Domain unfolding and the stability of thermolysin in guanidine hydrochloride

Equilibrium and kinetic studies of the unfolding and autolysis of the two domain protein thermolysin in guanidine hydrochloride are described. Enzyme activity, circular dichroism, fluorescence, sedimentation, size exclusion chromatography, and viscosity measurements were used to monitor conformational transitions and characterize the native and denatured states. The observation of biphasic transitions for the unfolding of apothermolysin and the spectroscopic changes associated with each phase of the overall unfolding process suggest unfolding of the N-terminal domain at less than 1 M guanidine hydrochloride, followed by the unfolding of the C-terminal domain, with the transition midpoint at 3 M guanidine hydrochloride. The refolding of the C-terminal domain is reversible; however, refolding of the N-terminal domain could not be demonstrated owing to protein aggregation. A quantitative analysis of the two transitions suggest that the unfolding of the two structural domains of thermolysin is not completely independent. Attempts to measure the unfolding of holothermolysin were hampered by autolysis. However, it was possible to show that at least three calcium ions serve to stabilize thermolysin against autolysis or unfolding in guanidine hydrochloride. Similar stabilization was observed for thermolysin with a single terbium ion bound at calcium site S(1). This result is consistent with our earlier findings, which suggest that calcium bound at sites S(1)-S(2) are located at a critical point on the unfolding pathway of thermolysin and serve to act as an interdomain lock.     

Autolysis of thermolysin. Isolation and characterization of a folded three-fragment complex

Incubation of the neutral metalloendopeptidase thermolysin at pH 7.2 in the presence of EDTA and/or low concentrations of calcium ions produces fast enzyme inactivation as a result of autolysis. The 'nicked' protein is a folded species composed of three tightly associated protein fragments. Dissociation of this complex can be achieved under denaturing conditions, such as gel filtration on a column equilibrated with 5 M guanidine hydrochloride or reverse-phase high-performance liquid chromatography (HPLC) at acidic pH. The positions of the peptide bond cleavages were defined by isolation of the individual fragments by HPLC and their characterization by amino acid analysis after acid hydrolysis, end-group determination and partial amino acid sequencing. The results of these analyses indicated that the nicked protein is composed of fragments 1-196, 197-204 and 205-316 and thus that the corresponding sites of limited proteolysis occur at the polypeptide chain loop involved in the binding of Ca(4) in native thermolysin [Matthews, B. W., Weaver, L. H. and Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The overall conformational properties of nicked thermolysin are quite similar to those of the intact protein, as judged by spectroscopic measurements and by the fact that rabbit antibodies against native thermolysin recognize and precipitate the nicked protein in immunodiffusion assays. The nicked protein was much less stable to heat and unfolding agents than intact thermolysin. These results contribute to a better knowledge of the molecular mechanism of stabilization of native thermolysin by the four bound calcium ions and demonstrate that the function of Ca(4) is to stabilize the loop 190-205 on the surface of the molecule against autolysis.     

Folding of thermolysin fragments. Correlation between conformational stability and antigenicity of carboxyl-terminal fragments

Circular dichroism (CD) and immunochemical measurements have been used to examine conformational properties of COOH-terminal fragments 121-316, 206-316 and 225(226)-316 of thermolysin, and to compare these properties to those of native thermolysin and thermolysin S, the stable partially active two-fragment complex composed of fragments 5-224(225) and 225(226)-316. In aqueous solution at neutral pH, all the COOH-terminal fragments attain a native-like conformation, as judged both by the content of secondary structure deduced from far-ultraviolet CD spectra and by the recognition of rabbit polyclonal antibodies specific for the COOH-terminal region in native thermolysin. The three fragments showed reversible cooperative unfolding transitions mediated by both heat and guanidine hydrochloride (Gdn X HCl). The phase transition curves were analyzed for Tm (temperature of half-denaturation) and Gibbs free energies (delta GD) of unfolding from native to denatured state. The observed order of thermal stability is 225(226)-316 less than or equal to 206-316 less than 121-316 less than thermolysin S less than thermolysin. The ranking of delta GD values for the three fragments correlates with the size of each fragment. Competitive binding studies by radioimmunoassay using 14C-labeled thermolysin and affinity purified antibodies specific for native antigenic determinants in segment 206-316 of native thermolysin indicate that the COOH-terminal fragments adopt native-like conformations which are in equilibrium with non-native conformations. These equilibria are shifted towards the native state as the fragment size increases from 225(226)-316, to 206-316, to 121-316. Fragment 225(226)-316, when combined with fragment 5-224(225) in the thermolysin S complex, adopts a more stable native-like conformation and becomes much more antigenic. It has been shown that the degree of antigenicity of COOH-terminal fragments towards thermolysin antibodies correlates directly with their conformational stability. The results of this study are discussed in relation to the recently proposed correlation between antigenicity and segmental mobility of globular proteins.     

