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PubMed2001

Comparison of the amino acid residues in the sixth transmembrane domains accessible in the binding-site crevices of mu, delta, and kappa opioid receptors.

We have mapped the residues in the sixth transmembrane domains (TMs 6) of the mu, delta, and kappa opioid receptors that are accessible in the binding-site crevices by the substituted cysteine accessibility method (SCAM). We previously showed that ligand binding to the C7.38S mutants of the mu and kappa receptors and the wild-type delta receptor was relatively insensitive to methanethiosulfonate ethylammonium (MTSEA), a positively charged sulfhydryl-specific reagent. These MTSEA-insensitive constructs were used as the templates, and 22 consecutive residues in TM6 (excluding C6.47) of each receptor were mutated to cysteine, 1 at a time. Most mutants retained binding affinities for [3H]diprenorphine, a nonselective opioid antagonist, similar to that of the template receptors. Treatment with MTSEA significantly inhibited [3H]diprenorphine binding to 11 of 22 mutants of the rat mu receptor and 9 of 22 mutants of the human delta receptor and 10 of 22 mutants of the human kappa receptor. Naloxone or diprenorphine protected all sensitive mutants, except the A6.42(287)C mu mutant. Thus, V6.40, F6.44, W6.48, I6.51, Y6.54, V6.55, I6.56, I6.57, K6.58, and A6.59 of the mu receptor; F6.44, I6.51, F6.54, V6.55, I6.56, V6.57, W6.58, T6.59, and L6.60 of the delta receptor; and F6.44, W6.48, I6.51, F6.54, I6.55, L6.56, V6.57, E6.58, A6.59, and L6.60 of the kappa receptor are on the water-accessible surface of the binding-site crevices. The accessibility patterns of residues in the TMs 6 of the mu, delta, and kappa opioid receptors are consistent with the notion that the TMs 6 are in alpha-helical conformations with a narrow strip of accessibility on the intracellular side of 6.54 and a wider area of accessibility on the extracellular side of 6.54, likely due to a proline kink at 6.50 that bends the helix in toward the binding pocket and enables considerable motion in this region. The wider exposure of residues 6.55-6.60 to the binding-site crevice, combined with the divergent amino acid sequences, is consistent with the inferred role of residues in this region in determining ligand binding selectivity. The conservation of the accessibility pattern on the cytoplasmic side of 6.54 suggests that this region may be important for receptor activation. This accessibility pattern is similar to that of the D2 dopamine receptor, the only other GPCR in which TM6 has been mapped by SCAM. That opioid receptors and the remotely related D2 dopamine receptor have similar accessibility patterns in TM6 suggest that these segments of GPCRs in the rhodopsin-like subfamily not only share secondary structure but also are packed similarly into the transmembrane bundle and thus have similar tertiary structure.

Amino Acid Substitution↗
PubMed2001

Enthalpic and entropic contributions to the mutational changes in the reduction potential of azurin.

The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins following point mutations and residue ionizations were factorized into the enthalpic and entropic contributions through variable temperature direct electrochemistry experiments. The effects on the reduction enthalpy due to changes in the first coordination sphere of the copper ion, as in the Met121Gln and Met121His variants of A. denitrificans azurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met44Lys and His35Leu variants of P. aeruginosa azurin, respectively, and proton uptake/release in wild-type and mutated species could invariably be accounted for on the basis of simple coordination chemistry and/or electrostatic considerations. The concomitant changes in reduction entropy were found in general to contribute to the E degrees ' variation to a lesser extent as compared to the enthalpy changes. However, their effects were by no means negligible and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential. Several lines of evidence indicate that the entropic effects are notably influenced by reduction-induced solvent reorganization effects. In particular, protein reduction tends to be favored on entropic grounds with increasing exposure of the copper site to the solvent. Moreover, enthalpy-entropy compensation phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site or alter the H-bonding network in the hydration shell of the molecule. Therefore, in these cases, caution must be used in making predictions of E degrees ' changes simply based on Coulombic or coordination chemistry arguments.

