Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 2. 1H NMR studies

Structural transitions of the protein Streptomyces subtilisin inhibitor (SSI) from the native state to the cold-denatured and heat-denatured states were studied by 1H NMR spectroscopy in the temperature range from -10 to 60 degrees C in the acidic pH range. Assignments of some of the 1H NMR signals of SSI in the cold-denatured and heat-denatured states were performed by a combined use of selective deuteration and site-directed mutagenesis. Throughout the pH range from 2.1 to 3.1, both transitions were cooperative and basically only three distinct spectra corresponding to structures in the cold-denatured, native, and heat-denatured states were detected. In the cold-denatured state, the side-chain signals of Met73, His106, at least one Val, and two Leu were observed at distinctly shifted positions from those for a random-coiled structure, suggesting the formation of a tertiary structure, while those of Met70, His43, and Ala2 were observed at positions for a random-coiled structure. This tertiary structure in the cold-denatured state is entirely different from that in the native state, as some amino acid residues exposed to the solvent in the native state (e.g., Met73, His106) are buried while those sequestered in the native state (e.g., His43) are exposed. In the heat-denatured state, however, most 1H NMR signals were observed at random-coiled positions, indicating that there is much less tertiary structure in the heat-denatured state than in the cold-denatured state. At pH values below 2.09, a structural transition was observed from the cold-denatured state to the heat-denatured state without passing through the native state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonessential role for methionines in the productive association between calmodulin and the plasma membrane Ca-ATPase.

To investigate the role of hydrophobic interactions involving methionine side chains in facilitating the productive association between calmodulin (CaM) and the plasma membrane (PM) Ca-ATPase, we have substituted the polar amino acid Gln for Met at multiple positions in both the amino- and carboxyl-terminal domains of CaM. Conformationally sensitive fluorescence signals indicate that these mutations have little effect on the backbone fold of the carboxyl-terminal domain of CaM. The insertion of multiple Gln in either globular domain results in a decrease in the apparent affinity of CaM for the PM-Ca-ATPase. However, despite the multiple substitution of Gln for four methionines at positions 36, 51, 71, and 72 in the amino-terminal domain or for three methionines at positions 124, 144, and 145 in the carboxyl-terminal domain, these mutant CaMs are able to fully activate the PM-Ca-ATPase. Thus, although these CaM mutants have a decreased affinity for the CaM-binding site on the Ca-ATPase, they retain the ability to fully activate the Ca-ATPase at saturating concentrations of CaM. The role of individual methionines in modulating the affinity between the carboxyl terminus and the PM-Ca-ATPase was further investigated through the substitution of individual Met with Gln. Upon substitution of Met(124) and Met(144) with Gln, there is a 5- and 10-fold increase in the amount of CaM necessary to obtain half-maximal activation of the PM-Ca-ATPase, indicating that these methionine side chains participate in the high-affinity association between CaM and the PM-Ca-ATPase. However, substitution of Gln for Met(145) results in no change in the apparent affinity between CaM and the PM-Ca-ATPase, indicating that in contrast to all other known CaM targets, Met(145) does not participate in the interaction between CaM and the PM-Ca-ATPase. These results emphasize differences in the binding interactions between individual methionines in CaM and different target enzymes, and suggest that hydrophobic interactions between methionines in CaM and the binding site on the PM-Ca-ATPase are not necessary for enzyme activation. Calculation of the binding affinities of individual CaM domains associated with activation of the PM-Ca-ATPase suggests that mutations of methionines located in either domain of CaM can decrease the initial high-affinity association between CaM and the PM-Ca-ATPase, but have little effect upon the subsequent binding of the opposing globular domain. These results suggest that the initial associations between CaM and the CaM-binding sequence in the PM-Ca-ATPase are guided by nonspecific hydrophobic interactions involving both domains of CaM.     

The interaction of a tyrosyl residue and carboxyl groups in the specific interaction between Streptomyces subtilisin inhibitor and subtilisin BPN'. A chemical modification study.

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.     

