Conformational adjustments of SNase R and its N-terminal fragments probed by monoclonal antibodies

Two monoclonal antibodies specific for staphylococcal nuclease R (SNase R) (McAb2C9 and McAb1B8) were prepared and used to probe protein folding during peptide elongation, by measuring antibody binding to seven N-terminal fragments (SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52) of SNase R. Comparative studies of the conformations of the N-terminal fragments have shown that all seven fragments of SNase R have a certain amount of residual structure, indicating that folding may occur during elongation of the nascent peptide chain. We show that the binding abilities of the intact enzyme and its seven fragments to the monoclonal antibodies are not simply proportional to the length of the peptide chain, suggesting that there may be continuous conformational adjustment in the nascent peptide chain as new C-terminal amino acids are added. A folding intermediate close in structure to the native state but with structural features in common with SNR121 is highly populated in 0.6 M GuHCl, and is also formed transiently during folding.     

Searching for folding initiation sites of staphylococcal nuclease: a study of N-terminal short fragments

The N-terminal short fragments of staphylococcal nuclease (SNase), SNase20, SNase28, and SNase36, corresponding to the sequence regions, Ala1-Gly20, Ala1-Lys28, and Ala1-Leu36, respectively, as well as an 8-residue peptide (Ala17-Ile18-Asp19-Gly20-Asp21-Thr22-Val23-Lys24) have been synthesized. The conformational states of these fragments were investigated using CD and NMR spectroscopy in aqueous solution and in trifluoroethanol (TFE)-H(2)O mixture. SNase20 containing a sequence corresponding to a bent peptide in native SNase shows a transient population of bend-like conformation around Ala12-Thr13-Leu14 in TFE-H(2)O mixture. The sequence region of Ala17-Thr22 of SNase28 displays a localized propensity for turn-like conformation in both aqueous solution and TFE-H(2)O mixture. The conformational ensemble of SNase36 in aqueous solution includes populated turn-like conformations localized in sequence regions Ala17-Thr22 and Tyr27-Gln30. The analysis suggests that these sequence regions, which form the regular secondary structures in native protein, may serve as the folding nucleation sites of SNase fragments of different chain lengths starting from the N-terminal end. Thus, the formation of bend- and turn-like conformations of these sequence regions may be involved in the early folding events of the SNase polypeptide chain in vitro.     

汉滩病毒核蛋白的分段表达及抗原表位分析

在大肠杆菌中对汉滩病毒Ｓ基因４种不同长度片段的重组表达质粒进行诱导表达。结果表明表达的４种ＧＳＴ－ＮＰ融合蛋白均以不溶性包含体形式存在于茵体细胞内,表达量分别占菌体蛋白总量的２９－３６％,分子量分别约为７２ｋＤ、６６ｋＤ、５４ｋＤ和４４ｋＤＤ。Ｗｅｓｔｅｒｎ ｂｌｏｔ显示５４ｋＤ和７２ｋＤ融合蛋白用酶标记汉滩病毒ＮＰＭｃＡｂｌＡ８和抗ＧＳＴ ＭｃＡｂ ３Ｃ１１染色呈阳反应。６６ｋＤ和４４ｋＤ融合蛋     

SNase R与SNase酶学性质的比较研究

金黄色葡萄球菌核酸酶(SNase)的一种类似物SNase R在E.coli DH5x细胞中高效表达, 经磷酸纤维素离子交换柱层析纯化后.在SDS-PAGE上为一条分子量17.5KDa的蛋白带.本文对SNase R和SNase的某些酶学性质.酶动力学性质进行了比较研究.为蛋白质结构与功能及肽链折叠研究提供了又一种材料.     