Acid-Base Equilibrium↗
PubMed1998

Stability effects of increasing the hydrophobicity of solvent-exposed side chains in staphylococcal nuclease.

A total of fifty single site surface phenylalanine substitution mutants have been made in the model protein staphylococcal nuclease. The fifty residues that were replaced with phenylalanine were chosen to give a broad sampling of solvent accessibility, secondary structure, and backbone conformations. The change in the stability of each mutant protein relative to wild type was measured by guanidine hydrochloride denaturation. These results were compared to previous results obtained when these same sites were substituted with an alanine and a glycine. By this means, changes in the stability due to the loss of interactions of the wild-type side chain can be separated from the effects of introducing the bulky, hydrophobic phenylalanine in these solvent-exposed positions. In general, our results agree with the conventional wisdom that placing a hydrophobic residue in a solvent-exposed position is destabilizing in most cases, but less destabilizing than most changes in the hydrophobic core of the protein. However, the degree to which a hydrophobic surface substitution destabilizes or stabilizes a globular protein is highly context-dependent, with some mutations being as destabilizing as those in the core. This indicates that steric and packing considerations are also important on the surface of a globular protein but generally are not as important as in the interior. No evidence for the widespread occurrence of the so-called reverse hydrophobic effect at solvent-exposed sites was found. In addition, this survey of numerous sites suggests that previous measurements of alpha-helix "propensities" often seriously underestimate the importance of the environment of the side chain.

Amino Acid Sequence↗
PubMedDate not supplied

Comparative molecular field analysis (CoMFA) of a series of symmetrical bis-benzamide cyclic urea derivatives as HIV-1 protease inhibitors.

A 3D-QSAR study using CoMFA methodology was conducted on a series of 29 symmetrical bis-benzamide cyclic urea derivatives having anti-HIV-1-protease activities. Active site minimization of the ligands was used to exclude conformations which are not sterically accessible within the active site. A significant cross validated correlation coefficient q2 (0.724) was obtained indicating the predictive potential of the model for untested compounds of this class. A significant non-cross-validated correlation coefficient (r2) of 0.971 with a low standard error estimate (S) of 0.119 was obtained indicating that the model reliably predicted the ant-protease activities of poorly to highly active compounds. The model was used to predict the anti-protease activities of eight test-set compounds, and the predicted values were in good agreement with the experimental values. The CoMFA coefficient contour plots identified several key features which explain the wide range of activities. The already reported 2D-QSAR along with the CoMFA model presented here may help in designing effective HIV-1 protease inhibitors.

Computer Simulation↗
PubMed2003

A versatile set of aminooxy amino acids for the synthesis of neoglycopeptides.

Four N-alkylaminooxy amino acids have been synthesized in 22-56% overall yield from readily available amino acid precursors. Each amino acid can be efficiently incorporated into peptides using Boc-chemistry-based solid-phase peptide synthesis, and in three of the four cases the resulting peptides can be chemoselectively glycosylated at the aminooxy side chains to generate neoglycopeptides. The range of N-alkylaminooxy amino acids prepared allows attachment of sugars at two-, three-, or four-atom distances from the peptide backbone, and each ensures that attached sugars adopt cyclic conformations. These derivatives provide convenient access to arrays of biologically relevant neoglycopeptides that may be used to probe the influence of attached sugars on the structure and function of peptides and proteins.

Amino Acids↗
PubMed2003

Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation.

Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47(phox), a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47(phox) with phosphoinositides. Although the isolated phox homology (PX) domain of p47(phox) can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47(phox) is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47(phox) to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47(phox), because protein kinase C treatment of the wild-type p47(phox) but not of a mutant protein with the S303304328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47(phox) translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47(phox) nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47(phox) enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47(phox) from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.

Amino Acid Substitution↗
PubMed1988

Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: the amino-terminal residue of the large subunit is acetylated proline.