Effect of inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI

The protein Streptomyces subtilisin inhibitor, SSI, efficiently inhibits a bacterial serine protease, subtilisin BPN'. We recently demonstrated that functional change in SSI was possible simply by replacing the amino acid residue at the reactive P1 site (methionine 73) of SSI. The present paper reports the additional effect of replacing methionine 70 at the P4 site of SSI (Lys73) on inhibitory activity toward two types of serine proteases, trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversion of methionine 70 at the P4 site of SSI(Lys73) to glycine or alanine resulted in increased inhibitory activity toward trypsin and lysyl endopeptidase, while replacement with phenylalanine weakened the inhibitory activity toward trypsin. This suggests that steric hindrance at the P4 site of SSI(Lys73) is an obstacle for its binding with trypsin. In contrast, the same P4 replacements had hardly any effect on inhibitory activity toward subtilisin BPN'. Thus the subsite structure of subtilisin BPN' is tolerant to these replacements. This contrast in the effect of P4 substitution might be due to the differences in the S4 subsite structures between the trypsin-like and the subtilisin-like proteases. These findings demonstrate the importance of considering structural complementarity, not only at the main reactive site but also at subsites of a protease, when designing stronger inhibitors.     

Inhibition of subtilisin BPN' by reaction site P1 mutants of Streptomyces subtilisin inhibitor

It has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the P1 site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site P1 (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the P1 site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or a beta-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (Ki) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/Ki) of mutated SSIs and log(1/Km) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydration-coupled dynamics in proteins studied by neutron scattering and NMR: the case of the typical EF-hand calcium-binding parvalbumin

The influence of hydration on the internal dynamics of a typical EF-hand calciprotein, parvalbumin, was investigated by incoherent quasi-elastic neutron scattering (IQNS) and solid-state 13C-NMR spectroscopy using the powdered protein at different hydration levels. Both approaches establish an increase in protein dynamics upon progressive hydration above a threshold that only corresponds to partial coverage of the protein surface by the water molecules. Selective motions are apparent by NMR in the 10-ns time scale at the level of the polar lysyl side chains (externally located), as well as of more internally located side chains (from Ala and Ile), whereas IQNS monitors diffusive motions of hydrogen atoms in the protein at time scales up to 20 ps. Hydration-induced dynamics at the level of the abundant lysyl residues mainly involve the ammonium extremity of the side chain, as shown by NMR. The combined results suggest that peripheral water-protein interactions influence the protein dynamics in a global manner. There is a progressive induction of mobility at increasing hydration from the periphery toward the protein interior. This study gives a microscopic view of the structural and dynamic events following the hydration of a globular protein.     

H n.m.r. spectrum from the flexible N-terminal segment of Streptomyces subtilisin inhibitor

The 1H n.m.r. spectrum of Streptomyces subtilisin inhibitor shows a limited number of unusually sharp signals at room temperature. Some of these signals are assigned uniquely to protons of the side chains of the N-terminal segment, Aspl-Ala2-Pro3-Ser4-Ala5-Leu6-Tyr7-based on experiments of spin decoupling, pH titration, and enzymatic cleavage of the protein. Quantitative examination of these signals indicates that the N-terminal end of this protein is heterogeneous in that the protein contains a considerable fraction whose sequence starts with Ala2 rather than with Asp1. The pKa values for the amino groups of Asp1 and Ala2 exposed at the N-terminus are determined to be 8.9 +/- 0.4 and 9.0 +/- 0.1, respectively. Furthermore, examination of the line-widths of the methyl proton resonances of Ala2 and Ala5 residues indicates that the N-terminal peptide segment is free and undergoes rapid segmental motions in the order of 10(-9) s.     

Surface exposure of the methionine side chains of calmodulin in solution. A nitroxide spin label and two-dimensional NMR study

Binding of calcium to calmodulin (CaM) causes a conformational change in this ubiquitous calcium regulatory protein that allows the activation of many target proteins. Met residues make up a large portion of its hydrophobic target binding surfaces. In this work, we have studied the surface exposure of the Met residues in the apo- and calcium-bound states of CaM in solution. Complexes of calcium-CaM with synthetic peptides derived from the CaM-binding domains of myosin light chain kinase, constitutive nitric-oxide synthase, and CaM-dependent protein kinase I were also studied. The surface exposure was measured by NMR by studying the effects of the soluble nitroxide spin label, 4-hydroxyl-2,2,6, 6-tetramethylpiperidinyl-1-oxy, on the line widths and relaxation rates of the Met methyl resonances in samples of biosynthetically 13C-methyl-Met-labeled CaM. The Met residues move from an almost completely buried state in apo-CaM to an essentially fully exposed state in Ca2+4-CaM. Binding of two Ca2+ to the C-terminal lobe of CaM causes full exposure of the C-terminal Met residues and a partial exposure of the N-terminal Met side chains. Binding of the three target peptides blocks the access of the nitroxide surface probe to nearly all Met residues, although the mode of binding is distinct for the three peptides studied. These data show that calcium binding to CaM controls the surface exposure of the Met residues, thereby providing the switch for target protein binding.     