Identification of second arginine‐glycine‐aspartic acid motif of ovine vitronectin as the complement C9 binding site and its implication in bacterial infection

Vitronectin (Vn), a multifunctional protein of blood and extracellular matrix, interacts with complement C9. This interaction may modulate innate immunity. Details of Vn–C9 interactions are limited. Vn–C9 interactions were assessed by employing a goat homologous system and observing Vn binding to C9 in three different assays. Using recombinant fragments, C9 binding was mapped to the N‐terminus of Vn. Site directed mutagenesis was performed to alter the second arginine glycine aspartic acid (RGD) sequence (RGD‐2) of Vn. Changing R to G or D to A in RGD‐2 caused significant decrease in Vn binding to C9 whereas changing of R to G in the first RGD motif (RGD‐1) had no effect on Vn binding to C9. These results imply that the RGD‐2 of goat Vn is involved in C9 binding. In a competitive binding assay, the presence of soluble RGD peptide inhibited Vn binding to C9 whereas heparin had no effect. Vn binding to C9 was also evaluated in terms of bacterial pathogenesis. Serum dependent inhibition of Escherichia coli growth was significantly reverted when Vn or its N‐fragment were included in the assay. The C‐fragment, which did not support C9 binding, also partly nullified serum‐dependent inhibition of bacterial growth, probably through other serum component(s).     

Effect of a specific hydrogen bond (N138ND2-Q106O) on conformational integrity, stability, and activity of staphylococcal nuclease

There are two hydrogen bonding interactions (N138ND2-Q106O and Y54OH-S141OG) between the C-terminal region and the main body of staphylococcal nuclease (SNase). To examine the role of these hydrogen bonds, SNase(141) and its three mutants, SNase(141)N138D, SNase(141)S141A, and SNase(141)N138D/S141A, were created. The N138D mutation has the N138ND2-Q106O interaction deleted and the S141A mutation has the Y54OH-S141OG and S141OG-N138O interactions deleted. The conformational features, stability, and activity of the proteins have been compared by using circular dichroism, intrinsic and ANS-binding fluorescence, GdnHCl-induced denaturation, and activity assay. The results clearly show that the N138D mutation significantly alters the secondary and tertiary structures of the protein, producing a partially unfolding state; in contrast, the S141A mutation has no such effect on structure. These results strongly suggest that the specific hydrogen bond, N138ND2-Q106O, plays an important role in maintaining the conformational integrity and stability of the nuclease.     

Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of staphylococcal nuclease

The high-resolution X-ray crystal structure of staphylococcal nuclease (SNase) suggests that the guanidinium groups of Arg 35 and Arg 87 participate as electrophilic catalysts in the attack of water on the substrate phosphodiester. Both arginine residues have been replaced with "conservative" lysine residues so that both the importance of these residues in catalysis and the effect of changes in electrostatic interactions on active site conformation can be assessed. The catalytic efficiencies of R35K and R87K are decreased by factors of 10(4) and 10(5) relative to wild-type SNase, with R87K showing a very significant reduction in its affinity for both DNA substrate and the competitive inhibitor thymidine 3',5'-bisphosphate (pdTp). The thermal denaturation behavior of both mutant enzymes differs from that of wild type both in the absence and in the presence of the active site ligands Ca2+ and pdTp. Both the 1H NMR chemical shifts and interresidue nuclear Overhauser effects (NOEs) of residues previously assigned to be in the hydrophobic core of SNase are altered in R35K and R87K. These observations, similar to those recently reported by our laboratories for substitutions for Glu 43 [Hibler, D. W., Stolowich, N. J., Reynolds, M. A., Gerlt, J. A., Wilde, J. A., & Bolton, P. H. (1987) Biochemistry 26, 6278; Wilde, J. A., Bolton, P. H., Dell'Acqua, M., Hibler, D. W., Pourmotabbed, T., & Gerlt, J. A. (1988) Biochemistry 27, 4127], suggest that lysine substitutions are not conservative in SNase and disrupt the conformation of the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct screening of recombinants in gram-positive bacteria using the secreted staphylococcal nuclease as a reporter.