Trypsin rapidly inactivated the catalytic activities of spinach ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], but the stoichiometry of binding of the reaction-intermediate analogue carboxyarabinitol bisphosphate was only slightly reduced after proteolysis. Electrophoretic analysis indicated that several forms of the large subunit were generated during proteolysis but that the small subunit was resistant. Three tryptic peptides were isolated and characterized after digestion of the activated enzyme; the tryptic-sensitive sites were identified at Lys-8, Lys-14, and Lys-466 of the large subunit. Tryptic digestion of the enzyme complexed with the reaction-intermediate analogue released only two peptides by hydrolysis at Lys-8 and at Lys-14. The loss of susceptibility of Lys-466 to trypsin may be the result of a conformational change that limits the accessibility of the carboxyl-terminal region after binding of the reaction-intermediate analogue. Analysis of the amino-terminal tryptic peptide by composition and fast atom bombardment mass spectrometry demonstrated that the actual amino-terminal residue is proline at position 3 of the DNA-deduced sequence and that this proline is blocked with an N-acetyl moiety. Thus, posttranslational processing of the chloroplast-encoded large subunit of the enzyme must occur to remove Met-1 and Ser-2 and to acetylate the amino terminus.

Acetylation↗
PubMed1995

Molecular determinants conferring alpha-toxin resistance in recombinant DNA-derived acetylcholine receptors.

Sequences of the alpha-subunits of the nicotinic acetylcholine receptor from the snake and mongoose contain several differences in the region between amino acids 183 and 200. Receptors from both of these species reveal resistance to the snake alpha-toxins presumably arising as a protective evolutionary mechanism. Sequence differences include the added glycosylation signals at residue 187 in the mongoose and at residues 189 and 111 in snake. Although previous observations with peptides and fusion proteins either synthesized chemically or in a bacterial expression system indicate that certain amino acid residues may contribute to the resistance, our findings with the intact receptor in an eukaryotic expression system indicate the major role for glycosylation. In this study, we show that addition of glycosylation signals gives rise to virtually complete glycosylation at the added sites, although heterogeneity of oligosaccharide processing is evident. By analysis of combinations of mutants, we document that glycosylation exerts the predominant influence on alpha-toxin binding. Substitutions at other residues are largely without influence as single mutations but appear to decrease affinity further in multiple mutants, particularly where the receptor is glycosylated at the 187 and 189 positions. Glycosylation exerts a major influence on the dissociation as well as the association rates of the alpha-toxin-receptor complex, suggesting that the decrease for alpha-toxin affinity is not simply a consequence of restricted diffusional access, rather glycosylation affects the conformation and stability of the bound complex.

Amino Acid Sequence↗
PubMed2000

Opposite effects of Ca(2+) and GTP binding on tissue transglutaminase tertiary structure.

Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides. The protein activity is known to be finely tuned by Ca(2+) and GTP binding. In this study we report the effects of these ligands on the enzyme structure, as revealed by circular dichroism, and steady-state and dynamic fluorescence measurements. We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure. In particular the metal ions were responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS). Unlike Ca(2+), the nucleotide binding increased the protein dynamics, reducing its rotational correlation lifetime from 32 to 25 ns, preventing also the binding of ANS into the protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a three-state denaturation mechanism, involving an intermediate species with the characteristics of the so-called "molten globule" state. The effect of GTP binding (but not that of Ca(2+)) had an important consequence on the stability of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least approximately 0.7 kcal/mol. Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP. These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein conformational change which, decreasing the accessibility of the protein matrix to the solvent, renders more difficult the exposure of the active site.

Anilino Naphthalenesulfonates↗
PubMed2003

Translocation of N-WASP by nuclear localization and export signals into the nucleus modulates expression of HSP90.

