The effect of evolution on homologous proteins: a comparison between the chromophore microenvironments of Italian water buffalo (Bos bubalus, L.) and sperm whale apomyoglobin.

The perturbing effect of guanidium hydrochloride and pH on the molecular structure of water buffalo apomyoglobin has been investigated by circular dichroism in the far and near ultraviolet and by fluorescence. In the wavelength region between 320 and 260 nm the circular dichroic spectrum of the globin is highly structured and the contributions of the aromatic chromophores have been resolved. Buffalo apomyoglobin undergoes a structural transition at neutral pH which involves elements of the secondary and tertiary structure, as indicated by changes of dichroic activity of the peptide and aromatic chromophores and the fluorescence of the two tryptophanyl residues. The possibility of charge-transfer complex between indole and imidazole is discussed. A major structural transition with abrupt unfolding takes place in the pH region between 5.6 and 4.3. Below pH 4.3 the peptide helical residues, which survive the acid transition, appear to be resistent to further acidification to pH 2.0 while tryptophanyl emission is quenched and shifted to longer wavelengths. A structural transition occurs also in alkali above pH 10, which has been detected by the same techniques. The relationships between buffalo and sperm whale apomyoglobin are discussed.     

Allosteric serine hydroxymethyltransferase from monkey liver: Correlation of conformational changes caused by denaturants with the alterations in catalytic activity

The far-ultraviolet region circular dichroic spectrumof serine hydroxymethyltransferase from monkey liver showed that the protein is in an α-helical conformation. The near ultraviolet circular dichoric spectrum revealed two negative bands originating from the tertiary conformational environment of the aromatic amino acid residues. Addition of urea or guanidinium chloride perturbed the characteristic fluorescence and far ultraviolet circular dichroic spectrum of the enzyme. The decrease in (θ)₂₂₂ and enzyme activity followed identical patterns with increasing concentrations of urea, whereas with guanidinium chloride, the loss of enzyme activity preceded the loss of secondary structure. 2-Chloroethanol, trifluoroethanol and sodium dodecyl sulphate enhanced the mean residue ellipticity values. In addition, sodium dodecyl sulphate also caused a perturbation of the fluorescence emission spectrum of the enzyme. Extremes of pH decreased the — (θ)₂₂₂ value. Plots of — (θ)₂₂₂and enzyme activity as a function of pH showed maximal values at pH 7.4–7.5. These results suggested the prevalence of “conformational flexibility” in the structure of serine hydroxymethyltransferase.     

Self-association of apo-C-I from the human high density lipoprotein complex.

The molecular properties of apo-C-I, isolated from the human high density lipoprotein complex, have been evaluated as a function of pH, solvent composition, and protein concentration by sedimentation equilibrium and circular dichroic measurements. This protein self-associates in aqueous solution at neutral pH with concomitant changes in secondary structure. In contrast, in the acid pH range, apo-C-I is monomeric and its ellipticity is independent of protein concentration. The results are discussed in terms of the interpretation of experiments where changes in the physical properties of apolipoproteins have been used to monitor ligand binding and lipid-apolipoprotein recombination.     

Purification and properties of an auxin-binding protein from maize shoot membranes

An auxin-binding protein was purified from membranes of maize shoots including the coleoptiles, leaf rolls and mesocotyls. The method of Ca2+-promoted sedimentation of membrane particles was adopted for large-scale preparation. The auxin-binding protein was solubilized from the acetone-washed membranes, and purified by successive chromatographies on DEAE-Sephacel, 1-naphthylacetic acid-linked AH-Sepharose 4B, and Sephadex G-100 columns. The yield of the purified protein was about 0.2 mg from 1 kg of shoots. The binding protein exists as a dimer with a subunit molecular weight of 21,000, and possesses one auxin-binding site per dimer. The preparation also contains a minor molecular form with a subunit molecular weight of 20,000. The auxin-binding protein is not a hydrophobic protein, as judged from its amino acid composition and solubility. The circular dichroic (CD) spectrum of the binding protein resembles the spectrum anticipated from the beta-structures, and shows no alpha-helix characteristic in the secondary structure. The CD spectral changes induced on the binding of auxin and its antagonist seem to be related to the receptor function. The affinity of the binding protein for auxin is dependent on pH, with an optimum at pH 5.0, while the binding protein is unstable below pH 6. We discuss here the intracellular localization of the auxin-binding protein from the view point of the controversial pH-dependence of the binding affinity and stability.     

