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PubMed1995

Rheumatoid-factor-reactive sites on CH3 established by overlapping 7-mer peptide epitope analysis.

Polyclonal IgM rheumatoid factors (RF) from ten patients with rheumatoid arthritis and six monoclonal IgM RF were isolated from monomeric IgG affinity columns and studied for their reactivity with the entire CH3 domain of IgG synthesized as overlapping 7-mers using a pin-ELISA assay. All ten polyclonal IgM RF showed similar profiles of reactivity which included peptides with solvent accessible residues PREPQVY (residues 343-349), PQVYTLP (residues 346-352), TLPPRSE (350-356), DGSFFLY (401-407), WQQGNVF (417-423), CSVMHEG (425-430), EGLHNHY (430-436) and KSLSLSP (439-446) of the CH3 domain. Substitution of a neutral glycine or alanine for each residue within these RF-reactive epitopes indicated that tyrosine at position 349, prolines at 343, 346 and 352, glutamine 347, valine 348, threonine 350, leucine 351, arginine 354, aspartic acid 401, tyrosine 407, serine 426, histidine 429, leucine 432, tyrosine 436 and lysine 439 represented important single amino acids within CH3 for RF reactivity. Regions of CH3 primary sequence with and without the single allotype-specific amino acid substitutions of glycine for alanine 431 (Gmx) or aspartic acid for glutamic acid (356) and leucine for methionine (358) (Gma) often showed considerable differences in reactivity with individual polyclonal and monoclonal RF. However, these differences in RF reactivity did not correlate with the individual anti-Gm RF specificity. Assays using monoclonal IgM RF produced from RA synovial B cells or peripheral blood B cells frequently showed a much more restricted spectrum of reactive CH3 epitopes. 7-mer peptides representing RF-reactive sites on CH3 preincubated with polyclonal IgM RF showed strong inhibition (55-66%) of RF binding to whole IgG on the ELISA plate. These studies indicate that it is possible to define portions of the IgG CH3 domain participating in the reaction with IgM RF using reactive epitope-mapping with sequential linear peptides derived from the primary IgG CH3 sequence.

Amino Acid Sequence↗

PubMed1989

UV spectroscopic studies of human erythrocyte superoxide dismutase.

Using UV absorption spectroscopy, first derivative spectroscopy, and UV difference spectroscopy, the active site of human superoxide dismutase is probed. First derivative spectra (dA/d lambda versus lambda) show the HESOD spectrum to be a composite of Phe and Trp absorbance. The 278 and 288 nm Trp absorbance peaks are sensitive to solvent polarity. A 5-10% decrease in these peaks accompanies copper removal from the active site indicating greater solvent access to Trp in the apoenzyme than the holoenzyme. A Trp UV difference peak at 305-310 nm documents the presence or absence of copper at the active site, and documents also the movement of a nonbridging copper-binding His (His 46 or 120) when HESOD is inhibited by azide or when the copper moiety is reduced. Trp absorbances indicate that neither cyanide nor KCl inhibition affects the Cu(II)-His bonds. Phe UV absorbance is increased by the presence of copper at the active site and increased further by the addition of cyanide or azide. Neither Trp nor Phe responds to the presence of zinc in the active site. A molecular graphics program, FRODO, shows Trp and the four Phe residues lying in an approximate ring around the active site of HESOD and thus excellently placed to report on active site perturbations.

Apoenzymes↗

PubMed1991

Protein interactions with surface-immobilized metal ions: structure-dependent variations in affinity and binding capacity with temperature and urea concentration.