A system for direct screening of recombinant clones in Lactococcus lactis, based on secretion of the staphylococcal nuclease (SNase) in the organism, was developed. The nuc gene (encoding SNase) was cloned on both rolling-circle and theta-replicating plasmids. L. lactis strains containing these nuc+ plasmids secrete SNase and are readily detectable by a simple plate test. A multicloning site (MCS) was introduced just after the cleavage site between leader peptide and the mature SNase, without affecting nuclease activity. Cloning foreign DNA fragments into any site of the MCS interrupts nuc and thus results in nuc mutant clones which are easily distinguished fron nuc+ clones on plates. The utility of this system for L. lactis was demonstrated by cloning an antibiotic resistance marker and Escherichia coli chromosomal DNA fragments into the MCS of the nucMCS cassette. Both cloning vectors containing the nucMCS cassette were also introduced into Streptococcus salivarius subsp. thermophilus, in which direct screening of nuc mutant recombinant clones was also achieved. The potential uses of nuc as a secretion reporter system are discussed.     

Probing the folding capacity and residual structures in 1-79 residues fragment of staphylococcal nuclease by biophysical and NMR methods

1-79 residues SNase fragment (SNase79) has chain length containing a sequence for helix alpha(1), omega-loop, beta(I)-sheet, and partial beta(II)-sheet of native SNase. The incomplete "beta-barrel" structural region of SNase79 makes this fragment to be interested in investigation of its conformation. For this study, we use CD, fluorescence, and NMR spectroscopy to probe the folding capacity and the residual structures in SNase79. The optical spectra obtained for SNase79 and its mutants reveal the presence of retained capacity for folding of the fragment. The NMR derived (13)C(alpha) secondary chemical shifts, (3)J(NH-Halpha) coupling constants, amide-proton temperature coefficients, interresidue NOEs, and (15)N relaxation data determine the intrinsic propensities for helix- and turn- or beta-sheet-like conformations of SNase79, which is not the result of stabilizing inter-molecular interactions by oligomerization effects. The residual turn- and helix-like structures may serve as potential local nucleation sites, whereas the residual beta(I)-sheet-like structure can be regarded as a potential non-local nucleation site in the folding of SNase79. The intrinsic local and non-local interactions in these potential initiation sites are insufficient to stabilize the folding of SNase79 due to the shortage of relevant long-range interactions from other part of the fragment. The conformational ensemble of SNase79 is a highly heterogeneous collection of interconverting conformations having transiently populated helix- and beta-sheet- or turn-like structures.     

Restricted backbone conformational and motional flexibilities of loops containing peptidyl-proline bonds dominate the enzyme activity of staphylococcal nuclease

The role of cis-trans isomerizations of peptidyl-proline bonds in the enzyme activity of staphylococcal nuclease (SNase) was examined by mutation of proline residues. The proline-free SNase ([Pro-]SNase), namely, P11A/P31A/P42A/P47T/P56A/P117G-mutant SNase, was adopted for elucidating the correlation between the nuclease activity and the backbone conformational and dynamic states of SNase. The 3D solution structure of [Pro-]SNase has been determined by heteronuclear NMR experiments. Comparing the structure of [Pro-]SNase with the structure of SNase revealed the conformational differences between the two proteins. In the structure of [Pro-]SNase, conformational rearrangements were observed for the loop of residues Ala112-His121 containing a trans Lys116-Gly117 peptide bond and for the C-terminal alpha-helical loop of residues Leu137-Glu142. Mutation of proline at position 117 also caused the conformational rearrangement of the p-loop (Asp77-Leu89), which is remote from the Ala112-His121 loop. The Ala112-His121 loop and p-loop are placed closer to each other in [Pro-]SNase than in SNase. The backbone dynamic features of the omega-loop (Pro42-Pro56) of SNase are different from those of [Pro-]SNase. The backbone of the omega-loop exhibits restricted flexibility with slow conformational exchange motions in SNase, but is highly flexible in [Pro-]SNase. The analysis indicates that the restrained backbone conformation of the Ala112-His121 loop and restricted flexibility of the omega-loop are two dominant factors determining the enzyme activity of SNase. Of the two factors, the former is correlated with the strained cis Lys116-Pro117 peptide bond and the latter is correlated with the cis-trans isomerizations of the His46-Pro47 peptide bond.     