A spectroscopic and conformational study of pertussis toxin

The conformation of native pertussis toxin has been investigated by secondary structure prediction and by circular dichroism, fluorescence and second-derivative ultraviolet absorption spectroscopy. The far-ultraviolet circular dichroic spectrum is characteristic of a protein of high beta-sheet and low alpha-helix content. This is also shown by an analysis of the circular dichroic spectrum with the Contin programme which indicates that the toxin possesses 53% beta-sheet, 10% alpha-helix and 37% beta-turn/loop secondary structure. Second-derivative ultraviolet absorption spectroscopy suggests that 34 tyrosine residues are solvent-exposed and quenching of tryptophan fluorescence emission has shown that 4 tryptophan residues are accessible to iodide ions. One of these tryptophans appears to be in close proximity to a positively charged side-chain, since only 3 tryptophans are accessible to caesium ion fluorescence quenching. When excited at 280 nm, the emission spectrum contains a significant contribution from tyrosine fluorescence, which may be a consequence of the high proportion (55%) of surface-exposed tyrosines. No changes in the circular dichroic spectra of the toxin were found in the presence of the substrate NAD. However, NAD did quench both tyrosine and tryptophan fluorescence emission but did not change the shape of the emission spectrum, or the accessibility of the tryptophans to either the ionic fluorescence quenchers or the neutral quencher acrylamide.     

The subunit characterization of Callinectes sapidus hemocyanin.

Hemocyanin from the blue crab, Callinectes sapidus, sediments at 25.7 S and has a native molecular weight of 940 000 +/- 20 000. Under solution conditions of increased pH (approximately 10) or ionic strength, the native molecule dissociates to a 17 S species. Reversal of this dissociation was unsuccessful. At pH 10 and with the removal of Mg2+, the 17 S species reversibly dissociates to form a subunit species which sediments at 6 S. A comparison of the circular dichroic spectra of the 25.7 S and 6 S hemocyanins suggests that little happens to the structural integrity of the polypeptide backbone upon the two dissociations. Molecular weight estimations under reducing and denaturing conditions indicate that the 6 S hemocyanin species represents the constituent polypeptide chain of the protein molecule. Chemical analysis suggests the presence of a small amount, less than 3%, of carbohydrate bound to the polypeptide chain. Electrophoresis of the hemocyanin in the presence of sodium dodecyl sulfate or urea reveals two major electrophoretic species of either slightly different chemical composition or slightly different polypeptide chain length.     

Effect of pH on oligomeric equilibrium and saccharide-binding properties of peanut agglutinin

The conformation and saccharide-binding properties of peanut agglutinin (PNA) depend on pH as studied by analytical ultracentrifugation, fluorescence, circular dichroism, equilibrium dialysis, and absorption spectroscopy. PNA is tetrameric in neutral solution and dissociates reversibly into dimers below pH 5.1. Below pH 3.4, the lectin is totally dimeric. Lowering of the pH induces reversible changes in the tertiary and secondary structures of PNA. Binding of saturating amounts of lactose to tetrameric (pH 6.9) or dimeric (pH 3.2) PNA resulted in identical ultraviolet difference spectra. Fluorescence studies of PNA as a function of pH in the presence of lactose indicated that tryptophanyl residues, present at or near the saccharide binding site, are more accessible to the ligand in dimeric than in tetrameric PNA. For solutions of dimeric PNA, containing only minor amounts of tetramers (pH 3.6), equilibrium dialysis with MeUmb-beta Gal beta(1----3)GalNac showed that the binding capacity of PNA was the same as for tetrameric PNA (one binding site per protomer) but the apparent association constant was one order of magnitude lower than for tetrameric PNA. The enhancement of MeUmb-beta Gal beta(1----3)GalNac fluorescence upon binding to PNA was pH dependent: 50% at neutrality, 16% at pH 3.7, and unobservable at pH 3.0, suggesting that the microenvironment of this PNA-bound chromophore changed progressively with pH and was dependent on ionization of an acidic amino acid residue.     

Structure and stability of human thyroxine-binding globulin.

