (M)F195H: Rhodobacter capsulatus reaction center symmetry mutants which affect the initial electron donor.

The redox properties, absorption, electroabsorption, CD, EPR, and P+QA- recombination kinetics have been measured for the special pairs of two mutants of Rhodobacter capsulatus reaction centers involving amino acid changes in the vicinity of the special pair, P. Both mutants symmetrize amino acid residues so that portions of the M-sequence are replaced with L-sequence: sym1 symmetrizes all residues between M187 and M203, whereas (M)F195H is a single amino acid subset of the sym1 mutation. (M)F195H introduces a His residue in a position where it is likely to form a hydrogen bond to the acetyl group of the M-side bacteriochlorophyll of P. For both mutants compared with wild-type, (i) the redox potential is at least 100 meV greater, (ii) the P+QA- recombination rate is about twice as fast at room temperature, and (iii) the large electroabsorption feature for the QY band of P is shifted relative to the absorption spectrum. The comparison of the properties observed for the sym1 and (M)F195H reaction center mutants and the differences between these mutants and wild-type suggest that residue M195 is an important determinant of the properties of the special pair.

Circular Dichroism↗
PubMed1991

Rapid-flow resonance Raman spectroscopy of bacterial photosynthetic reaction centers.

Rapid-flow resonance Raman vibrational spectra of bacterial photosynthetic reaction centers from the R-26 mutant of Rhodobacter sphaeroides have been obtained by using excitation wavelengths (810-910 nm) resonant with the lowest energy, photochemically active electronic absorption. The technique of shifted excitation Raman difference spectroscopy is used to identify genuine Raman scattering bands in the presence of a large fluorescence background. The comparison of spectra obtained from untreated reaction centers and from reaction centers treated with the oxidant K3Fe(CN)6 demonstrates that resonance enhancement is obtained from the special pair. Relatively strong Raman scattering is observed for special pair vibrations with frequencies of 36, 94, 127, 202, 730, and 898 cm-1; other modes are observed at 71, 337, and 685 cm-1. Qualitative Raman excitation profiles are reported for some of the strong modes, and resonance enhancement is observed to occur throughout the near-IR absorption band of the special pair. These Raman data determine which vibrations are coupled to the optical absorption in the special pair and, thus, probe the nuclear motion that occurs after electronic excitation. Implications for the interpretation of previous hole-burning experiments and for the excited-state dynamics and photochemistry of reaction centers are discussed.

Kinetics↗
PubMed1991

Stark effect spectroscopy of carotenoids in photosynthetic antenna and reaction center complexes.

The effects of electric fields on the absorption spectra of the carotenoids spheroidene and spheroidenone in photosynthetic antenna and reaction center complexes (wild-type and several mutants) from purple non-sulfur bacteria are compared with those for the isolated pigments in organic glasses. In general, the field effects are substantially larger for the carotenoid in the protein complexes than for the extracted pigments and larger for spheroidenone than spheroidene. Furthermore, the electrochromic effects for carotenoids in all complexes are much larger than those for the Qx transitions of the bacteriochlorophyll and bacteriopheophytin pigments which absorb in the 450-700 nm spectral region. The underlying mechanism responsible for the Stark effect spectra in the complexes is found to be dominated by a change in permanent dipole moment of the carotenoid upon excitation. The magnitude of this dipole moment change is found to be considerably larger in the B800-850 complex compared to the reaction center for spheroidene; it is approximately equivalent in the two complexes for spheroidenone. These results are discussed in terms of the effects of differences in the carotenoid functional groups, isomers and perturbations on the electronic structure from interactions with the organized environment in the proteins. these data provide a quantitative basis for the analysis of carotenoid bandshifts which are used to measure transmembrane potential, and they highlight some of the pitfalls in making such measurements on complex membranes containing multiple populations of carotenoids. The results for spheroidenone should be useful for studies of mutant proteins, since mutant strains are often grown semi-aerobically to minimize reversion.

Bacterial Proteins↗
PubMed1991

Large protein-induced dipoles for a symmetric carotenoid in a photosynthetic antenna complex.