We have used equilibrium binding analyses to evaluate the influence of temperature and urea on the affinity of hen egg white lysozyme and bovine pancreatic ribonuclease A for surface-immobilized Cu(II) ions. Linear Scatchard plots suggested that these model proteins were interacting with immobilized metal ions via a single class of intermediate-affinity (Kd = 10-40 microM) binding sites. Alterations in temperature had little or no effect on the immobilized Cu(II) binding capacity of either protein. Temperature effects on the interaction affinity, however, were protein-dependent and varied considerably. The affinity of lysozyme for immobilized Cu(II) ions was significantly decreased with increased temperature (0 degree C-37 degrees C), yet the affinity of ribonuclease did not vary measurably over the same temperature range. The van 't Hoff plot (1n K vs 1/T) for lysozyme suggests a straight line relationship (single mechanism) with a delta H of approximately -5.5 kcal/mol. Urea effects also varied in a protein-dependent manner. A 10-fold reduction in the affinity of lysozyme for the immobilized Cu(II) was observed with the urea concentrations up to 3 M; yet urea had no effect on the affinity of ribonuclease for the immobilized metal ions. Although the interaction capacity of lysozyme with the immobilized Cu(II) ions was decreased by 50% in 3 M urea, ribonuclease interaction capacity was not diminished in urea. Thus, temperature- and urea-dependent alterations in protein-metal ion interactions were observed for lysozyme but not ribonuclease A. The complete, yet reversible, inhibition of lysozyme- and ribonuclease-metal ion interactions by carboxyethylation with low concentrations of diethylpyrocarbonate provided direct evidence of histidyl involvement. The differential response of these proteins to the effects of temperature and urea was, therefore, interpreted based on calculated solvent-accessibilities and surface distributions of His residues, individual His residue pKa values, and specific features of the protein surface structure in the immediate environment of the surface-exposed histidyl residues. Possible interaction mechanisms involved in protein recognition of macromolecular surface-immobilized metal ions are presented.

Animals↗

PubMed1990

Localisation of amino acid sequence stretches containing a continuous epitope on the surface of the two Lathyrus ochrus isolectins.

A series of 66 overlapping heptapeptides covering the complete amino acid sequences of the heavy subunits (beta chains) of the two Lathyrus ochrus isolectins (LoLI and LoLII) were synthesised and coupled to bovine serum albumin used as a carrier protein, and the conjugates were allowed to react with polyclonal antibodies directed against the whole isolectins. A similar approach was previously carried out with a series of 21 peptides corresponding to the light subunits (alpha chains) of the two isolectins. Of this total of 87 conjugates, 54 reacted significantly with the polyclonal antibodies. They corresponded to 8 linear peptides laid out along the entire sequences of both subunits, most of them being localised along the heavy subunits. All these continuous/sequential epitopes fall into regions predicted to be putative determinants, according to their hydrophilicity, flexibility and solvent accessibility.

Amino Acid Sequence↗

PubMed1992

Footprinting of EcoRI endonuclease at high pressure.

Hydroxyl radicals generated by irradiation with gamma rays have been used to footprint EcoRI endonuclease with single base pair resolution at pressures up to 144 MPa. At atmospheric pressure (0.1 MPa) a 10 base pair footprint was found. With increasing pressure three types of responses were observed: (1) bases distant from the recognition sequence showed a moderate increase in solvent exposure; (2) the bases at the point of enzymatic activity showed a large increase in cleavage by the hydroxyl radicals; and (3) the two center-most bases exhibited no pressure-induced change in solvent accessibility. The results are interpreted in terms of localized conformational changes of EcoRI.

Autoradiography↗

PubMed1984

Co(II) derivatives of Cu,Zn-superoxide dismutase with the cobalt bound in the place of copper. A new spectroscopic tool for the study of the active site.

Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substituted for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent pKa = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region (epsilon = 75 M-1 X cm-1 at 456 nm; epsilon = 90 M-1 X cm-1 at 520 nm; epsilon = 70 M-1 X cm-1 at 600 nm) and three bands in the near infrared region, at 790 nm (epsilon = 18 M-1 X cm-1), 916 nm (epsilon = 27 M-1 X cm-1) and 1045 nm (epsilon = 25 M-1 X cm-1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity (epsilon = 225 M-1 X cm-1 at 544 nm; epsilon = 315 M-1 X cm-1 at 575 nm; epsilon = 330 M-1 X cm-1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm (epsilon = 44 M-1 X cm-1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN- gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN- complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN- provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.

Animals↗

PubMed1984

Conformational changes in human urokinase induced by a specific reduction of disulfide bond in Cys-194-Cys-222 associated with exhibition of enzymatic activity.

