Effects of pH and urea on the conformational properties of subtilisin DY

Subtilisin DY is very resistant to the denaturing action of urea: the conformational properties are not affected up to 4.5 M-urea, and even in the presence of 8 M-urea there is only a slow loss of ordered structure and caseinolytic activity. C.d. and fluorescence-emission studies also show that this proteinase is stable in the 5.5-10.0 pH range, whereas below pH 5.5 a sharp denaturation occurs that is complete at pH 4.5. Protein denaturation leads to a change of the emission quantum yield; in particular, in the native protein, indole fluorescence is quenched by some amino groups. Moreover, subtilisin DY possesses two classes of tyrosine residues: one class of exposed residues titrates normally, with pKapp. = 10.24, whereas one class of partially buried or hydrogen-bonded residues ionizes with pKapp. = 11.58. In general, such conformational properties resemble those of other subtilisins. However, some differences occur: e.g., subtilisin DY is less stable at acidic pH values and its tyrosine residues are more accessible to the solvent. Such differences are probably due to small variations of the three-dimensional structure; e.g., subtilisin DY has a slightly lower alpha-helix content.     

Topography of all tyrosine residues in subtilisin DY

The extracellular alkaline proteinase subtilisin DY was nitrated with increasing amounts of tetranitromethane. At 2-fold molar excess of the reagent with respect to the tyrosine residues in the enzyme, when 1.3 residues were modified, a peak of the caseinolytic activity (13% increase) was observed. Evidence is provided that the diminishing of the pK of the phenolic hydroxyl group in Tyr(3NO2)104 causes this phenomenon. The products obtained after nitration of the enzyme with 5-fold and 200-fold molar excess of tetranitromethane were cleaved by trypsin and cyanogen bromide and the peptides obtained were studied by analysis with respect to the tyrosine and 3-nitrotyrosine residues. Their degree of substitution was established. Tyrosine-104 was the first modified residue, then follow the residues with numbers 57, 143, 206, 262 and somewhat later 21, 209, 263, all fully modified by 200-fold molar excess of the reagent. Partial modification was observed at numbers 91, 167, 214, 238 and no modification at numbers 6 and 171. It has been established that the nonmodified residues are buried inside the molecule and the partially modified residues are screened by the side chains of lysine, valine, leucine, and tryptophan as seen on a working video three-dimensional model of subtilisin Carlsberg. The approach for characterization of tyrosyl groups in proteins based on peptide sequencing and HPLC quantitation of the phenylthiohydantoin derivatives of tyrosine and 3-nitrotyrosine was further developed with respect to the quantitation of the HPLC-separated peptides using fragments of the protein studied.     

Reductive alkylation of lysine residues in subtilisin DY

Only lysine epsilon-amino groups (and the N-terminal alpha-amino group) in native subtilisin DY were reductively alkylated by glyceraldehyde in the presence of sodium cyanoborohydride. The modified protein molecule was cleaved by TosPheCH2Cl-trypsin or cyanogen bromide and the two sets of peptides obtained were fractionated and purified by gel filtration and HPLC. For determination of the degree of modification of each lysine residue, selected peptides were subjected to sequence analysis combined with quantitative estimation of the containing PTH-Lys and PTH-epsilon-DHP-Lys. The data obtained showed that the lysine residues in positions 12, 15, 27, 43, 136, 141, 265 were entirely modified, those in positions 170, 184, 237 were partially modified, and Lys22 and Lys94 were unaccessible for the reagent. The caseinolytic activity decreased by 23% when the maximum number of lysine residues (8.6 of the total 12 residues) in subtilisin DY were modified. The CD-spectra of native and modified enzyme showed only slight differences. Both these experiments suggest that the lysine residues do not take part directly in the catalytic reaction but are responsible for maintaining the native three-dimensional enzyme structure. The data obtained for the accessibility of the different lysine residues in subtilisin DY correlated very well with the positions of these residues in a video model of the structure of subtilisin Carlsberg, thus suggesting that the spatial structures of these two enzymes are very similar.     