Unusually large electric field effects have been measured for the absorption spectra of carotenoids (spheroidene) in the B800-850 light-harvesting complex from the photosynthetic bacterium Rhodobacter sphaeroides. Quantitative analysis shows that the difference in the permanent dipole moment between the ground state and excited states in this protein complex is substantially larger than for pure spheroidene extracted from the protein. The results demonstrate the presence of a large perturbation on the electronic structure of this nearly symmetric carotenoid due to the organized environment in the protein. This work also provides an explanation for the seemingly anomalous dependence of carotenoid band shifts on transmembrane potential and a generally useful approach for calibrating electric field-sensitive dyes that are widely used to probe potentials in biological systems.

Bacterial Proteins↗
PubMed1990

X-ray crystal structure of a recombinant human myoglobin mutant at 2.8 A 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.

Humans↗
PubMed1990

Characterization of the overall and internal dynamics of short oligonucleotides by depolarized dynamic light scattering and NMR relaxation measurements.

The dynamics of three synthetic oligonucleotides d(CG)4, d(CG)6, and d(CGCGTTGTTCGCG) of different length and shape were studied in solution by depolarized dynamic light scattering (DDLS) and time-resolved nuclear Overhauser effect cross-relaxation measurements. For cylindrically symmetric molecules the DDLS spectrum is dominated by the rotation of the main symmetry axis of the cylinder. The experimental correlation times describe the rotation of the oligonucleotides under hydrodynamic stick boundary conditions. It is shown that the hydrodynamic theory of Tirado and Garcia de la Torre gives good predictions of the rotational diffusion coefficients of cylindrically symmetric molecules of the small axial ratios studied here. These relations are used to calculate the solution dimensions of the DNA fragments from measured correlation times. The hydrodynamic diameter of the octamer and dodecamer is 20.5 +/- 1.0 A, assuming a rise per base of 3.4 A. The tridecamer, d(CGCGTTGTTCGCG), adopts a hairpin structure with nearly spherical dimensions and a diameter of 23.0 +/- 2.0 A. The DDLS relaxation measurements provide a powerful method for distinguishing between different conformations of the oligonucleotides (e.g., DNA double-helix versus hairpin structure). Furthermore, the rotational correlation times are a very sensitive probe of the length of different fragments. The NMR results reflect the anisotropic motion of the molecules as well as the amount of local internal motion present. The experimental correlation time from NMR is determined by the rotation of both the short and long axes of the oligonucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Stark spectroscopy of the Rhodobacter sphaeroides reaction center heterodimer mutant.

The effect of an electric field has been measured on the absorption spectrum (Stark effect) of the heterodimer mutant (M)H202L of Rhodobacter sphaeroides reaction centers, where the primary electron donor consists of one bacteriochlorophyll alpha and one bacteriopheophytin alpha. The electronic absorption spectrum of the heterodimer mutant from 820-950 nm is relatively featureless in a poly(vinyl alcohol) film, but it exhibits some structure in a glycerol/water glass at 77 K. A feature is seen in the Stark effect spectrum of the heterodimer at 77 K centered at 927 and 936 nm in poly(vinyl alcohol) and a glycerol/water glass, respectively. This feature has approximately the same shape and width as the Stark effect for the primary electron donor of the wild type, which consists of a pair of bacteriochlorophyll alpha molecules. The angle zeta A between the transition moment at the frequency of absorption and the difference dipole delta muA is 36 +/- 2 degrees in the wild type and 32 +/- 2 degrees for that feature in the heterodimer. A range of values for [delta muA] = (13-17)/f Debye units (where f is the local field correction) is obtained for the 936-nm feature in glycerol/water, depending on analysis method. This feature is interpreted as arising from a transition to the lower exciton state of the heterodimer, which is more strongly mixed with a low-lying charge transfer transition than in the wild type.

Bacterial Proteins↗
PubMed1989

Ligand and proton exchange dynamics in recombinant human myoglobin mutants.

Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively conservative change of a surface residue can substantially perturb ligand and proton exchange dynamics in a manner that is not readily predicted from the static structures.

Amino Acids↗