The disulfide bond in Cys-194-Cys-222 of the isolated B chain (UK X B) of human urinary urokinase was selectively carboxamidomethylated (R X CAM-UK X B) after specific reduction with dithiothreitol in the presence of the competitive inhibitor N-alpha-benzoyl-L-argininamide (BzArgNH2) and 0.3 M guanidine with inactivation. There were no differences between UK X B and R X CAM-UK X B in CD spectra of 200-250 nm and infrared spectra in the amide I and II regions, whereas two out of five negative CD bands of 250-310 nm were slightly reduced in R X CAM-UK X B. R X CAM-UK X B was more susceptible than UK X B to guanidine denaturation, H-2H exchange in peptide protons and tyrosine-residue ionization. These results indicate that Cys-194-Cys-222 contributes to conformational stabilization against perturbants, but not to static conformation in the resting state, except for a slight disruption in the amino-acid side-chains. BzArgNH2 and 0.3 M guanidine, corresponding to the threshold point toward denaturing transition, enhanced more markedly all the five CD bands of 250-310 nm in UK X B than in R X CAM-UK X B, but did not alter CD spectra of 200-250 nm. The deletion of either BzArgNH2 or guanidine did not induce the inactivation or the cleavage of Cys-194-Cys-222 upon the reduction of UK X B, nor did it induce a marked CD enhancement in the near-ultraviolet upon the addition to UK X B. The results suggest that the BzArgNH2-UK X B interaction enhances the solvent accessibility to the disulfide bond with aid of critical guanidine-denaturation.

Arginine↗

PubMed1985

Ionization of tyrosine residues in horse-heart ferricytochrome c and its guanidinated and acetylated-guanidinated derivatives.

Spectrophotometric titration curves were obtained at 242 nm for native and fully guanidinated horse-heart ferricytochrome c. The cytochrome c data were fit over the pH range 9-12 (I = 0.35) by a theoretical curve with pK' values of 10.35 and 11.70. The slope of the experimental data increases sharply above pH 12.5 suggesting that two tyrosine residues with pK' values greater than 12.5 are exposed by conformation change. The guanidinated cytochrome c data after correction for the alkaline spin-state transition were fit over the entire pH range 9-13.6 (I = 0.35) by a theoretical curve with pK' values 10.37, 10.78, 11.50, and 13.60. These results along with viscosity measurements indicate that the unfolding transition occurs at higher pH in the guanidinated derivative. N-Acetylimidazole was used to acetylate specific tyrosyl groups of guanidinated cytochrome c. Assignments of acetylated tyrosine residues were confirmed by peptide mapping of 14C-labelled derivatives. Spectrophotometric titrations with rapid data acquisition of two monoacetylated derivatives allowed assignments of pK'1 (10.37) to Tyr-67 and pK'4 (13.60) to Tyr-97. The basis for the large differences in acidity and chemical reactivity of these two residues is not obvious from the crystallographic structure and may arise from differences in solvent access due to motions of the polypeptide chain.

Acetylation↗

PubMed1988

Conformational study of the antitumor protein alpha-sarcin.

The antitumor protein alpha-sarcin is a single polypeptide chain produced by the mold Aspergillus giganteus. It inhibits protein synthesis in some tumor cells by inactivating the larger ribosomal subunit. The secondary structure of the molecule has been studied by circular dichroism and predictive methods. The protein contains about 40% of periodic structures, mainly located at both extremes of the polypeptide chain. beta-Turns and aperiodic conformation appear at the central part of the molecule. Two different tyrosine populations have been observed in alpha-sarcin. Attempts to correlate solvent accessibility and particular protein regions have been carried out by using CD in the near-ultraviolet region, fluorescence and absorbance spectroscopies as well as acrylamide quenching and hydropathy profiles. Five different pH-induced conformational transitions are detected. Two of them, at pH 2.5 and 10.2, are denaturing transitions. These results are explained in terms of the structural features of this molecule, and related to its ribonucleolytic activity and ability to cross cell membranes.

Acrylamide↗

PubMed1990

A physicochemical comparison of the isozymes of creatine kinase from rabbit brain and muscle.

