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1.
Characterization of functional domains of equine infectious anemia virus Rev suggests a bipartite RNA-binding domain
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Lee JH Murphy SC Belshan M Sparks WO Wannemuehler Y Liu S Hope TJ Dobbs D Carpenter S 《Journal of virology》2006,80(8):3844-3852
Equine infectious anemia virus (EIAV) Rev is an essential regulatory protein that facilitates expression of viral mRNAs encoding structural proteins and genomic RNA and regulates alternative splicing of the bicistronic tat/rev mRNA. EIAV Rev is characterized by a high rate of genetic variation in vivo, and changes in Rev genotype and phenotype have been shown to coincide with changes in clinical disease. To better understand how genetic variation alters Rev phenotype, we undertook deletion and mutational analyses to map functional domains and to identify specific motifs that are essential for EIAV Rev activity. All functional domains are contained within the second exon of EIAV Rev. The overall organization of domains within Rev exon 2 includes a nuclear export signal, a large central region required for RNA binding, a nonessential region, and a C-terminal region required for both nuclear localization and RNA binding. Subcellular localization of green fluorescent protein-Rev mutants indicated that basic residues within the KRRRK motif in the C-terminal region of Rev are necessary for targeting of Rev to the nucleus. Two separate regions of Rev were necessary for RNA binding: a central region encompassing residues 57 to 130 and a C-terminal region spanning residues 144 to 165. Within these regions were two distinct, short arginine-rich motifs essential for RNA binding, including an RRDRW motif in the central region and the KRRRK motif near the C terminus. These findings suggest that EIAV Rev utilizes a bipartite RNA-binding domain. 相似文献
2.
The solution structure of the capsid protein (CA) from the human T-cell leukemia virus type one (HTLV-I), a retrovirus that causes T-cell leukemia and HTLV-I-associated myelopathy in humans, has been determined by NMR methods. The protein consists of independent N and C-terminal domains connected by a flexible linker. The domains are structurally similar to the N-terminal "core" and C-terminal "dimerization" domains, respectively, of the human immunodeficiency virus type one (HIV-1) and equine infectious anemia virus (EIAV) capsid proteins, although several important differences exist. In particular, hydrophobic residues near the major homology region are partially buried in HTLV-I CA, which is monomeric in solution, whereas analogous residues in HIV-1 and EIAV CA project from the C-terminal domain and promote dimerization. These differences in the structure and oligomerization state of the proteins appear to be related to, and possibly controlled by, the oxidation state of conserved cysteine residues, which are reduced in HTLV-I CA but form a disulfide bond in the HIV-1 and EIAV CA crystal structures. The results are consistent with an oxidative capsid assembly mechanism, in which CA oligomerization or maturation is triggered by disulfide bo nd formation as the budding virus enters the oxidizing environment of the bloodstream. 相似文献
3.
Archaeal Group II chaperonins (Cpns) are strongly conserved, considering that their growth temperatures range from 23 to 122 °C. The C-terminal 15–25 residues are hypervariable, and highly charged in thermophilic species. Our hypothesis is that the C-terminal is a key determinant of stabilization of the Cpn complex. The C-terminus of the Cpn from the hyperthermophile Pyrococcus furiosus was mutated to test this hypothesis. C-terminal deletions and replacement of charged residues resulted in destabilization. The stability of ATPase activity declined in proportion to the reduction in charged residues with Ala or Gly. An EK-rich motif (528EKEKEKEGEK537) proved to be a key domain for stabilization at or near 100 °C. Mutations “tuned” the Cpn for optimal protein folding at lower optimal temperatures, and Glu substitution was more potent than Lys replacement. Pf Cpn stability was enhanced by Ca2+, especially in the mutant Cpn lacking C-terminal Lys residues. This suggests that Glu-Glu interactions between C termini might be mediated by Ca2+. The C-terminal of a Cpn from the psychrophilic archaeon Methanococcoides burtonii was replaced by a domain from the hyperthermophile, resulting in increased thermostability and thermoactivity. We conclude that localized evolutionary variation in the C-terminus modulates the temperature range of archaeal Cpns. 相似文献
4.