N-WASP regulates the actin cytoskeleton through activation of the Arp2/3 complex. N-WASP localizes at the cell periphery, where it controls actin polymerization downstream of signal molecules such as adapter proteins, Cdc42, Src family kinases, and phosphoinositides. N-WASP also localizes in the nucleus; however, the role of N-WASP in the nucleus is unclear. Here, we show that localization of N-WASP is controlled through phosphorylation by Src family kinases in which phosphorylated N-WASP is exported from the nucleus in a nuclear export signal (NES) and leptomycin B-dependent manner. N-WASP had nuclear localization signal (NLS) at its basic region and NES close to the phosphorylation site by Src family kinases, indicating that phosphorylation controls the accessibility to the NES through conformational changes. Increased levels of unphosphorylated N-WASP in the nucleus suppressed expression of HSP90 and transcription from a heat shock element (HSE). N-WASP bound heat shock transcription factor (HSTF) and enhanced the HSTF association with HSE. In addition, nuclear N-WASP was present in the protein complex that associates with HSE, suggesting that N-WASP participates in suppression of HSP90 transcription. Increased levels of unphosphorylated N-WASP also decreased the activities of Src family kinases in cells but not in experiments in vitro with pure N-WASP and Fyn. Because HSP90 is essential for the activities of Src family kinases, these results suggest that localization of N-WASP modulates Src kinase activity by regulating HSP90 expression.

Active Transport, Cell Nucleus↗
PubMed2006

Phosphorylation of focal adhesion kinase (FAK) on Ser732 is induced by rho-dependent kinase and is essential for proline-rich tyrosine kinase-2-mediated phosphorylation of FAK on Tyr407 in response to vascular endothelial growth factor.

Focal adhesion kinase (FAK) is phosphorylated on tyrosine and serine residues after cell activation. In the present work, we investigated the relationship between tyrosine and serine phosphorylation of FAK in promoting endothelial cell migration in response to vascular endothelial growth factor (VEGF). We found that VEGF induces the activation of the Rho-dependent kinase (ROCK) downstream from vascular endothelial growth factor receptor (VEGFR) 2. In turn, activated ROCK directly phosphorylates FAK on Ser732. Proline-rich tyrosine kinase-2 (Pyk2) is also activated in response to VEGF. Its activation requires the clustering of integrin alphavbeta3 and triggers directly the phosphorylation of Tyr407 within FAK, an event necessary for cell migration. Interestingly, ROCK-mediated phosphorylation of Ser732 is essential for Pyk2-dependent phosphorylation of Tyr407, because the latter is abrogated in cells expressing a FAK mutant that is nonphosphorylatable on Ser732. We suggest that VEGF elicits the activation of the VEGFR2-ROCK pathway, leading to phosphorylation of Ser732 within FAK. In turn, phosphorylation of Ser732 would change the conformation of FAK, making it accessible to Pyk2 activated in response to its association with integrin beta3. Then, activated Pyk2 triggers the phosphorylation of FAK on Tyr407, promoting cell migration.

Animals↗
PubMed2001

Tolerance of point substitution of methionine for isoleucine in hen egg white lysozyme.

X-ray structure determination of proteins by using the multiple-wavelength anomalous dispersion method targeting selenomethionine is now widely employed. Isoleucine was examined for the second choice of the substitution of methionine next to leucine. We performed a systematic mutational study of the substitutions of methionine for isoleucine. All mutated lysozymes were less stable than the wild-type by about 1 kcal/mol and it is suggested that this instability was caused by the change in residual hydrophobicity from isoleucine to methionine. The X-ray structures of all mutant lysozymes were very similar to that of the wild-type. In addition, both the accessible surface areas and the conformation of the side chain of methionine in all mutant lysozymes were similar to those of the side chain at the respective isoleucine in the wild-type. Therefore, it is suggested that the mutation from isoleucine to methionine in a protein can be considered as a "safe" substitution.

Amino Acid Substitution↗
PubMed1988

Identification of an intracellular peptide segment involved in sodium channel inactivation.