An engineered amino-terminal domain of yeast phosphoglycerate kinase with native-like structure.

Previous studies have suggested that the carboxy-terminal peptide (residues 401-415) and interdomain helix (residues 185-199) of yeast phosphoglycerate kinase, a two-domain enzyme, play a role in the folding and stability of the amino-terminal domain (residues 1-184). A deletion mutant has been created in which the carboxy-terminal peptide is attached to the amino-terminal domain (residues 1-184) plus interdomain helix (residues 185-199) through a flexible peptide linker, thus eliminating the carboxy-terminal domain entirely. CD, fluorescence, gel filtration, and NMR experiments indicated that, unlike versions described previously, this isolated N-domain is soluble, monomeric, compactly folded, native-like in structure, and capable of binding the substrate 3-phosphoglycerate with high affinity in a saturable manner. The midpoint of the guanidine-induced unfolding transition was the same as that of the native two-domain protein (Cm approximately 0.8 M). The free energy change associated with guanidine-induced unfolding was one-third that of the native enzyme, in agreement with previous studies that evaluated the intrinsic stability of the N-domain and the contribution of domain-domain interactions to the stability of PGK. These observations suggest that the C-terminal peptide and interdomain helix are sufficient for maintaining a native-like fold of the N-domain in the absence of the C-domain.     

Semisynthesis of carboxy-terminal fragments of thermolysin

Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205-316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu302 residue into fragments 205-302 and 303-316. Upon incubation for 2-5 days of fragment 205-302 with a 5-fold excess of peptide 303-316, prepared by solid phase synthesis, with V8-protease in 0.1 M ammonium acetate, pH 6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to approximately 90% (based on fragment 205-302). The same procedure was used to prepare also the thermolysin fragments 205-315 and 205-311 by enzymatic coupling of fragment 205-302 to peptide 303-315 or 303-311, these last prepared by proteolytic digestion of the synthetic peptide 303-316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205-316 of thermolysin at the level of its helical segment encompassing residues 301-312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.     

Complete amino acid sequence of Scytalidium lignicolum acid protease B

The acid protease B (SLB) of Scytalidium lignicolum was reduced and carboxymethylated and then subjected to tryptic digestion. Five fragments were isolated and some of them were further digested with alpha-chymotrypsin, thermolysin, and dilute acetic acid. The sequence analysis of these fragments and the peptides by conventional methods established the complete amino acid sequence of SLB. The enzyme was composed of 204 amino acid residues with threonine and valine as its amino- and carboxyl-termini, respectively. Locations of three disulfide bridges were also established to be Cys47-126, Cys140-163, and Cys192-201 by enzymatic fragmentation of the denatured and unmodified SLB. Only a slight homology was found in the sequences of SLB and other acid proteases hitherto reported.     

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance.

Streptococcus equisimilis streptokinase (SK) is a single-chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al., 1996, Protein Sci 5:693-704) that SK is composed of three structural domains (A, B, and C) with a C-terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two-chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46 degrees C and its stability is largely independent of the presence of the other domains. The high-temperature transition in intact SK (at approximately 63 degrees C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63-Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (A1 and A2) adopt unstructured conformations when separated. A1 binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native-like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.     