Role of surfactant on the proteolysis of aqueous bovine serum albumin

Nonionic and ionic surfactants diminish the initial rate of proteolysis of aqueous bovine serum albumin (BSA) by subtilisin Carlsberg. Surfactants studied include: nonionic tetraethylene glycol monododecyl ether (C12E4); anionic sodium dodecyl sulfate (SDS), anionic sodium dodecylbenzenesulfonate (SDBS), and cationic dodecyltrimethylamonium bromide (DTAB). Kinetic data are obtained using fluorescence emission. Special attention is given to enzyme kinetic specificity determined by fitting initial-rate data to the Michaelis-Menten model. All surfactants reduce the rate of proteolysis, most strongly at concentrations near and above the critical micelle concentration (CMC). Circular dichroism (CD), tryptophan/tyrosine fluorescence spectra, and tryptophan fluorescence thermograms indicate that BSA partially unfolds at ionic surfactant concentrations near and above the CMC. Changes in BSA conformation are less apparent at ionic surfactant concentrations below the CMC and for the nonionic surfactant C12E4. Subtilisin Carlsberg activity against the polypeptide, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, decreased due to enzyme-surfactant interaction. At the concentrations and time frames studied, there was no enzyme autolysis. Importantly, aqueous proteolysis rates are significantly reduced at high surfactant concentrations where protein-micellar-surfactant aggregates occur. To explain the negative effect of surfactant on subtilisin Carlsberg proteolytic activity against BSA, we propose that micelle/protein complexes hinder enzyme access.     

A study of subtilisin types Novo and Carlsberg by circular polarization of fluorescence.

The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.     

Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus

The complete amino-acid sequence of subtilisin DY, an extracellular alkaline proteinase produced by Bacillus subtilis strain DY was determined. This included automated sequence analysis of the whole molecule and its large fragments such as tryptic peptides obtained from the inactivated enzyme, peptides generated by cyanogen bromide, by o-iodosobenzoic acid and by hydroxylamine. The peptides were isolated by gel filtration and by reversed-phase high performance liquid chromatography. The amino-acid sequence of subtilisin DY was determined by overlapping the isolated peptides. It consists of 274 amino-acid residues, like that of subtilisin Carlsberg. By comparison with the structures of the subtilisins Carlsberg, amylosacchariticus and BPN' 32, 80 and 82 amino-acid substitutions were found, which are caused by 37, 102 and 106 nucleotide mutations, respectively. It was found also that 62.5% of the amino-acid residues in the molecules of these four subtilisins are identical with respect to kind and position of the residue, which suggests that these molecules have had a common ancestral precursor. The amino-acid replacement analysis of the four subtilisins leads to the conclusion that they have evolved almost independently.     

Intramolecular distances between tryptophan residues and the active-site serine residue in alkaline bacterial proteinases as measured by fluorescence energy-transfer studies.

Singlet-singlet energy transfer from the tryptophan residues to an active-site-serine-bound 5-dimethylaminonaphthalene-1-sulphonyl group was investigated in four subtilisins. The transfer distances for subtilisin Novo and mesentericopeptidase are 1.93 +/- 0.20 nm (19.3 +/- 2.0 A) and 1.81 +/- 0.20 nm (18.1 +/- 2.0 A) respectively. The positions of the indole groups in the three-dimensional structures of the two pairs of proteinases, namely subtilisin Novo and mesentericopeptidase on the one hand and subtilisins Carlsberg and DY on the other, are essentially identical.     

THE INTRINSIC FLUORESCENCE CHARACTERISTICS OF THE MYELIN BASIC PROTEIN

—The encephalitogenic basic protein has been isolated from the myelin sheath of ox brain white matter and the purity and amino acid composition have been verified. The intrinsic fluorescence characteristics of the purified basic protein have been determined and the results interpreted in terms of current ideas on the structure of the protein. Fluorescence data obtained from the basic protein in aqueous solution indicate that the tyrosine and tryptophan residues are largely exposed to the solvent and that resonance energy transfer from tyrosine to tryptophan is very inefficient. Denaturing conditions in 8 m -urea have little effect on the fluorescence properties of the protein. The ionic detergent, sodium dodecyl sulphate, interacts with the basic protein and alters the fluorescence properties in a manner which indicates that the tryptophan residue is in the hydrocarbon chain region of the detergent while the local positive charge around the tyrosine residues is neutralized by the negatively charged sulphate head-groups. The fluorescence results suggest that the basic protein can be used as a natural, non-perturbing probe which will report on its environment after it has reacted with other membrane components.     

Fluorescence of human liver alanine aminopeptidase.

Fluorescence of human liver alanine aminopeptidase has been attributed to tryptophan fluorescence. The fluorescence maximum is at 330 nm, 20 nm lower than that for free tryptophan, suggesting that most of the enzyme tryptophans are in a nonpolar environment and are shielded from solvent. Quenching of enzyme fluorescence by iodide, pyridine, and N-methyl nicotinamide also demonstrates that enzyme tryptophan residues are largely buried and inaccessible to solvent. Those accessible are in negatively charged environments. 8-(1'-dimethylaminonaphthalene-5'-sulfonylamido-octanoic acid (8-DNS-octanoic acid) and epsilon-DNS-L-Lys inhibit aminopeptidase. One molecule of inhibitor when bound to the enzyme quenched 57% and 63% of enzyme fluorescence, respectively. Such efficient quenching may indicate a degree of segregation of tryptophan toward the active center.     

Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana

The location of tryptophan residues in the actin macromolecule was studied on the basis of the known 3D structure. For every tryptophan residue the polarity and packing density of their microenvironments were evaluated. To estimate the accessibility of the tryptophan residues to the solvent molecules it was proposed to analyze the radial dependence of the packing density of atoms in the macromolecule about the geometric center of the indole rings of the tryptophan residues. The proposed analysis revealed that the microenvironment of tryptophan residues Trp-340 and Trp-356 has a very high density. So these residues can be regarded as internal and inaccessible to solvent molecules. Their microenvironment is mainly formed by non-polar groups of protein. Though the packing density of the Trp-86 microenvironment is lower, this tryptophan residue is apparently also inaccessible to solvent molecules, as it is located in the inner region of macromolecule. Tryptophan residue Trp-79 is external and accessible to the solvent. All residues that can affect tryptophan fluorescence were revealed. It was found that in the close vicinity of tryptophan residues Trp-79 and Trp-86 there are a number of sulfur atoms of cysteine and methionine residues that are known to be effective quenchers of tryptophan fluorescence. The most essential is the location of SG atom of Cys-10 near the NE1 atom of the indole ring of tryptophan residue Trp-86. On the basis of microenvironment analysis of these tryptophan residues and the evaluation of energy transfer between them it was concluded that the contribution of tryptophan residues Trp-79 and Trp-86 must be low. Intrinsic fluorescence of actin must be mainly determined by two other tryptophan residues--Trp-340 and Trp-356. It is possible that the unstrained conformation of tryptophan residue Trp-340 and the existence of aromatic rings of tyrosine and phenylalanine and proline residues in the microenvironments of tryptophan residues Trp-340 and Trp-356 are also essential to their blue fluorescence spectrum.     

The time resolved fluorescence and anisotropy of subtilisins BPN' and Carlsberg

Time-resolved emission and anisotropy have been measured for the tryptophan (Trp) residues of two closely related subtilisin proteins. The single Trp of subtilisin Carlsberg shows complex lifetime properties, and anisotropy consistent with a fast (ca. 200 ps) segmental motion, on the "wobbling in a cone model" the semi angle is in the range 38 to 47 degrees. The lifetime and anisotropy properties for this single Trp residue suggest that the predominant state is that of an effectively non-emitting statically quenched fluorophore. This fast component is also resolved in the anisotropy of subtilisin BPN' but with relatively low amplitude, due to the dominant emission of the other Trp residues. The diversity of the photophysical properties is not readily correlated with the structure of the proteins, though the observed complexity is consistent with the likely heterogeneity of environment due to the surface location of all the Trp residues.     

Investigation of the membrane-active peptides melittin and glucagon by photochemically induced dynamic-nuclear-polarization (photo-CIDNP) NMR

The photochemically induced dynamic-nuclear-polarization (photo-CIDNP) NMR technique was used to investigate the membrane-active peptides melittin and glucagon. The experiments were performed both in the absence and presence of phospholipid vesicles in order to study the topography of the membrane-bound state. From the results it can be concluded that the melittin peptide chain is oriented in such a way that the single tryptophan residue (Trp19) reaches into the membrane. In the case of glucagon, a binding interaction with vesicle membranes is indicated within the pH range 2-10, whereby the single tryptophan residue (Trp25) is buried in the lipid bilayer and the tyrosine and histidine residues are exposed to the aqueous solvent.     

Photo-CIDNP studies of the influence of ligand binding on the surface accessibility of aromatic residues in dihydrofolate reductase

The surface accessibility of the histidine, tyrosine, and tryptophan residues of Lactobacillus casei dihydrofolate reductase has been determined from 360-MHz 1H photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR experiments. In the absence of ligands, four (or perhaps five) of the seven histidine residues and at least one of the four tryptophan residues are accessible to a flavin dye molecule. One of the five tyrosine residues is also slightly accessible. Of the accessible histidine residues, one becomes inaccessible on the binding of NADP+ and one on the binding of p-aminobenzoyl glutamate. These have been assigned to residues which interact directly with these two ligands. One histidine residue (probably His-22) shows an increase in accessibility on addition of folate or methotrexate to the enzyme . NADP+ complex. In addition, the binding of several ligands, notably trimethoprim, leads to an increase in the accessibility of a tryptophan residue. This is clear evidence for ligand-induced conformational changes in dihydrofolate reductase and allows us to identify some of the residues involved.     