A comparison of specific structural features of creatine kinase from rabbit muscle and brain was undertaken to determine if the observed isozyme specific differences in catalytic cooperativity are related to conformational differences, particularly differences in packing density. The intrinsic fluorescence of the brain isozyme is 2-fold higher than the muscle isozyme. In the denatured state, both proteins display the characteristic red shift in emission maximum; however, the emission intensity of the brain isozyme increases only 5% upon denaturation compared to nearly 100% increase for the muscle protein. The fluorescence lifetimes are 2.65 ns (67%) and 0.48 ns for native muscle enzyme and 4.38 ns (65%) and 0.80 ns for brain enzyme. Upon denaturation, the lifetimes are 3.98 ns (77%) and 0.99 ns for muscle protein and 3.82 ns (79%) and 0.86 ns for brain protein. Stern-Volmer plots of quenching by acrylamide are essentially the same for both native isozymes indicating that the differences of the intrinsic fluorescence of the native proteins are not due to differences in solvent accessibility. The spectral and lifetime differences in the isozymes in the native state and changes accompanying denaturation are consistent with the occurrence of energy transfer in native muscle isozyme. The rotational correlation times of 5-[2-(iodoacetyl)aminoethyl]aminonaphthalene-1-sulfonate conjugated proteins, derivatized at the active site reactive thiol, are best described by two term decay laws. The slower rotations, 45.1 ns (75%) and 40.6 ns (71%) reflect overall macromolecular rotation for the muscle and brain isozymes, respectively. The faster motions, 2.4 ns for muscle isozyme and 0.4 ns for the brain isozyme, are attributed to the probe or probe associated segmental motions and indicate these motions are more restricted in the muscle protein. Reactivity of creatine kinase (2.5-10 microM) with the amino-specific reagent trinitrobenzene sulfonate (0.4-2 mM) was analyzed by pseudo-first-order and second order models, neither of which was adequate for the entire range of data. However, in every case, the rate constants were faster for brain creatine kinase but the extent of reaction was greater for muscle creatine kinase. The faster initial reactivity of the brain isozyme is consistent with greater accessibility for lysine derivatization.(ABSTRACT TRUNCATED AT 400 WORDS)

Acrylamide↗

PubMed1991

Secondary structure perturbations in salt-induced protein precipitates.

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na2SO4, were studied for twelve different proteins. alpha-helix and beta-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of beta-sheet structures with a concomitant loss of alpha-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of alpha-helix and beta-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting beta-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in alpha-helix content were influenced by the initial helical content and the dipole moment. The enhanced beta-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.

Chemical Precipitation↗

PubMed1991

1H-NMR studies of bovine platelet factor 4: histidine assignments and interactions with heparin.

1H-NMR spectroscopy has been used to assign and to characterize the two histidine C2H resonances of the heparin binding protein, bovine platelet factor 4. One histidine has a pKa value of 6.51 at 27 degrees C; the second histidine exhibits 2 pKa values of 5.52 and 5.66 at 27 degrees C. The two histidine resonances have been assigned by an analysis of their deuterium exchange kinetics. Both resonances are solvent accessible with half-times of exchange at pH 8.8 of 3.3 and 4.0 days. These two resonances have been assigned by digesting partially deuterated protein with Staphylococcus aureus V-8 proteinase, separating and purifying the resulting peptides, and determining their relative and residual hydrogen content by NMR. The results indicate that His-38 has the lower pKa value and the slower deuterium exchange rate, whereas His-50 has the higher pKa value and the faster deuterium exchange rate at pH 8.8. The 1H-NMR resonance of His-38 of bovine platelet factor 4 is preferentially perturbed by the introduction of heparin. This observation and the presence of His-38 within the belt of positively charged residues around the platelet factor 4 tetramer supports the model of the platelet factor 4-heparin complex in which the polysaccharide crosses over each of the 2 alpha-helices of the 2 dimers at right angles.

Animals↗

PubMed1993

A comparative study of the conformational properties of Escherichia coli-derived rat intestinal and liver fatty acid binding proteins.

Fourier transform infrared spectroscopy has been used to examine the conformation in aqueous solution of Escherichia coli-expressed rat intestinal and liver fatty-acid binding proteins (I-FABP and L-FABP, respectively). While I-FABP is known from X-ray analysis to have a predominantly beta-structure with 10 antiparallel beta-strands forming two orthogonal sheets that surround the ligand binding pocket, no structural data are available for L-FABP. As expected for homologous proteins with related functions, the secondary structures of I-FABP and L-FABP are very similar. In both proteins, the conformation-sensitive amide-I band shows the maximum absorption at around 1630 cm-1, proving that beta-sheet is the major structural element. However, there are three critical differences between I-FABP and L-FABP; (i), a different solvent accessibility of the protein backbone; (ii), a different pH sensitivity and (iii), a different thermostability, with L-FABP being thermally more stable than I-FABP. These results suggest that, in spite of having a similar overall conformation, the architecture of these proteins is stabilized by slightly different interactions. Such dissimilarities, well-paralleled by fatty-acid binding studies, may provide a structural basis for their functional diversification.