Physiologic inactivation of fluid phase C3b: isolation and structural analysis of C3c, C3d,g (alpha 2D), and C3g

The fragments that result from the inactivation of C3b have not been completely characterized. Initial inactivation is catalyzed by the protease factor I, which, in the presence of its cofactor (factor H), cleaves two peptide bonds in the alpha'-chain of C3b. This results in the release of a small peptide (C3f, Mr 3000) from iC3b, which consists of the C3 beta chain covalently bonded to two alpha'-chain-derived peptides (Mr 68,000 and Mr 43,000). Surface-bound iC3b is cleaved at a third site by factor I to produce C3c and C3d,g (or alpha 2D). The factor I cofactor for this cleavage is the C3b receptor that is present on erythrocyte and leukocyte membranes. This report describes the isolation and initial structural characterization of C3c and C3d,g generated in whole blood after complement activation with cobra venom factor. These fragments were compared with the C3 fragments isolated from the serum and plasma of a patient with complement activation in vivo. The fragments were isolated with two solid phase monoclonal antibodies, one of which recognizes a determinant on C3g (clone 9) and one of which recognizes a determinant on C3c (clone 4). C3c isolated from normal blood showed three polypeptides that had apparent m.w. of 75,000, 43,000, and 27,000. The C3d,g consisted of a single polypeptide chain with a m.w. of 40,000. Amino terminal sequence analysis showed that the Mr 27,000 peptide from C3c is derived from the amino terminal portion of the alpha'-chain of C3b, whereas the Mr 43,000 peptide is derived from the carboxy terminus of the same chain. Amino terminal sequence analysis showed also that C3g is derived from the amino terminus of C3d,g. The C3 fragments isolated from a patient with partial lipodystrophy, nephritic factor activity, low serum C3 levels, and circulating C3 cleavage products showed a more complicated pattern on SDS-PAGE. The fragment isolated with clone 9 had an apparent m.w. of 40,000, identical to C3d,g generated in vitro, and it had the same amino terminal sequence as C3d,g generated in vitro. The eluate from insolubilized clone 4, however, showed prominent bands with Mr of 75,000, 56,000, 43,000, and 27,000, together with a triple-banded pattern at 68,000 and a minor band at 80,000. This eluate thus appears to contain C3c, and iC3b or an iC3b-like product. The origin of the Mr 56,000 and Mr 80,000 peptides have not yet been determined. These studies, with previous data, definitively order the C3c and C3d,g peptides in the alpha-chain of C3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum.

The gene for staphylococcal nuclease (SNase), an extracellular enzyme of Staphylococcus aureus, was introduced into Corynebacterium glutamicum. The heterologous gene was expressed in this host organism, and SNase was efficiently exported to the culture medium. Amino-terminal sequencing of SNase secreted by C. glutamicum revealed that the signal peptide was apparently cleaved off at precisely the same position as in the original host, S. aureus. As with S. aureus, a second smaller form of SNase (A form), whose appearance is presumably the result of a secondary processing step, was found in the culture medium of the recombinant C. glutamicum strain. The A form was one residue shorter than the mature nuclease A produced by S. aureus. Variation of the sodium chloride concentration in the growth medium had a marked influence on the location and the processing of SNase by C. glutamicum. In a complex growth medium containing 4% sodium chloride, SNase was exclusively located in the supernatant, but a significant amount of the enzyme remained cell associated if the strain was grown in a low-salt medium. Also, high salt concentrations seemed to inhibit processing of the high-molecular-weight form of SNase (B form) to the smaller A form. Similarities and differences in the export and modes of processing of SNase by three different, nonrelated gram-positive host organisms are discussed. Finally, a versatile Escherichia coli-C. glutamicum tac-lacIq expression shuttle vector was constructed. With this vector, it was possible to achieve isopropyl-beta-D-galactopyranoside (IPTG)-inducible overexpression and secretion of SNase in C. glutamicum, whereby the expression level was dependent on the concentration of the inducer.     