The secondary and tertiary structure of human plasma thyroxine-binding globulin (TBG) was investigated by circular dichroism and fluorescence properties. The relaxation time of TBG indicated that it is a compact, symmetric molecule. It was calculated from the far ultraviolet CD spectrum that about one-half of the peptide groups are equally distributed in alpha helical and beta structures. In the near ultraviolet, the CD spectrum of TBG was modified when thyroxine was bound. TBG was stable at temperatures below 50 degrees at pH 9 and below 35 degrees at pH 10.5. Below pH 5 tryptophanyl fluorescence revealed a molecular transition which followed first order kinetics. The transition resulted in an irreversible loss of binding of the hormone. Acidification to pH 3.4 produced only a minor change in the CD spectrum, in which some of the alpha helical peptides were converted to beta structure.     

Purification and properties of the polymeric fatty acid synthetase from a filamentous fungus.

Fatty acid synthetase was purified from the filamentous fungus, Aspergillus fumigatus to a specific activity of 4000--5000 munits/mg protein. Its purity was established by its appearance in electron micrographs, on sodium dodecyl sulphate polyacrylamide gels and by analytical ultracentrifugation, and also by its behaviour upon sucrose gradient centrifugation. This enzyme comprises two large polypeptides with molecular weights of 190 000 and 186 000. Evidence from electron microscopy indicates that it consists of three equivalent loops of protein. It dissociates into different-sized circular subunits on ageing or upon dissolution in buffer of low ionic strength. Differences in properties between this fungal synthetase and that found in yeast have been noted and relate, for example, to inhibition by acetyl CoA and malonyl-CoA, cold-lability and pH optimum. The synthetase from A. fumigatus, purified by different procedures, consistently exists in two forms of similar specific activity, with sedimentation coefficients approx. 40 S and 60 S. Synthetase activity present in crude extracts has been identified as a very heavy component with sedimentation coefficient greater than 100 S.     

Conformational prediction and circular dichroism studies on ribosomal protein S4 from Escherichia coli.

The conformation of ribosomal protein S4 from Escherichia coli has been studied by circular dichroism (CD) and shown to possess unique conformation free in solution. The near ultraviolet spectrum suggests the existence of unique tertiary structural environment for the aromatic amino acid residues. The far ultraviolet spectrum gives an estimation of its secondary structure which is 32% alpha-helix and 14% beta-structure in reconstitution buffer at 25 degrees C. The conformation of S4 has been predicted from its sequence, and two models are presented here. An attempt is made to correlate these two molecular models with the available physicochemical data concerning the shape, conformation, and possible RNA binding site of protein S4.     

Retention of helical structure of pBR322 covalently closed circular duplex DNA at high alkaline pH

Denaturation of covalently closed circular duplex replicative form (RF) I at high pH yields a form with high sedimentation coefficient even after neutralization. This form allowed less ethidium bromide to be intercalated but yielded a circular dichroic spectrum which had reduced magnitude of both positive circular dichroism at 273 nm and negative circular dichroism at 245 nm. The circular dichroic spectrum of this form is similar to that of RFV DNA. Gel electrophoretic analysis of this DNA revealed that, although part is retained in the groove, another part appeared as a faster-moving band, which we designated as RF I_d. This faster-moving form is cleaved by the restriction endonuclease BamHI at a single site giving a single RF III, comigrating with the RF III obtained from RF I by BamHI cleavage. This signifies that the two strands of RF I did not slide over one another during the formation of RF I_d as suggested previously.     

The circular dichroism of synaptosomal membranes: studies of interactions with neuropharmacological agents

The circular dichroic spectrum of synaptosomal membranes was highly reproducible and qualitatively similar to that of erythrocyte membranes. The spectrum exhibited a minimum at 224 nm, a shoulder at 212 nm and a maximum at 195 nm. The mean molar ellipticity at the maximum and minimum was approximately +8000 and –8000 respectively. The protein components were the dominant source of the CD signal. Quantitative estimates showed negligible contributions to the spectrum from cholesterol, phosphatidyl serine and TV-acyl sugars. Phospholine iodide, eserine, decamethonium, tetramethyl ammonium chloride and acetylcholine at concentrations of 10^-3 and 10^-4M did not produce detectable perturbations of the membrane circular dichroism. The circular dichroic spectrum of d-tubocurarine exhibited a maximum at 198 nm and a minimum at 212 nm. Addition of d-tubocurarine to membrane suspensions in the cuvette yielded a complex spectrum representing the result of a simple additive combination of the circular dichroic spectra of d-tubocurarine and the membranes. However, preincubation of the membranes with 3 x 10^-3m d-tubocurarine at 0-4°C for 5-10 min followed by sedimentation, several washes and resuspension resulted in a circular dichroic spectrum which appeared to involve no change in the membrane contribution, but there was a substantial decrease in the molar ellipticity of the d-tubocurarine remaining with the membranes.     