Dynamics of fd coat protein in the bacteriophage

The dynamics of the coat protein in fd bacteriophage are described with solid-state 15N and 2H NMR experiments. The virus particles and the coat protein subunits are immobile on the time scales of the 15N chemical shift anisotropy (10(3) Hz) and 2H quadrupole (10(6) Hz) interactions. Previously we have shown that the Trp-26 side chain is immobile, that the two Tyr and three Phe side chains undergo only rapid twofold jump motions about their C beta-C gamma bond axis [Gall, C. M., Cross, T. A., DiVerdi, J. A., & Opella, S. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 101-105], and that most of the backbone peptide linkages are highly constrained but do undergo rapid small amplitude motions [Cross, T. A., & Opella, S. J. (1982) J. Mol. Biol. 159, 543-549] in the coat protein subunits in the virus particles. In this paper, we demonstrate that the four N-terminal residues of the coat protein subunits are highly mobile, since both backbone and side-chain sites of these residues undergo large amplitude motions that are rapid on the time scales of the solid-state NMR experiments. In addition, the dynamics of the methyl-containing aliphatic residues Ala, Leu, Val, Thr, and Met are analyzed. Large amplitude jump motions are observed in nearly all of these side chains even though, with the exception of the N-terminal residue Ala-1, their backbone peptide linkages are highly constrained. The established information about the dynamics of the structural form of fd coat protein in the virus particle is summarized qualitatively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydration dependence of backbone and side chain polylysine dynamics: a 13C solid-state NMR and IR spectroscopy study

The molecular dynamics of solid poly-L-lysine has been studied by the following natural abundance (13)C-NMR relaxation methods: measurements of the relaxation times T(1) at two resonance frequencies, off-resonance T(1rho) at two spin-lock frequencies, and proton-decoupled T(1rho). Experiments were performed at different temperatures and hydration levels (up to 17% H(2)O by weight). The natural abundance (13)C-CPMAS spectrum of polylysine provides spectral resolution of all types of backbone and side chain carbons and thus, dynamic parameters could be determined separately for each of them. At the same time, the conformational properties of polylysine were investigated by Fourier transform infrared spectroscopy. The data obtained from the different NMR experiments were simultaneously analyzed using the correlation function formalism and model-free approach. The results indicate that in dry polylysine both backbone and side chains take part in two low amplitude motions with correlation times of the order of 10(-4) s and 10(-9) s. Upon hydration, the dynamic parameters of the backbone remain almost constant except for the amplitude of the slower process that increases moderately. The side chain dynamics reveals a much stronger hydration response: the amplitudes of both slow and fast motions increase significantly and the correlation time of the slow motion shortens by about five orders of magnitude, and at hydration levels of more than 10% H(2)O fast and slow side chain motions are experimentally indistinguishable. These changes in the molecular dynamics cannot be ascribed to any hydration-dependent conformational transitions of polylysine because IR spectra reveal almost no hydration dependence in either backbone or side chain absorption domains. The physical nature of the fast and slow motions, their correlation time distributions, and hydration dependence of microdynamic parameters are discussed.     

Enhancing oxidative resistance of Agrobacterium radiobacter N-carbamoyl D-amino acid amidohydrolase by engineering solvent-accessible methionine residues

N-Carbamoyl D-amino acid amidohydrolase (D-NCAase) that catalyzes the stereospecific hydrolysis of N-carbamoyl D-amino acids to their corresponding D-amino acids is valuable in pharmaceutical industry. Agrobacterium radiobacter D-NCAase is sensitive to oxidative damage by hydrogen peroxide. To investigate the role of methionine residues in oxidative inactivation, each of the nine methionine residues in A. radiobacter D-NCAase was substituted with leucine, respectively, by site-directed mutagenesis. Except for two mutants (Met5Leu and Met31Leu) with similar activities, seven mutants (Met73Leu, Met167Leu/Met169Leu, Met184Leu, Met220Leu, Met239Leu, Met244Leu, and Met239Leu/Met244Leu) were found to have reduced activities. In the presence of H(2)O(2), three mutants (Met239Leu, Met244Leu, and Met239Leu/Met244Leu) with substitution of highly solvent-accessible methionines by leucines retained their activities. The other mutants were also considerably resistant to chemical oxidation than was the wild-type enzyme. Thus, substitution of solvent-accessible methionine residues with leucine to enhance oxidative stability of D-NCAase is practical but might be with compromised activity.     