Catalytic activation of human glucokinase by substrate binding: residue contacts involved in the binding of D-glucose to the super-open form and conformational transitions

alpha-D-Glucose activates glucokinase (EC 2.7.1.1) on its binding to the active site by inducing a global hysteretic conformational change. Using intrinsic tryptophan fluorescence as a probe on the alpha-D-glucose induced conformational changes in the pancreatic isoform 1 of human glucokinase, key residues involved in the process were identified by site-directed mutagenesis. Single-site W-->F mutations enabled the assignment of the fluorescence enhancement (DeltaF/F(0)) mainly to W99 and W167 in flexible loop structures, but the biphasic time course of DeltaF/F(0) is variably influenced by all tryptophan residues. The human glucokinase-alpha-D-glucose association (K(d) = 4.8 +/- 0.1 mm at 25 degrees C) is driven by a favourable entropy change (DeltaS = 150 +/- 10 J.mol(-1).K(-1)). Although X-ray crystallographic studies have revealed the alpha-d-glucose binding residues in the closed state, the contact residues that make essential contributions to its binding to the super-open conformation remain unidentified. In the present study, we combined functional mutagenesis with structural dynamic analyses to identify residue contacts involved in the initial binding of alpha-d-glucose and conformational transitions. The mutations N204A, D205A or E256A/K in the L-domain resulted in enzyme forms that did not bind alpha-D-glucose at 200 mm and were essentially catalytically inactive. Our data support a molecular dynamic model in which a concerted binding of alpha-D-glucose to N204, N231 and E256 in the super-open conformation induces local torsional stresses at N204/D205 propagating towards a closed conformation, involving structural changes in the highly flexible interdomain connecting region II (R192-N204), helix 5 (V181-R191), helix 6 (D205-Y215) and the C-terminal helix 17 (R447-K460).     

Two separate 18-amino acid domains of tau promote the polymerization of tubulin

Tau is a heat-stable microtubule-associated protein which promotes tubulin polymerization. The assembly promoting region of tau was localized using synthetic peptides modeled after domains found in both human and mouse tau. The design of these synthetic peptides was based on the triple repeat motif found in mouse tau. The first peptide, Tau-(187-204), and the second peptide, Tau-(218-235), are capable of promoting the polymerization of tubulin into microtubules, at concentrations above 100 microM. Two other peptides tested, TauR and Tau-(250-267), were not able to promote the assembly of tubulin over a range of concentrations up to 800 microM. TauR is a random analog of Tau-(187-204). Although TauR is unable to promote polymerization, it can modify Tau-(187-204)-induced tubulin assembly.     

Domain characteristics of the carboxyl-terminal fragment 206-316 of thermolysin: immunochemical studies

The extent of nativeness of the stable conformation of the thermolysin fragment containing the carboxyl-terminal third of the protein (from residues 206 to 316, denoted fragment FII) was examined by its immunogenic and antigenic characteristics. Antisera elicited in rabbits by either intact thermolysin or fragment FII were fractionated serially on two affinity columns, containing either the isolated fragment or intact protein. Both sera gave rise to substantial antibody populations which recognized the fragment FII region in native thermolysin. The relative affinities of these specific antibodies for isolated fragment FII and intact thermolysin were evaluated by radioimmunoassay, by assessing the relative extents of competition by these for binding of either 14C-labeled thermolysin or 14C-labeled fragment FII to each antibody population. Competition by fragment FII was substantial, though generally weaker than that for intact thermolysin, for antibody binding of both labeled antigens. The data demonstrate that the stable structure of fragment FII as observed spectroscopically likely is one which possesses conformational features similar to those of this region in intact thermolysin, but with perhaps less conformational rigidity. The results support the view that the region of thermolysin composed primarily of residues 206-316 is a conformational domain of the intact protein and that isolated fragment FII retains domain-like characteristics of stable and native-like conformation.     