Nuclear magnetic resonance evidence for a flexible region at the C-terminus of alpha-tropomyosin 总被引:1,自引:0,他引:1
The 1H nuclear magnetic resonance spectrum of alpha-tropomyosin contains a number of sharp peaks indicative of the presence of small regions of high flexibility in the molecule. Removal of 9 to 11 residues from the C-terminus by digestion with carboxypeptidase A causes a marked decline in the intensity of these peaks. The difference is consistent with at least the C-terminal four residues of the sequence (-Met-Thr-Ser-Ile) being highly mobile. The conformation of the C-terminus is thus radically different from the alpha-helical coiled-coil from which the bulk of the molecule is constructed. 相似文献
5.
Lambda cro repressor complex with OR3 DNA: 15N NMR observations 总被引:1,自引:0,他引:1
15N NMR studies of the coliphage lambda cro repressor are presented. The protein has been uniformally labeled with 15N, and individual amino acids have been incorporated. Although the four C-terminal residues (63-66) were not located in the original crystallographic studies of the protein [Anderson, W.F., Ohlendorf, D.H., Takeda, Y., & Matthews, B.W. (1981) Nature (London) 290, 754], it has been proposed that the C-terminus is involved in DNA binding [Ohlendorf, D.H., Anderson, W.F., Fisher, R.G., Takeda, Y., & Matthews, B.W. (1982) Nature (London) 298, 718]. These experiments give direct verification of that proposal. [15N]Amide resonances are assigned for residues 56, 62, 63, and 66 in the C-terminus by enzymatic digestion and by 13C-15N double-labeling experiments. 15N[1H] nuclear Overhauser effects show that the C-terminus is mobile on a nanosecond time scale. Exchange experiments using distortionless enhancement via polarization transfer, which is sensitive to proton exchange on the 1/JNH (10 ms) time scale, indicate that the amide protons in the C-terminus are freely accessible to solvent. It is thus a flexible arm in solution. The binding of both specific operator and nonspecific DNA is shown to reduce both the mobility and the degree of solvent exposure of this arm. Two-dimensional 15N-1H correlation experiments using 15N-labeled cro reveal inconsistencies with previously reported 1H NMR assignments for the lysine amides [Weber, P.L., Wemmer, D.E., & Reid, B.R. (1985) Biochemistry 24, 4553]. This result suggests that those assignments require reexamination, illustrating the utility of 15N labeling for obtaining 1H resonance assignments of biomolecules. Furthermore, isomerization of the peptide bond of Pro-59, which has been previously suggested (Weber et al., 1985) and which would significantly affect the properties of the C-terminal arm, is shown to not occur. 相似文献
6.
Functional roles of equine infectious anemia virus Gag p9 in viral budding and infection 总被引:8,自引:0,他引:8
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Previous studies utilizing Gag polyprotein budding assays with transfected cells reveal that the equine infectious anemia virus (EIAV) Gag p9 protein provides a late assembly function mediated by a critical Y(23)P(24)D(25)L(26) motif (L-domain) to release viral particles from the plasma membrane. To elucidate further the role of EIAV p9 in virus assembly and replication, we have examined the replication properties of a defined series of p9 truncation and site-directed mutations in the context of a reference infectious molecular proviral clone, EIAV(uk). Characterization of these p9 proviral mutants revealed new functional properties of p9 in EIAV replication, not previously elucidated by Gag polyprotein budding assays. The results of these studies demonstrated that only the N-terminal 31 amino acids of a total of 51 residues in the complete p9 protein were required to maintain replication competence in transfected equine cells; proviral mutants with p9 C-terminal truncations of 20 or fewer amino acids remained replication competent, while mutants with truncations of 21 or more residues were completely replication defective. The inability of the defective p9 proviral mutations to produce infectious virus could not be attributed to defects in Gag polyprotein expression or processing, in virion RT activity, or in virus budding. While proviral replication competence appeared to be associated with the presence of a K(30)K(31) motif and potential ubiquitination of the EIAV p9 protein, mutations of these lysine residues to methionines produced variant proviruses that replicated as well as the parental EIAV(uk) in transfected ED cells. Thus, these observations reveal for the first time that EIAV p9 is not absolutely required for virus budding in the context of proviral gene expression, suggesting that other EIAV proteins can at least in part mediate late budding functions previously associated with the p9 protein. In addition, the data define a function for EIAV p9 in the infectivity of virus particles, indicating a previously unrecognized role for this Gag protein in EIAV replication. 相似文献
7.