Dynamics of methyl groups in proteins as studied by proton-detected 13C NMR spectroscopy. Application to the leucine residues of staphylococcal nuclease.

This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having Mr = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-13C2]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the 13C T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau f (5-80 ps) and tau s (less than 2 ns). The fast motion is identified with the rapid rotation of the methyl group about the C gamma-C delta bond axis, while the slow motion is associated with reorientation of the C gamma-C delta bond axis itself. The mean squared order parameters associated with the latter motion, Ss2, lie in the range 0.34-0.92. The values of Ss2 correlate reasonably well with the temperature factors of the leucine methyl carbons obtained from the crystal structures, but some are smaller than anticipated on the basis of the fact that nearly all leucine methyl carbons are buried and have temperature factors no larger than that of the leucine backbone atoms. Five leucine residues in liganded SNase and eight in unliganded SNase have values of Ss2 less than 0.71. These order parameters correspond to large amplitude motions (angular excursions of 27-67 degrees) of the C gamma-C delta bond axis. These results indicate that, in solution, the internal motions of the leucine side chains of SNase are significantly larger than suggested by the X-ray structures or by qualitative analysis of NOESY spectra. Comparison of Ss2 values obtained from liganded and unliganded SNase reveals a strong correlation between delta Ss2 and distance between the leucine methyl carbon and the ligands.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel double-headed proteinaceous inhibitor for metalloproteinase and serine proteinase

A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI (Streptomyces caespitosus neutral proteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K(i) value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN' (K(i) = 1.4 nm) (EC ). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M(r) = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN' was identified to be Met(71)-Tyr(72) bond by specific cleavage. To identify the reactive site for ScNP, Tyr(33) and Tyr(72), which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN' but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN' was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN'.     

Activation of constitutive nitric oxide synthases by oxidized calmodulin mutants

Several calmodulin (CaM) mutants were engineered in an effort to identify the functional implications of the oxidation of individual methionines in CaM on the activity of the constitutive isoforms of nitric oxide synthase (NOS). Site-directed mutagenesis was used to substitute the majority of methionines with leucines. Substitution of all nine methionine residues in CaM with leucines had minimal effects on the binding affinity or maximal enzyme activation for either the neuronal (nNOS) or endothelial (eNOS) isoform. Selective substitution permitted determination of the functional consequences of the site-specific oxidation of Met(144) and Met(145) on the regulation of electron transfer within nNOS and eNOS. Site-specific oxidation of Met(144) and Met(145) resulted in changes in the CaM concentration necessary for half-maximal activation of nNOS and eNOS, suggesting that these side chains are involved in stabilizing the productive association between CaM and NOS. However, the site-specific oxidation of Met(144) and Met(145) had essentially no effect on the maximal extent of eNOS activation in the presence of saturating concentrations of CaM. In contrast, the site-specific oxidation of Met(144) (but not Met(145)) resulted in a reduction in the level of nNOS activation that was associated with decreased rates of electron transfer within the reductase domain. Thus, nNOS and eNOS exhibit different functional sensitivities to conditions of oxidative stress that are expected to oxidize CaM. This may underlie some aspects of the observed differences in the sensitivities of proteins in vasculature and neuronal tissues to nitration that are linked to NOS activation and the associated generation of peroxynitrite.     

From Powder to Solution: Hydration Dependence of Human Hemoglobin Dynamics Correlated to Body Temperature

A transition in hemoglobin (Hb), involving partial unfolding and aggregation, has been shown previously by various biophysical methods. The correlation between the transition temperature and body temperature for Hb from different species, suggested that it might be significant for biological function. To focus on such biologically relevant human Hb dynamics, we studied the protein internal picosecond motions as a response to hydration, by elastic and quasielastic neutron scattering. Rates of fast diffusive motions were found to be significantly enhanced with increasing hydration from fully hydrated powder to concentrated Hb solution. In concentrated protein solution, the data showed that amino acid side chains can explore larger volumes above body temperature than expected from normal temperature dependence. The body temperature transition in protein dynamics was absent in fully hydrated powder, indicating that picosecond protein dynamics responsible for the transition is activated only at a sufficient level of hydration. A collateral result from the study is that fully hydrated protein powder samples do not accurately describe all aspects of protein picosecond dynamics that might be necessary for biological function.     