States of tryptophan residues in castor bean hemagglutinin: the perturbing effects of solvents on tryptophans in hemagglutinin and its complexes as analyzed by UV-difference spectroscopy

The states of tryptophan residues in castor bean hemagglutinin (CBH) were analyzed by solvent perturbation studies employing ultraviolet difference spectroscopy. Eight out of 22 tryptophan residues in CBH were exposed to ethylene glycol and glycerol, suggesting that the remaining 14 tryptophan residues are buried in the interior of the CBH molecule. The fraction of tryptophan residues accessible to the perturbant decreased with increase in the molecular size of the perturbant, and only 2 tryptophan residues were exposed to polyethylene glycol 600. Upon binding with raffinose, 2 tryptophan residues were shielded from the perturbing effect of the solvent, and binding of lactose reduced the number of tryptophan residues accessible to the perturbant by 1 mol per mol of protein. Binding of galactose, however, did not change the accessibility of tryptophan to the perturbant. On the other hand, the accessibility of tyrosine to the perturbant remained unchanged after binding with raffinose and lactose, suggesting that tyrosine is not directly involved in the saccharide binding of CBH. Based on these results, it is proposed that one tryptophan residue at the saccharide-binding site on each B-chain of CBH lies on the surface of the protein molecule and is located at a subsite which is accessible to a glucopyranoside moiety in the lactose molecule or a glycopyranosyl-fructofuranosyl moiety in the raffinose molecule, whereas such a residue is not present at the galactopyranoside-recognition site.     

Subtilisin 72: a serine protease from Bac. subtilis strain 72 - an enzyme similar to subtilisin Carlsberg]

Subtilisin 72, a serine proteinase secreted by Bac. subtilis strain 72 was purified by covalent chromatography on Sepharose sorbent containing p-(omega-aminomethyl)phenylboronic acid as a ligand. The homogeneity of subtilisin 72 was confirmed by isoelectrofocusing in a thin layer of polyacrylamide gel (pl 8.6). The amino acid composition of this enzyme is different from that of other subtilisins, e. g. subtilisin Carlsberg. The N = terminal amino acid sequence of subtilisin 72 traced up to the 35th residue turned to be the same as that of subtilisin Carlsberg with the exception of the 21st (Tyr) and the 30th (Ile) residues. This very pronounced extent of homology shows that subtilisin 72 is very similar although not identical to subtilisin Carlsberg.     

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.     

An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.     

A spectrophotometric assay for the characterization of the S'' subsite specificity of alpha-chymotrypsin

Pig and horse colipases contain three tyrosine residues. In addition, horse colipase possesses a tryptophan residue. Some of the tyrosine residues are involved in the association of colipase and a bile salt micelle. The present report demonstrates that the aromatic residues responsible for colipase fluorescence are in an aqueous environment. In the presence of bile salt micelles, changes in colipase fluorescence properties indicate that the intrinsic fluorophores are located in a more hydrophobic environment upon colipase-micelle complex formation. In addition, the fluorescence of an NBD group fixed on lysine 60, which is very close to the aromatic region in the pig colipase, is also altered in the presence of micelles. These results show that the micelle binding site is not limited to the tyrosine residues but may be broadened to adjacent residues such as lysine 60 and also tryptophan 52 in horse colipase.     

Active-site titration of serine proteases in organic solvents

Calculation of kinetic constants of an enzymatic reaction in organic solvents requires knowledge of the functional active-site concentration in organic solvents, and this can be significantly different than that in water. An experimental method for active-site titration of serine proteases in organic media has been developed based on the kinetics of inhibition by phenylmethanesulfonyl fluoride (PMSF), a serine-specific inhibitor (or suicide substrate). This kinetic approach is fundamentally different from other techniques that require complete titration of all accessible enzyme active sites. This active site titration method was applied to subtilisins BPN' and Carlsberg and alpha-chymotrypsin and resulted in fractions of active sites that ranged from 8 to 62% (of the fraction active in water) depending on the enzyme, the method of enzyme preparation, and the organic solvent used. The active-site concentration of subtilisin BPN' and Carlsberg increased with increasing hydrophobicity of the solvent and with increasing solvent hydration in tetrahydrofuran. The dependence of the fraction of active sites on the nature of the organic solvent appears to be governed largely by solvent-induced inactivation caused by direct interaction of a hydrophilic solvent with the enzyme. (c) 1996 John Wiley & Sons, Inc.     

Methionine residue accessibility in native subtilisin DY

The three methionine residues of subtilisin DY were specifically modified into methionine sulfoxide using increasing amounts of chloramine T. By means of subsequent treatment with cyanogen bromide, gel chromatography, Edman degradation of the obtained peptides and the known structure of subtilisin DY it was established that Met222 is exposed to the surrounding solution, Met124 is partially exposed and Met199 is buried. The data obtained were confirmed on a computer graphics space movable model of subtilisin Carlsberg where Met222 was seen to be on the surface of the molecule and Met199 shielded by Tyr262, Ala179 and Leu196. Upon oxidation of Met222 of subtilisin DY by chloramine T, 25% of its caseinolytic activity was lost. This can be explained by the immediate adjacency to the active-site Ser221. An additional 5% loss of activity was observed at each subsequent methionine modification.