Discrete soluble forms of middle and high molecular weight neurofilament proteins in dilute aqueous buffers

Neurofilament proteins purified from bovine spinal cord were characterized by sedimentation studies in aqueous buffers. In 10 mM Tris, pH 8, the middle molecular weight neurofilament protein (NF-M) has a sedimentation coefficient, S20,w, of 2.6 S. Sedimentation equilibrium data shows considerable nonideality; extrapolation to infinite dilution and correction for the primary charge effect yield a molecular weight of 1.09 X 10(5), indicative of a monomeric structure. When the ionic strength was increased, the sedimentation coefficient increased slightly, and the protein began to form larger aggregates. Reconstitution of short intermediate filaments was observed upon dialysis of denatured NF-M versus a reconstitution buffer. A circular dichroism spectrum of NF-M in 10 mM Tris was typical of alpha + beta proteins. High molecular weight neurofilament protein (NF-H) showed a considerable tendency to aggregate in 10 mM Tris, but a principal species with a sedimentation coefficient of 3.2 S was observed, and sedimentation equilibrium data also suggest a monomeric structure.     

Corynebacterium sarcosine oxidase: a unique enzyme having covalently-bound and noncovalently-bound flavins

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC, 1.5.3.1) was purified to homogeneity from Corynebacterium sp. U-96 by the use of ionic exchange chromatographies and gel filtrations. The enzyme contained two mol FAD per mol enzyme (one covalently-bound and one noncovalently-bound; mol. wt., 174,000). The “semiapoenzyme”, which contains the covalently-bound FAD alone, was prepared by the acid-ammonium sulfate treatment. The semiapoenzyme had practically no activity for sarcosine oxidation, but retained intact back-bone structure judging from the circular dichroic spectrum in the far ultraviolet region. On the contrary, the circular dichroic spectrum of the semiapoenzyme in the visible region (a large negative band around 443 nm) was quite distinct from that of the holoenzyme (positive bands at 387, 456 and 489 nm).     

Circular dichroism and the conformational properties of soybean beta-amylase

The conformational properties of soybean β-amylase were investigated by the circular dichroism probe and measurement of enzyme activity. The enzyme exhibited a positive circular dichroism band at 192 nm, a negative band at 222 nm, and a shoulder near 210 nm. Analysis of the spectrum in the far ultraviolet zone indicated the presence of approximately 30% of α helix and 5–10% of β-pleated sheet, the rest of the polypeptide main chain possessing aperiodic structure. In the near ultraviolet reagion, the enzyme protein showed at least six positive peaks at 259, 265, 273, 281, 292, and 297 nm. The positive bands at 292 and 297 nm remained unaltered on acetylation of the enzyme by N-acetylimidazole and were assigned to tryptophanyl chromophores. These bands were affected in intensity in the presence of maltose or cycloheptaamylose, which indicates that some tryptophan residues are situated at the binding sites. The native conformation of soybean β-amylase was found to be sensitive to pH variation (below pH 5 and above pH 10), sodium dodecyl sulfate, guanidine hydrochloride, and heating to 50–55 °C. Complete disorganization of the secondary structure was attained by 6 m guanidine hydrochloride. Sodium dodecyl sulfate was effective in disturbing the tertiary structure of the enzyme but did not affect significantly the secondary structure. Enzymatic inactivation was paralleled by the decrease of circular dichroism bands in the near ultraviolet region as produced by the denaturants. It is concluded that the uniquely folded structure of the enzyme contains some less rigid domains and a rigid core stabilized by hydrophobic interactions, electrostatic interactions, and hydrogen bonds.     

Spectroscopic studies on proteinase K and subtilisin DY. Relation to X-ray models.

Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin-like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0-11.0 (PMS-proteinase K) and 5.0-10.0 (PMS-subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65 degrees and 48 degrees, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.     