Nicked apomyoglobin: a noncovalent complex of two polypeptide fragments comprising the entire protein chain

Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysin results in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) and C-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformational and association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealed that both fragments in isolation acquire a high content of helical secondary structure, while near-UV CD indicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalent protein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similar to those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CD measurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region provided evidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intact protein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the 153-residue chain of the protein, is similar in both protein species, as given by fluorescence emission data. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments 1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelike conformational features that promote their specific association and mutual stabilization into a nicked protein species much resembling in its structural features intact apoMb. It is suggested that the formation of a noncovalent complex upon fragment complementation can mimic the protein folding process of the entire protein chain, with the difference that the folding of the complementary fragments is an intermolecular process. In particular, this study emphasizes the importance of interactions between marginally stable elements of secondary structure in promoting the tertiary contacts of a native protein. Considering that apoMb has been extensively used as a paradigm in protein folding studies for the past few decades, the novel fragment complementing system of apoMb here described appears to be very useful for investigating the initial as well as late events in protein folding.     

A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau

The protein domain responsible for the interaction of tau with tubulin has been identified. Biophysical studies indicated that the synthetic peptide Val187-Gly204 (VRSKIG-STENLKHQPGGG) from the repetitive sequence on tau binds to two sites on the tubulin heterodimer and to one site on each of the microtubule-associated protein-interacting C-terminal tubulin peptides alpha(430-441) and beta(422-434). The binding data showed a relatively stronger interaction of Val187-Gly204 with beta(422-434) as compared to that with alpha(430-441). The interaction of this tau peptide with either alpha or beta tubulin peptides appears to be associated with conformational changes in both the tau and the tubulin peptides. The beta tubulin peptide also appears to induce a structural change of tau fragment Val218-Gly235. Interestingly, tau peptides Val187-Gly204 and Val218-Gly235 induced tubulin self-assembly in a cold-reversible fashion, and incorporated into the assembled polymers. The specificity of the interaction of the tau peptide was supported by the competition of tau protein for the interaction with the tubulin polymer. In addition, the tau peptide appears to contain the principal antigenic determinant(s) recognized by anti-idiotypic antibodies that react with the tubulin binding domains on microtubule-associated proteins. The present findings together with the demonstration of the presence of multiple sites for the binding of the alpha(430-441) and beta(422-434) tubulin fragments to tau, and the existence of repetitive sequences on tau, strongly support the hypothesis that the region of tau defined by the repetitive sequences is involved in its interaction with tubulin.     

Origins of blood acetate in the rat.

1. Fluorimetric techniques were used to characterize the environment of tryptophan residues in thermolysin and apo-thermolysin. The apo-thermolysin was obtained by dissolving the enzyme in the presence of 10mm-EDTA, which removed the functional Zn(2+) ion and the four Ca(2+) ions/molecule from the enzyme. 2. At 25 degrees C in aqueous solution the fluorescence-emission spectrum of the native holoenzyme, on excitation at 290nm, was essentially characteristic of tryptophan, with an emission maximum at 333nm. The emission maximum of the apoenzyme is red-shifted to 338nm and the relative intensity of fluorescence is decreased by 10%, both effects indicating some unfolding of the protein molecule, with the indole groups being transferred to a more hydrophilic environment. 3. Fluorescence quenching studies using KI, N'-methylnicotinamide hydrochloride and acrylamide indicated a more open structure in the apoenzyme, with the tryptophan residues located in a negatively charged environment. 4. The thermal properties of the apoenzyme, as monitored by fluorescence-emission measurements, are dramatically changed with respect to the native holoenzyme. In fact, whereas the native enzyme is heat-stable up to about 80 degrees C, for the apoenzyme a thermal transition is observed near 48 degrees C. The apoenzyme is also unstable to the action of unfolding agents such as urea and guanidinium chloride, much as for other globular proteins from mesophilic organisms. 5. The functional Zn(2+) ion does not contribute noticeably to the stability of thermolysin. 6. It is concluded that a major role in the structural stability of thermolysin is played by the Ca(2+) ions, which have a bridging function within this disulphide-free protein molecule.     