马传染性贫血病毒(EIAV)减毒疫苗是世界首例慢病毒疫苗,但其作用机理尚不明了.研究发现,EIAV疫苗株EIAVFDDV12的跨膜蛋白gp45在马体内发生高频率261W位点翻译终止突变,使该蛋白质C端出现154个氨基酸的截短.为了探讨该截短对EIAV疫苗株生物学特性的作用,以EIAV弱毒疫苗株感染性克隆为骨干,构建了gp45截短型感染性病毒株,检测该截短突变对EIAV疫苗株在体外培养的马外周血单核细胞由来的巨噬细胞(MDM)、驴MDM和驴胎皮细胞(FDD)中的复制.实验结果表明,gp45截短型毒株在马和驴MDM中复制能力比未截短型毒株显著降低(P<0.01),特别是在马MDM中此差异更明显.相反,截短型毒株在FDD中的复制能力则显著高于未截短型毒株(P<0.01).此外,结果显示gp45截短型毒株在马MDM中的低水平复制降低了EIAV对其靶细胞诱导的凋亡.以上结果提示,EIAV疫苗的gp45截短型毒株是适应在体外FDD细胞中传代致弱的变异,该变异导致疫苗株在EIAV体内主要靶细胞巨噬细胞中复制能力的降低,导致毒力进一步减弱. 相似文献
8.
N Izadi-Pruneyre N Wolff V Redeker C Wandersman M Delepierre A Lecroisey 《European journal of biochemistry》1999,261(2):562-568
HasA is a haem-binding protein which is secreted under iron-deficiency conditions by the gram-negative bacterium Serratia marcescens. It is a monomer of 19 kDa (187 residues) able to bind free haem as well as to capture it from haemoglobin. HasA delivers haem to a specific outer-membrane receptor HasR and allows the bacteria to grow in the absence of any other source of iron. It is secreted by a signal peptide-independent pathway which involves a C-terminal secretion signal and an ABC (ATP-binding cassette) transporter. The C-terminal region of the secretion signal containing the essential secretion motif is cleaved during or after the secretion process by proteases secreted by the bacteria. In this work, we study by 1H NMR the conformation of the C-terminal extremity of HasA in the whole protein and that of the isolated secretion signal peptide in a zwitterionic micelle complex that mimicks the membrane environment. We identify a helical region followed by a random-coil C-terminus in the peptide-micelle complex and we show that in both the whole protein and the complex, the last 15 residues containing the motif essential for secretion are highly flexible and unstructured. This flexibility may be a prerequisite to the recognition of HasA by its ABC transporter. We determine the cleavage site of the C-terminal extremity of the protein and analyse the effect of the cleavage on the haem acquisition process. 相似文献
9.
Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus. 总被引:20,自引:17,他引:3
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N R Rice L E Henderson R C Sowder T D Copeland S Oroszlan J F Edwards 《Journal of virology》1990,64(8):3770-3778
The transmembrane (TM) envelope protein of lentiviruses, including equine infectious anemia virus (EIAV), is significantly larger than that of other retroviruses and may extend in the C-terminal direction 100 to 200 amino acids beyond the TM domain. This size difference suggests a lentivirus-specific function for the long C-terminal extension. We have investigated the synthesis and processing of the EIAV TM protein by immune precipitation and immunoblotting experiments, by using several envelope-specific peptide antisera. We show that the TM protein in EIAV particles is cleaved by proteolysis to an N-terminal glycosylated 32- to 35-kilodalton (kDa) segment and a C-terminal nonglycosylated 20-kDa segment. The 20-kDa fragment was isolated from virus fractionated by high-pressure liquid chromatography, and its N-terminal amino acid sequence was determined for 13 residues. Together with the known nucleotide sequence, this fixes the cleavage site at a His-Leu bond located 240 amino acids from the N terminus of the TM protein. Since the 32- to 35-kDa fragment and the 20-kDa fragment are not detectable in infected cells, we assume that cleavage occurs in the virus particle and that the viral protease may be responsible. We have also found that some cells producing a tissue-culture-adapted strain of EIAV synthesize a truncated envelope precursor polyprotein. The point of truncation differs slightly in the two cases we have observed but lies just downstream from the membrane-spanning domain, close to the cleavage point described above. In one case, virus producing the truncated envelope protein appeared to be much more infectious than virus producing the full-size protein, suggesting that host cell factors can select for virus on the basis of the C-terminal domain of the TM protein. 相似文献
10.