Isolation and Characterization of A Temperature Sensitive Protein from dog Colostrum And Milk

A temperature sensitive protein (one of several) that was soluble at 2°C but reversibly precipitated upon warming to room temperature was isolated from dog milk by precipitation with 50% saturated ammonium sulfate, ion exchange chromatography, centrifugation at 30°C and preparative isoelectric focusing in polyacrylamide gel. The molecular weight by sedimentation equilibrium was 13, 400; however, by thin layer gel filtration it appeared to be much larger. Ultracentrifu-gation studies at conditions near those at which the protein precipitated revealed no evidence of aggregation. The protein had a fairly high content of nonpolar amino acids with proline being in the highest amount. The effects of pH, ionic strength, heat and ethanol on the solubility of the protein were studied. A circular dichroism spectrum of the temperature sensitive protein indicated that the protein is quite unordered. An antiserum against the temperature sensitive protein reacted with each of the other temperature sensitive proteins from milk but not with dog serum or saliva.     

Analysis of the near-ultraviolet absorption and circular dichroic spectra of parsley plastocyanin for the effects of pH and copper center conformation changes

The absorption and circular dichroic (CD) spectra of parsley plastocyanin (PC) were measured in order to determine the effects of changes in primary amino acid sequence on both the copper center and protein components of the PC molecule. The near-ultraviolet (uv) absorption and CD spectra of parsley PC were found to be qualitatively similar to those of spinach, poplar, and lettuce PC, except for the near-uv CD spectrum of the reduced form at low pH (ca. pH 5.0). The CD spectrum of reduced parsley PC in the 250-265 nm wavelength region changes from positive to negative ellipticity upon reduction of pH, and is characterized by a pKa value of 5.7. This pKa value is the same as that for the protonation of the histidine 87 copper ligand, observed by NMR, and the change in conformation of the copper center. Similar processes are believed to occur in the other PC species at lower pH values. Thus, the pH-dependent perturbations of the near-uv CD spectra of reduced PC are interpreted as due to transitions in the reduced copper center. The increase in the near-uv absorption spectrum of reduced PC can be divided into pH-independent and pH-dependent portions. The pH-independent portion resembles the absorption spectrum of tetrahedral Cu(I) metallothionein, suggesting the presence of Cu(I)-Cys 84 and/or Cu(I)-Met 92 charge transfer transitions in the near-uv absorption spectra of reduced PC. The pH dependence of the absorption spectrum changes and the pH difference absorption spectrum indicate that tyrosine residues may contribute to at least a part of the pH-dependent portion of the absorption increase of reduced PC.     

Tetanus toxin conformation

Summary The circular dichroic spectrum of highly purified tetanus toxin has been determined between 200–310 nm. A comparison of the ellipticity between 207–243 nm and of the rotational strengths of the major resolved bands between 200–250 nm with the corresponding values from proteins of known conformation indicates that tetanus toxin contains about 20% -helix and 23% -structure. Above 250 nm the resolved spectrum showed contributions from tryptophanyl, tyrosyl, and phenylalanyl groups. The rotational strengths of the major near ultraviolet circular dichroic bands were significantly higher in the toxin than in low molecular weight peptides containing aromatic residues. This indicates that tetanus toxin has a stable tertiary structure.Andrew W. Mellon Foundation Teacher-Scholar Awardee.Camille and Henry Dreyfus Foundation Teacher-Scholar Awardee.     

Rabbit cardiac myosin. I. Physical and chemical characterization of the native molecule.

Rabbit cardiac myosin, isolated from frozen tissue, was effectively purified by batchwise treatment with DEAE-cellulose in addition to suing cilution-precipitation techniques. An extensive experimental program was subsequently carried out with respect to the enzymic amino acid, optical and physicochemical properties of native cardiac myosin. This program has included the following: examination of the effects of pH and varying concentrations of ATP, CaCl2, MgCl2, and PCMB on its ATPase activity; measurement of its circular dichroic spectrum in solvent buffers, at different pH or containing ATP in the absence or presence of Ca-2+ or Mg-2+ ions; study of the concentration dependence of its viscosity and sedimentation velocity at low temperatures; and investigation of its molecular weight by the Archibald method and low- and high-speed sedimentation equilibrium. The results of these studies were consistent with the interpretation that cardiac myosin is comprised of highly asymmetric, semi-rigid molecules with a molecular weight in the order of 4.7 times 10-5, which display non-ideality even in solvent buffers of high ionic strength at neurtal pH. In addition, computer analysis of the high-speed sedimentation equilibrium data has provided evidence for the presence of a self-association reaction at low protein concentration. Even though the specif ATPase activity of cardiac myosin was found to be approximately one-third that reported for skeletal myosin in all cases, it was concluded, on the the basis of the essentially analogous physical and chemical properties of rabbit cardiac and skeletal myosin, that the two proteins are very similar in terms of molecular size, shape, and secondary structure.