Antibodies directed against a conserved intracellular segment of the sodium channel alpha subunit slow the inactivation of sodium channels in rat muscle cells. Of four site-directed antibodies tested, only antibodies against the short intracellular segment between homologous transmembrane domains III and IV slowed inactivation, and their effects were blocked by the corresponding peptide antigen. No effects on the voltage dependence of sodium channel activation or of steady-state inactivation were observed, but the rate of onset of the antibody effect and the extent of slowing of inactivation were voltage-dependent. Antibody binding was more rapid at negative potentials, at which sodium channels are not inactivated; antibody-induced slowing of inactivation was greater during depolarizations to more positive membrane potentials. The peptide segment recognized by this antibody appears to participate directly in rapid sodium channel inactivation during large depolarizations and to undergo a conformational change that reduces its accessibility to antibodies as the channel inactivates.

Amino Acid Sequence↗
PubMed2003

Interaction of pulmonary surfactant protein C with CD14 and lipopolysaccharide.

In addition to their effects on alveolar surface tension, some components of lung surfactant also have immunological functions. We found recently that the hydrophobic lung surfactant protein SP-C specifically binds to the lipid A region of lipopolysaccharide (LPS). In this study, we show that SP-C also interacts with CD14. Four observations showed cross talk between the three molecules SP-C, LPS, and CD14. (i) Like LBP, SP-C allows the binding of a fluorescent LPS to cells expressing CD14 (the other surfactant components were ineffective). (ii) Recombinant radiolabeled CD14 and SP-C (or a synthetic analog of SP-C) interact in a dose-dependent manner. (iii) LPS blocks the binding of radiolabeled CD14 to SP-C-coated wells. (iv) SP-C enhances the binding of radiolabeled CD14 to LPS-coated wells. These results, obtained with native murine SP-C and with three synthetic analogs, suggest that LPS and CD14 interact with the same region of SP-C and that binding of SP-C modifies the conformation of CD14 or the accessibility of its LPS-binding site, allowing it to bind LPS. This ability of SP-C to interact with the pattern recognition molecule CD14 extends the possible immunological targets of SP-C to a large panel of microorganisms that can enter the airways.

Amino Acid Sequence↗
PubMed1990

Insulin-receptor tyrosine kinase and glucose transport.

We identified the earliest events in autophosphorylation of the insulin receptor after insulin addition. Insulin-stimulated autophosphorylation at specific sites in the tyrosine kinase domain of the receptor's beta-subunit is correlated kinetically with activation of kinase-catalyzed phosphorylation of a model substrate (reduced and carboxyamidomethylated lysozyme; RCAM-lysozyme). To identify these sites, the deduced amino acid sequence of the 3T3-L1 adipocyte insulin receptor of the mouse was determined. Insulin-induced activation of substrate phosphorylation was shown to require autophosphorylation of three neighboring tyrosines (Tyr1148, Tyr1152, and Tyr1153) in the mouse receptor. A search for cellular substrates of the receptor kinase revealed that insulin causes accumulation of a 15,000-Mr phosphorylated (on tyrosine) cytosolic protein (pp15) in 3T3-L1 adipocytes treated with oxophenylarsine (PAO). PAO blocks turnover of the phosphoryl group of pp15, causing its accumulation, and thereby appears to interrupt signal transmission from the receptor to the glucose-transport system. Two membrane-bound protein phosphotyrosine phosphatases that are inhibited by PAO and are apparently responsible for the turnover of the pp15 phosphoryl group have been purified from 3T3-L1 adipocytes and characterized. These and other results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin-receptor tyrosine kinase action, couples signal transmission to the glucose-transport system. [32P]pp15 was purified to homogeneity from 3T3-L1 adipocytes. Amino acid and radiochemical sequence analysis of the purified tryptic [32P]phosphopeptide revealed that pp15 is the phosphorylation product of 422(aP2) protein, a 15,000-Mr adipocyte protein whose cDNA we previously cloned and sequenced. 422(aP2) protein was found to bind fatty acids. When exposed to a free fatty acid, notably oleic acid, 422(aP2) protein becomes an excellent substrate of the isolated insulin-receptor tyrosine kinase. Compelling evidence indicates that on binding fatty acid, 422(aP2) protein undergoes a conformational change whereby Tyr19 becomes accessible to the receptor tyrosine kinase and undergoes O-phosphorylation. Adipose tissue and skeletal and heart muscle, which exhibit insulin-stimulated glucose uptake, express a specific insulin-responsive glucose transporter. A cDNA (GT2) that encodes this protein was isolated from a mouse 3T3-L1 adipocyte library and sequenced. We also isolated and characterized the corresponding mouse gene GLUT4. DNase I footprinting with nuclear extracts from 3T3-L1 cells revealed that a differentiation-specific nuclear factor binds to the GLUT4 promoter. The purified transcription factor C/EBP binds at the same position.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Characterization of guanosine diphospho-D-mannose dehydrogenase from Pseudomonas aeruginosa. Structural analysis by limited proteolysis.