Identification of residues in the CH2/CH3 domain interface of IgA essential for interaction with the human fcalpha receptor (FcalphaR) CD89

Cellular receptors for IgA (FcalphaR) mediate important protective functions. An extensive panel of site-directed mutant IgAs was used to identify IgA residues critical for FcalphaR (CD89) binding and triggering. Although a tailpiece-deleted IgA1 was able to bind and trigger CD89, antibodies featuring CH3 domain exchanges between human IgA1 and IgG1 could not, indicating that both domains but not the tailpiece are required for FcalphaR recognition. To further investigate the role of the interdomain region, numerous IgA1s, each with a point substitution in either of two interdomain loops (Leu-257-Gly-259 in Calpha2; Pro-440-Phe-443 in Calpha3), were generated. With only one exception (G259R), substitutions produced either ablation (L257R, P440A, A442R, F443R) or marked reduction (P440R) in CD89 binding and triggering. Further support for involvement of these interdomain loops was provided by interspecies comparisons of IgA. Thus a human IgA1 mutant, LA441-442MN, which mimicked the mouse IgA loop sequence through substitution of two adjacent residues in the Calpha3 loop, was found, like mouse IgA, not to bind CD89. In contrast, bovine IgA1, identical to human IgA1 within these interdomain loops despite numerous differences elsewhere in the Fc region, did bind CD89. We have thus identified motifs in the interdomain region of IgA Fc critical for FcalphaR binding and triggering, significantly enhancing present understanding of the molecular basis of the IgA-FcalphaR interaction.     

Primary structure of tyrosinase from Neurospora crassa. I. Purification and amino acid sequence of the cyanogen bromide fragments

Cyanogen bromide (CB) cleavage of Neurospora tyrosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionylthreonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein. The amino acid sequences of the cyanogen bromide fragments CB2, CB3, and CB4 were determined by a combination of automated and manual sequence analysis on peptides derived by chemical and enzymatic cleavage of the intact and the maleylated derivatives. The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids whose chemical structure will be presented in the following paper.     

pH-induced domain interaction and conformational transitions of lipoxygenase-1

The multidomain structure of soybean LOX1 was examined over the pH range 1-12. Lipoxygenase-1 activity was reversible over broad pH range of 4-10 due to the reversibility of conformational states of the molecule. Below pH 4.0, due to collapse in hydrophobic interactions, the enzyme unfolded to an irreversible conformation with the properties of molten globule state with a mid point of transition at pH 2.4. This intermediate state lost iron irreversibly. In alkaline pH at 11.5, LOX1 underwent partial unfolding with the exposure of cysteine residues with subsequent oxidation of a pair of cysteine residues in the C-terminal domain and this intermediate showed some properties of molten globule state and retained 35% of activity. Beyond pH 12.0, the enzyme was completely inactivated irreversibly due to irreversible conformational changes. The pH-dependent urea-induced unfolding of LOX1 suggested that LOX1 was more stable at pH 7.0 and least stable at pH 9.0. Furthermore, the urea-induced unfolding of LOX1 indicated that the unfolding was biphasic due to pH-dependent domain interactions and involved sequential unfolding of domains. The loss of enzyme activity at pH 4. 0 and 7.0 occurred much earlier to unfolding of the C-domain at all pHs studied. The combination of urea-induced unfolding measurements and limited proteolysis experiments suggested that at pH 4.0, the domains in LOX1 were less interactive and existed as tightly folded units. Furthermore, these results confirmed the contribution of ionic interactions in the interdomain contacts.     