金黄色葡萄球菌核酸酶基因的片段缺失与定点突变

金黄色葡萄球菌核酸酶R(SNase R)是金黄色葡萄球菌核酸酶(SNase A)的一种类似物,具有与SNaseA相同的酶活性,与SNase A唯一不同之处是在N端多出6个氨基酸残基。为了得到完整的SNase基因并使其在E.Coli中表达,我们利用单链U模板—单引物突变法,将为6个额外氨基酸残基编码的18个氨基酸残基删除,其突变率可达90％。进而,完整的SNase A基因被重组入表达载体pBV221。细菌表达产物的PAGE分析结果指出,SNase A在E.coli中得到高效表达。与此同时,我们利用两个不同的引物在单链U模板上同时介导两种不同类型的突变(片段缺失、碱基取代)其突变率可达83％以上,这为进行多种类型的高效突变提供一个有用的方法。本文也对影响突变率的某些实验因素进行了讨论。     

Structural analysis of the N‐terminal fragment of the antiangiogenic protein endostatin: A molecular dynamics study

Endostatin is a potent antiangiogenic protein derived from the noncollagenous domain 1 (NC1) of collagen XVIII. The mechanism by which endostatin exerts its antiangiogenic effect is still incompletely understood. It has been shown that the 27 amino acid N‐terminal fragment of murine endostatin has antitumor, antimigration, and antipermeability activities comparable to the full soluble protein. To understand how this peptide can exert such elaborate function, we performed structural analysis using molecular dynamics to evaluate the behavior of this fragment in aqueous environment. Here, we show that the N‐terminal peptide of murine endostatin is able to assume a well‐defined structure, folding into a zinc‐dependent β‐hairpin conformation. Analyzing the folding mechanism, we were able to understand why the N‐terminal peptide of human endostatin with the same length failed to acquire a stable conformation. Conversely, we were able to predict the successful folding of the R4Q mutant and of a shorter form of the human peptide with 25 residues. Finally, we show that the β‐hairpin conformation assumed by the zinc‐bound peptide of murine endostatin has a high structural similarity with fragments of another family of angiogenesis inhibitors: the integrin‐binding portion of the NC1 domain of collagen IV. Indeed, our docking simulations show that arresten, canstatin, and the endostatin peptide bind to the same spot of αVβ3 integrin, suggesting similar interactions via a common binding site on this receptor. Proteins 2011;. © 2011 Wiley‐Liss, Inc.     

Native-like partially folded conformations and folding process revealed in the N-terminal large fragments of staphylococcal nuclease: a study by NMR spectroscopy

The N-terminal large fragments of staphylococcal nuclease (SNase), SNase110 (1-110 residues), SNase121 (1-121 residues), and SNase135 (1-135 residues), and the fragment mutants G88W110, G88W121, V66W110 and V66W121 were studied by heteronuclear multidimensional NMR spectroscopy. Ensembles of co-existent native-like partially folded and unfolded states were observed for fragments. The persistent native-like tertiary interaction drives fragments to be in partially folded states, which reveal native-like beta-barrel conformations. G88W and V66W mutations modulate the extent of inherent native-like tertiary interaction in fragment molecules, and in consequence, fragment mutants fold into native-like beta-subdomain conformations. In cooperation with the inherent tertiary interaction, 2 M TMAO (trimethylamine N-oxide) can promote the folding reaction of fragments through the changes of unfolding free energy, and a native-like beta-subdomain conformation is observed when the chain length contains 135 residues. Heterogeneous partially folded conformations of 1-121 and 1-135 fragments due to cis and trans X-prolyl bond of Lys116-Pro117 make a non-unique folding pathway of fragments. The folding reaction of fragments can be characterized as a hierarchical process.     