Iwahashi A Ishii A Yamazaki N Hashimoto M Ohkura K Kataoka M Majima E Terada H Shinohara Y 《Mitochondrion》2008,8(2):196-204
Comparison of the amino acid sequence of yeast type 2 ADP/ATP carrier (yAAC2) with that of bovine type 1 AAC (bAAC1) revealed that the N- and C-terminus of yAAC2 are 15- and 6-amino acids longer, respectively, than those of bAAC1. In the present study, we focused on the difference in the C-terminal region between yAAC2 and bAAC1. Deletion of first six residues of C-terminus of yAAC did not markedly affect the function of yAAC2; however, further deletion of 1 amino acid (7th amino acid from the C-terminus) destroyed its function. On the contrary, deletion of the first amino acid residue of the C-terminus of bAAC1 caused failure of its functional expression in yeast mitochondria. Based on these results, we concluded that the 6-amino acid residue extension of the C-terminus of yAAC2 was not necessary for the function of this carrier and that the remainder of the C-terminal region of yAAC2, having a length conserved with that of bAAC1, is important for the transport function of AACs. We next prepared various single-Cys mutants in which each of 32 residues in the C-terminus of yAAC2 was replaced by a Cys residue. Since all mutants were successfully expressed in yeast mitochondria, we examined the reactivity of these cysteine residues with the membrane-impermeable sulfhydryl reagent eosin 5-maleimide (EMA). As a result, all cysteine residues that replaced the 9 continuous amino acids in Met310-Lys318 showed high reactivity with EMA regardless of the presence of carboxyatractyloside or bongkrekic acid; and so this region was concluded to be exposed to the water-accessible environment. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier in the presence of bongkrekic acid were discussed. 相似文献
11.
Yoo A Seo YS Jung JW Sung SK Kim WT Lee W Yang DR 《Journal of structural biology》2006,156(3):407-420
The 1-aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the last step in the biosynthesis of ethylene from ACC in higher plants. The complex structure of ACC oxidase/Fe(2+)/H(2)O derived from Petunia hybrida has recently been established by X-ray crystallography and it provides a vast structural information for ACC oxidase. Our mutagenesis study shows that both Lys296 and Arg299 residues in the C-terminal helix play important roles in enzyme activity. Both K296R and R299K mutant proteins retain only 30-15% of their enzyme activities with respect to that of the wild-type, implying that the positive charges of C-terminal residues are involved in enzymatic reaction. Furthermore, the sequence alignment of ACC oxidases from 24 different species indicates an existence of the exclusively conserved motif (Lys296-Glu301) especially in the C-terminus. The structure model based on our findings suggests that the positive-charged surface in the C-terminal helix of the ACC oxidase could be a major stabilizer in the spatial arrangement of reactants and that the positive-charge network between the active site and C-terminus is critical for ACC oxidase activity. 相似文献
12.
Krummenacher C Supekar VM Whitbeck JC Lazear E Connolly SA Eisenberg RJ Cohen GH Wiley DC Carfí A 《The EMBO journal》2005,24(23):4144-4153
Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion. 相似文献
13.