Internal motion of a tryptophan residue in Streptomyces subtilisin inhibitor: deuterium nuclear magnetic resonance in solution

Deuterium NMR spectroscopy was used to study internal motions of a deuterium-labeled single tryptophan (Trp) residue (per subunit) of Streptomyces subtilisin inhibitor (SSI) in solution. The free inhibitor with the five ring protons of the Trp replaced with deuterons showed a narrow resonance component (56 Hz) of about one-quarter of the total intensity, in addition to the broad resonance component (about 600 Hz) at 25 degrees C, showing that it exits in an equilibrium mixture of two conformers, in one of which the tryptophan side chain is highly mobile. In analogy to the two structures of SSI found in the crystal, these two conformers were attributed to the one in which the contact between the alpha-lobe and the beta-lobe of the subunit is tight and the other in which the same contact is loose. When SSI forms a complex with subtilisin BPN', the broad component becomes invisibly broad, but the narrow component increases with even further narrowing, suggesting that the binding to the enzyme favors the "loose" conformer over the "tight" conformer.     

Protein-water displacement distributions

The statistical properties of fast protein-water motions are analyzed by dynamic neutron scattering experiments. Using isotopic exchange, one probes either protein or water hydrogen displacements. A moment analysis of the scattering function in the time domain yields model-independent information such as time-resolved mean square displacements and the Gauss-deviation. From the moments, one can reconstruct the displacement distribution. Hydration water displays two dynamical components, related to librational motions and anomalous diffusion along the protein surface. Rotational transitions of side chains, in particular of methyl groups, persist in the dehydrated and in the solvent-vitrified protein structure. The interaction with water induces further continuous protein motions on a small scale. Water acts as a plasticizer of displacements, which couple to functional processes such as open-closed transitions and ligand exchange.     

Anomalous temperature dependence of the thermal expansion of proteins

The temperature dependence of the apparent expansibility of lysozyme and ovalbumin in solution has been measured as a function of pH. This temperature dependence is explained in terms of suppressed fluctuations in bound water due to the protein. It is shown that the thermal expansion coefficient of bound water is different from bulk water. The pH dependence can be explained by increased hydration of side chains at lower pH. The amount in volume of hydration water in a typical protein-water system varies from 0.16 to 0.7. How the intrinsic thermal expansion coefficient of proteins can be derived from the apparent quantity is discussed. Intrinsic values of the thermal expansion coefficient for lysozyme at room temperature are between 1.7 and 4.4 x 10(-4) K-1 for a 10% solution.     

The effect of sodium dodecyl sulfate on the structure and function of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) has been shown to exist as a dimer of molecular weight of 23,000 in 25 mm phosphate buffer, at pH 7.0 (the ionic strength 0.1 m with NaCl), 25.0 °C in the concentration range of 0.01–10 mg/ml. In the present paper, the effects of an anionic detergent, sodium dodecyl sulfate (SDS), on the structure and function of SSI has been examined, [a]The molecular weight of SSI was measured in the SDS solution with the sedimentation equilibrium method of the multicomponent-polydisperse system under the conditions described above, and thereby it has been shown that SSI dissociates into monomers with SDS of 0.03–0.12% ($\text{w}\text{v}$) when the concentration of SSI is 1.00 mg/ml (87.0 μm as monomer), [b]As SSI dissociates into monomers, there were observed blue-shift troughs at 293 nm and 300 nm due to a tryptophyl residue and a red-shift of phenylalanyl residues in the absorption difference spectrum induced by the binding of SSI and SDS. [c] The inhibitory activity of SSI against subtilisin BPN′-catalyzed hydrolysis of p-nitrophenyl acetate was measured under the conditions that SSI is in monomer in the SDS solution. Unexpectedly half of the inhibitory activity of SSI against subtilisin BPN′ is lost in the SDS solution.     

High-level expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus S-3253

A secretory expression system for Streptomyces subtilisin inhibitor (SSI) was established in a heterologous host, Streptomyces lividans 66, by introducing the 1.8-kbp BglII/SalI fragment containing SSI gene into the Streptomyces multicopy vector, pIJ 702. The expression of SSI did not depend on the orientation of the 1.8-kbp BglII/SalI fragment or on the promoter for tyrosinase gene (mel) in pIJ 702, which suggested that this fragment also carries the SSI promoter. The expressed SSI in S.lividans 66 was secreted into the culture medium in a large amount, as observed with the original strain, S. albogriseolus S-3253. Amino acid sequence analysis showed that the SSI secreted from S. lividans 66 contained three additional amino acid residues in the NH2-terminal region. The inhibitory activity toward subtilisin BPN' and the antigenic activity of the SSI secreted from S. lividans 66 were found to be identical with those of authentic SSI.