Folding of the C-terminal fragment V111-D143 of staphylococcal nuclease in aqueous solution

Studies of conformational features of fragments SNase(111-143) and SNase(118-143) and segment E122-K136 in 1-139 fragment (SNase139) suggest that the high intrinsic helical propensity can drive segment E122-K136 fold into a stable helix only when the segments V111-H121 and L137-D143 flanked on segment E122-K136 in staphylococcal nuclease (SNase) have stable folding.     

Coordination abilities of the 1-16 and 1-28 fragments of beta-amyloid peptide towards copper(II) ions: a combined potentiometric and spectroscopic study

Stoichiometry, stability constants and solution structures of the copper(II) complexes of the (1-16H), (1-28H), (1-16M), (1-28M), (Ac-1-16H) and (Ac-1-16M) fragments of human (H) and mouse (M) beta-amyloid peptide were determined in aqueous solution in the pH range 2.5-10.5. The potentiometric and spectroscopic data (UV-Vis, CD, EPR) show that acetylation of the amino terminal group induces significant changes in the coordination properties of the (Ac-1-16H) and (Ac-1-16M) peptides compared to the (1-16H) and (1-16M) fragments, respectively. The (Ac-1-16H) peptide forms the 3N [N(Im)(6), N(Im)(13), N(Im)(14)] complex in a wide pH range (5-8), while for the (Ac-1-16M) fragment the 2N [N(Im)(6), N(Im)(14)] complex in the pH range 5-7 is suggested. At higher pH values sequential amide nitrogens are deprotonated and coordinated to copper(II) ions. The N-terminal amino group of the (1-16) and (1-28) fragments of human and mouse beta-amyloid peptide takes part in the coordination of the metal ion, although, at pH above 9 the complexes with the 4N [N(Im), 3N(-)] coordination mode are formed. The phenolate -OH group of the Tyr(10) residue of the human fragments does not coordinate to the metal ion.     

ω-芋螺毒素MVIIC的N及C端修饰对折叠及活性影响

 合成了 ω-芋螺毒素 MVIIC的三种 N及 C端修饰肽 ,应用高压液相色谱、CD及生物体内活性实验 ,研究了其 N及 C端修饰对折叠及活性的影响 .结果表明 :MVIIC N端用 Phe及 Ser修饰后降低其线性肽形成正确折叠的比例及结构的稳定性 ,对小鼠的脑室给药活性也相应降低 ;C端酰胺转为电负性羧基端后活性降低 ,CD谱存在显著差异 .     

非洲猪瘟病毒j5R膜蛋白的电脑预测和实验证实

蛋白多肽二级结构的电脑预测表明,非洲猪瘟病毒（ Ａｆｒｉｃａｎ ｓｗｉｎｅ ｆｅｖｅｒ ｖｉｒｕｓ , Ａ Ｓ Ｆ Ｖ）ｊ５ Ｒ阅读框编码１２ ．９ ｋ Ｄａ 膜蛋白。该蛋白的 Ｃ 末端含有一个潜在抗原决定簇,针对其合成肽的抗体能在 Ａ Ｓ Ｆ Ｖ 感染细胞和病毒颗粒中检测到２３ 或２５ ｋ Ｄａ（ 取决于不同毒株） 特异蛋白。免疫荧光试验显示,ｊ５ Ｒ 蛋白主要位于感染细胞的病毒复制部位。油水两相分离和细胞分级分离试验结果证明ｊ５ Ｒ 蛋白是膜相关蛋白