Alginate is believed to be a major virulence factor in the pathogenicity of Pseudomonas aeruginosa in the lungs of patients suffering from cystic fibrosis. Guanosine diphospho-D-mannose dehydrogenase (GDPmannose dehydrogenase, EC 1.1.1.132) is a key enzyme in the alginate biosynthetic pathway which catalyzes the oxidation of guanosine diphospho-D-mannose (GDP-D-mannose) to GDP-D-mannuronic acid. In this paper, we report the structural analysis of GMD by limited proteolysis using three different proteases, trypsin, submaxillary Arg-C protease, and chymotrypsin. Treatment of GMD with these proteases indicated that the amino-terminal part of this enzyme may fold into a structural domain with an apparent molecular mass of 25-26 kDa. Multiple proteolytic cleavage sites existed at the carboxyl-terminal end of this domain, indicating that this segment may represent an exposed region of the protein. Initial proteolysis also generated a carboxyl-terminal fragment with an apparent molecular mass of 16-17 kDa which was further digested into smaller fragments by trypsin and chymotrypsin. The proteolytic cleavage sites were localized by partial amino-terminal sequencing of the peptide fragments. Arg-295 was identified as the initial cleavage site for trypsin and Tyr-278 for chymotrypsin. Catalytic activity of GMD was totally abolished by the initial cleavage. However, binding of the substrate, GDP-D-mannose, increased stability toward proteolysis and inhibited the loss of enzyme activity. GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.

Affinity Labels↗
PubMed1978

Hydrogen exchange kinetics of human hemoglobins. The pH dependence of solvent accessibility in cyanomet-, oxy-, and deoxyhemoglobin.

The hydrogen exchange kinetics of human oxy-, deoxy-, and cyanomethemoglobin have been measured as a function of pH by the tritium tracer method. At 5 degrees C and in phosphate buffer both liganded and unliganded forms of ferrohemoglobin exhibit deviations from the regular pH dependence of exchange that is characteristic of cyanomethemoglobin. In oxyhemoglobin, the deviation from the normal exchange pattern is centered at pH 7.4 and is in the direction of increased exchange or solvent accessibility. The effect in deoxyhemogloin, while occurring at the same pH and being of the same order of magnitude, is in the opposite direction, thus suggesting a pH-induced conformational transition leading to a less accessible structure. The width of these pH-induced deviations in solvent accessibility is approximately 1 pH unit in both cases. We propose a model in which specific interactions between charged groups in both froms of ferrohemoglobin account for these deviations.

Cyanides↗
PubMed1987

Ligand binding channels reflected in the resonance Raman spectra of cryogenically trapped species of myoglobin.

Variations in the v2 region of the Raman spectra of cryogenically trapped photoproducts of different liganded myoglobins as a function of ligand (CO, O2, and n-butyl isocyanide) and species (whale, tuna, elephant) are reported. These variations are attributed to differences in the population of "open" (ligand accessible) and "closed" (ligand inaccessible) conformations of the distal heme pocket. Based on these findings and those derived from other spectroscopies including x-ray crystallography, NMR, IR spectra, and ESR, a working model is presented which accounts for how the conformation of the distal heme pocket, the geometry of the bound ligand, the identity of the ligand, and the dynamics of the dissociated ligand are all interconnected.

Animals↗