红树植物秋茄中受盐胁迫抑制的cyclophilin基因的鉴定与表达分析

利用代表性差异分析方法获得秋茄中两个编码亲环素(cyclophilin)蛋白的cDNA片段(称为SRGKC2和SRGKC3),该片段大小分别为282bp和160bp;序列分析表明：SRGKC2和SRGKC3是同一基因区域的不同长度片段,SRGKC3是SRGKC2片段的一部分。SRGKC2在84个氨基酸范围内与大戟属cyclophilin蛋白的氨基酸序列的一致性达到90％,SRGKC3在47个氨基酸范围内与蚕豆cyclophilin蛋白的一致性达到93％。Northern分析表明：盐分抑制SRGKC2片段的表达。依赖SRGKC2片段的序列资料,利用cDNA快速末端扩增(RACE)技术获取秋茄中cyclophilin基因的全长cDNA片段(命名为KCCYP1)(GenBank登录号：AY150052)。该cDNA全长约为0．9kb,含有一个516个核苷酸的完整开放阅读框,编码172个氨基酸,等电点为8．57,分子量18．2KDa。42—49位氨基酸残基为推测的ATP／GTP结合位点A基序(P—loop),48—54位氨基酸残基是插入的7个氨基酸残基。文中还对SRGKC2在不同种中的表达状况进行了分析。     

Domain separation precedes global unfolding of rhodanese

The enzyme rhodanese was investigated for the conformational transition associated with its urea unfolding. When rhodanese was treated with 0 or 3 M urea, the activity was not significantly affected. 4.25 M urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely inactivated within 2 min in 6 M urea. The 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid fluorescence intensity was not significantly increased during 0, 3, and 6 M urea equilibrations, and the fluorescence was dramatically increased with 4.25 M urea, indicating that hydrophobic surfaces are exposed. After 0 and 3 M urea equilibration, rhodanese was not significantly proteolyzed with trypsin. Treatment with 4.25 M urea led to simultaneous formation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progressive emergence of smaller peptides. The N termini of the 17- and 21.2-kDa bands were those of intact rhodanese. The N terminus of the 15.9-kDa band starts at the end of the interdomain tether. The 12-kDa band begins with either residue 183 or residue 187. The size and sequence information suggest that the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage. It is concluded that urea disrupts interaction between the two domains, increasing the accessibility of the interdomain tether that can be digested by trypsin. The released domains have increased proteolytic susceptibility and produce smaller peptides, which may represent subdomains of rhodanese.     

Probing the unfolding region of ribonuclease A by site-directed mutagenesis.

Ribonuclease A contains two exposed loop regions, around Ala20 and Asn34. Only the loop around Ala20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacent beta-sheet around Thr45) is the first region of the ribonuclease A molecule that becomes susceptible to thermolysin and trypsin under unfolding conditions. This second region therefore has been suggested to be involved in early steps of unfolding and was designated as the unfolding region of the ribonuclease A molecule. Consequently, modifications in this region should have a great impact on the unfolding and, thus, on the thermodynamic stability. Also, if the Ala20 loop contributes to the stability of the ribonuclease A molecule, rigidification of this flexible region should stabilize the entire protein molecule. We substituted several residues in both regions without any dramatic effects on the native conformation and catalytic activity. As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/R33S/F46Y. The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in DeltaG can be mainly attributed to an increased unfolding rate. Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule.     

红树植物秋茄中受盐胁迫抑制的cyclophilin基因的鉴定与表达分析

利用代表性差异分析方法获得秋茄中两个编码亲环素(cyclophilin)蛋白的cDNA片段(称为SRGKC2和SRGKC3),该片段大小分别为282 bp和160 bp;序列分析表明:SRGKC2和SRGKC3是同一基因区域的不同长度片段,SRGKC3是SRGKC2片段的一部分。SRGKC2在84个氨基酸范围内与大戟属cyclophilin蛋白的氨基酸序列的一致性达到90%,SRGKC3在47个氨基酸范围内与蚕豆cyclophilin蛋白的一致性达到93%。Northem分析表明:盐分抑制SRGKC2片段的表达。依赖SRGKC2片段的序列资料,利用cDNA快速末端扩增(RACE)技术获取秋茄中cyclophilin基因的全长cDNA片段(命名为KCCYPl)(GenBank登录号:AY150052)。该cDNA全长约为0.9kb,含有一个516个核苷酸的完整开放阅读框,编码172个氨基酸,等电点为8.57,分子量18.2 KDa。42-49位氨基酸残基为推测的ATP/GTP结合位点A基序(P-loop),48-54位氨基酸残基是插入的7个氨基酸残基。文中还对SRGKC2在不同种中的表达状况进行了分析。