The diverse roles of tyrosine residues in proteins may be attributed to their dual hydrophobic and polar nature, which can result in hydrophobic and ring stacking interactions, as well as hydrogen bonding. The small homodimeric DNA binding protein, factor for inversion stimulation (FIS), contains four tyrosine residues located at positions 38, 51, 69, and 95, each involved in specific intra- or intermolecular interactions. To investigate their contributions to the stability, flexibility, and spectroscopic properties of FIS, each one was independently mutated to phenylalanine. Equilibrium denaturation experiments show that Tyr95 and Tyr51 stabilize FIS by about 2 and 1 kcal/mol, respectively, as a result of their involvement in a hydrogen bond-salt bridge network. In contrast, Tyr38 destabilizes FIS by about 1 kcal/mol due to the placement of a hydroxyl group in a hydrophobic environment. The stability of FIS was not altered when the solvent-exposed Tyr69 was mutated. Limited proteolysis with trypsin and V8 proteases was used to monitor the flexibility of the C-terminus (residues 71-98) and the dimer core (residues 26-70), respectively. The results for Y95F and Y51F FIS revealed a different proteolytic susceptibility of the dimer core compared to the C-terminus, suggesting an increased flexibility of the latter. DNA binding affinity of the various FIS mutants was only modestly affected and correlated inversely with the C-terminal flexibility probed by trypsin proteolysis. Deconvolution of the fluorescence contribution of each mutant revealed that it varies in intensity and direction for each tyrosine in WT FIS, highlighting the role of specific interactions and the local environment in determining the fluorescence of tyrosine residues. The significant changes in stability, flexibility, and signals observed for the Y51F and Y95F mutations are attributed to their coupled participation in the hydrogen bond-salt bridge network. These results highlight the importance of tyrosine hydrogen-bonding and packing interactions for the stability of FIS and demonstrate the varying roles that tyrosine residues can play on the structural and spectroscopic properties of even small proteins. 相似文献
14.
Chemical and immunological characterizations of equine infectious anemia virus gag-encoded proteins. 总被引:5,自引:16,他引:5
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The viral core proteins (p15, p26, p11, and p9) of equine infectious anemia virus (EIAV) (Wyoming strain) were purified by reverse-phase high-pressure liquid chromatography. Each purified protein was analyzed for amino acid content, N-terminal amino acid sequence, C-terminal amino acid sequence, and phosphoamino acid content. The results of N- and C-terminal amino acid sequence analysis of each gag protein, taken together with the nucleotide sequence of the EIAV gag gene (R. M. Stephens, J. W. Casey, and N. R. Rice, Science 231:589-594, 1986), show that the order of the proteins in the precursor is p15-p26-*-p11-p9, where a pentapeptide also found in the virus is represented by the asterisk. The data are in complete agreement with the predicted structure of the gag polyprotein and show the peptide bonds cleaved during proteolytic processing. The N-terminus of p15 is blocked to Edman degradation. The p11 protein is identical to the nucleic acid-binding protein of EIAV previously isolated (C. W. Long, L. E. Henderson, and S. Oroszlan, Virology 104:491-496, 1980). High-titer rabbit antiserum was prepared against each purified protein. These antisera were used to detect the putative gag precursor (Pr55gag) and intermediate cleavage products designated Pr49 (p15-p26-*-p11), Pr40 (p15-p26), and Pr35 (p26-*-p11) in the virus and in virus-infected cells. High-titer antisera to EIAV p15 and p26 showed cross-reactivity with the homologous protein of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus. 相似文献
15.
Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein. 总被引:17,自引:12,他引:5
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We have previously demonstrated that the Gag p9 protein of equine infectious anemia virus (EIAV) is functionally homologous with Rous sarcoma virus (RSV) p2b and human immunodeficiency virus type 1 (HIV-1) p6 in providing a critical late assembly function in RSV Gag-mediated budding from transfected COS-1 cells (L. J. Parent et al., J. Virol. 69:5455-5460, 1995). In light of the absence of amino acid sequence homology between EIAV p9 and the functional homologs of RSV and HIV-1, we have now designed an EIAV Gag-mediated budding assay to define the late assembly (L) domain peptide sequences contained in the EIAV p9 protein. The results of these particle budding assays revealed that expression of EIAV Gag polyprotein in COS-1 cells yielded extracellular Gag particles with a characteristic density of 1.18 g/ml, while expression of EIAV Gag polyprotein lacking p9 resulted in a severe reduction in the release of extracellular Gag particles. The defect in EIAV Gag polyprotein particle assembly could be corrected by substituting either the RSV p2b or HIV-1 p6 protein for EIAV p9. These observations demonstrated that the L domains of EIAV, HIV-1, and RSV were interchangeable in mediating assembly of EIAV Gag particles in the COS-1 cell budding assay. To localize the L domain of EIAV p9, we next assayed the effects of deletions and site-specific mutations in the p9 protein on its ability to mediate budding of EIAV Gag particles. Analyses of EIAV Gag constructs with progressive N-terminal or C-terminal deletions of the p9 protein identified a minimum sequence of 11 amino acids (Q20N21L22Y23P24D25L26S27E28I29K30) capable of providing the late assembly function. Alanine scanning studies of this L-domain sequence demonstrated that mutations of residues Y23, P24, and L26 abrogated the p9 late budding function; mutations of other residues in the p9 L domain did not substantially affect the level of EIAV Gag particle assembly. These data indicate that the L domain in EIAV p9 utilizes a YXXL motif which we hypothesize may interact with cellular proteins to facilitate virus particle budding from infected cells. 相似文献
16.