Animals↗

PubMed1994

Amide hydrogen exchange of the central B-chain helix within the T- and R-states of insulin hexamers.

Comparative analysis of the 1H-NMR spectra of human insulin shows that in the presence of the allosteric ligand, phenol, the tertiary structure of the protein is altered as evidenced by the decreased rate of amide hydrogen-deuterium exchange. In particular, exchange of amide protons in residues of the B-chain helix (B9-B20) are significantly affected suggesting either a stabilization of this helix or a reduction in the solvent accessibility of the helix in the R-state. This paper exemplifies the exchange rates of two amides (ValB18 and TyrB16) from this helix which decrease by approximately 400-fold as a result of this ligand induced conformational transition.

Allosteric Site↗

PubMed1996

Cleft containing reactive thiol of myosin closes during ATP hydrolysis.

The probe binding cleft of myosin subfragment 1 (S1) contains the reactive thiol, SH1, and tryptophan 510 (Trp-510). Solvent accessibility to Trp-510, measured using the acrylamide quenching of its fluorescence, is highest in rigor and decreases during the ATPase cycle prior to force generation. These data suggest the probe binding cleft closes during ATP hydrolysis and opens during force generation. The closing of the probe binding cleft may be the origin of the shape change of S1 during ATP hydrolysis.

Adenosine Triphosphate↗

PubMed1993

HLA-DR beta chain residues that are predicted to be located in the floor of the peptide-binding groove contribute to antibody-binding epitopes.

The purpose of this study was to identify polymorphic residues in HLA-DR beta chains that are involved in the epitopes recognized by DRw52-like mAbs. Flow cytometric analysis of antibody binding to mouse fibroblast transfectants expressing wild-type or mutant DRw52-associated DR beta chains demonstrated that the amino acid at position 77, which is predicted to be on a solvent-accessible surface of the alpha-helix, contributes to the epitopes recognized by the 7.3.19.1 and 21r5 mAbs. In contrast, residues that are not predicted to occupy accessible positions on the alpha-helix contribute to binding of the I-LR2 and UL-52 mAbs. Substitutions at positions 10, 12, and 51, but not 9, 11, and 13, affect these epitopes. It is interesting that antibody binding is influenced by amino acid substitutions at DR beta positions 10 and 12 because the class II model predicts that residues at these positions are located in the middle of the floor of the peptide-binding site, with their side chains pointing down. These findings emphasize the functional importance of polymorphic residues whose side chains are not predicted to point into or up from the antigen-binding site and they raise the possibility that peptides contribute to the generation of these epitopes.

Amino Acid Sequence↗

PubMed1989

The computer program MolPot: a useful tool in chemical reactivity analysis.

MolPot is a fast program for producing reactivity diagrams of molecular compounds, particularly for drugs. These diagrams are characteristic of the molecule envelope and the Molecular Electrostatic Potential (MEP) of the molecule surface (van der Waals surface or the portion of this surface that is solvent accessible). The input data includes only the atom names and their Cartesian coordinates.

Computer Graphics↗

PubMed1991

ANTHEPROT: an interactive graphics software for analyzing protein structures from sequences.

ANTHEPROT is a fully interactive program devoted to the analysis of protein structures using a graphics workstation. It presents four options: The first option can predict secondary structures using five methods, and hydrophobicity, solvent accessibility, flexibility and antigenicity profiles using eighteen scales. The user may introduce his own scales. The results displayed on the screen can be easily analyzed. The second option is for representing results concerning up to eight proteins by one method. To compare these proteins, it is possible to align the profiles or the predicted secondary structure according to various motifs. The secondary structure deduced from crystallographic data may also be introduced. The third option is designed to compare the primary structure of two proteins and to visualize on the screen regions that exhibit similarity. Six different comparison matrices may be used, but the user can also introduce his own matrices. The last option is for studying the proteolytic peptides resulting from a chemical or enzymatic digestion of a given protein. It is possible to analyze the protein cleavage using eleven chemical reagents or enzymes. The results are displayed on the screen as RP-HPLC chromatogram.

Amino Acid Sequence↗