Bestrophins are a new family of anion channels. Here, we examined the Cl channel activity of mBest4. Surprisingly, wild type mouse bestrophin-4 (mBest4) did not induce functional Cl channels when over-expressed in HEK293 cells. However, deletion of part of the C-terminus (residues 353-669) produced large Cl currents, suggesting the presence of a C-terminal motif that inhibited Cl channel function. Deletion of a short motif (356-364) or substitution of certain residues in this motif with alanines also resulted in expression of robust Cl currents. The channel activity of the mBest4 protein lacking the C-terminus (residues 353-669) was specifically inhibited by co-expression of C-terminal fragments of mBest4 having the inhibitory motif, suggesting that the C-terminal motif blocked mBest4 channel activity probably by interacting with the channel pore. 相似文献
17.
The ammonium transport (Amt) proteins are a highly conserved family of integral membrane proteins found in eubacteria, archaea, fungi and plants. Genetic, biochemical and bioinformatic analyses suggest that they have a common tertiary structure comprising eleven trans-membrane helices with an N-out, C-in topology. The cytoplasmic C-terminus is variable in length but includes a core region of some 22 residues with considerable sequence conservation. Previous studies have indicated that this C-terminus is not absolutely required for Amt activity but that mutations that alter C-terminal residues can have very marked effects. Using the Escherichia coli AmtB protein as a model system for Amt proteins, we have carried out an extensive site-directed mutagenesis study to investigate the possible role of this region of the protein. Our data indicate that nearly all mutations fall into two phenotypic classes that are best explained in terms of two distinct effects of the C-terminal region on AmtB activity. Residues within the C-terminus play a significant role in normal AmtB function and the C-terminal region might also mediate co-operativity between the three subunits of AmtB. 相似文献
18.
15N NMR relaxation rates of mini-IGFBP-5, an N-terminal insulin-like growth factor binding domain of the insulin-like growth factor binding protein 5 (IGFBP-5), were analysed at three field strengths using the Lipari-Szabo procedure (see below) and reduced spectral density methods. Isotropic and anisotropic Lipari-Szabo models were analysed and an analytical formula for the overall correlation time for anisotropic molecules is presented. Mini-IGFBP-5 was found to be mainly rigid on fast ps time scales except for 11 unstructured flexible residues at the C-terminus. The insulin-like growth factor binding loop in the apo-protein exhibits small amounts of flexibility on fast time scales (ps to ns) but several loop residues show significant exchange broadening. These loop residues display no exchange broadening in the complex of IGF-II/mini-IGFBP-5. The isotropic overall tumbling time in solution at 31 degrees C of mini-IGFBP-5 complexed to IGF-II is tauc = 18.4 +/- 0.2 ns indicating a strong tendency for aggregation. 相似文献
19.
Sequence-specific 1H NMR assignments for the 36 residue bovine pancreatic polypeptide (bPP) have been completed. The secondary and tertiary structure of bPP in solution has been determined from experimental NMR data. It is shown that bPP has a very well-defined C-terminal alpha-helix involving residues 15-32. Although regular secondary structure cannot be clearly defined in the N-terminal region, residues 4-8 maintain a rather ordered conformation in solution. This is attributed primarily to the hydrophobic interactions between this region and the C-terminal helix. The two segments of the structure are joined by a turn which is poorly defined. The four end residues both at the N-terminus and the C-terminus are highly disordered in solution. The overall fold of the bPP molecule is very closely similar to that found in the crystal structure of avian pancreatic polypeptide (aPP). The RMS deviation for backbone atoms of residues 4-8 and 15-32 between the bPP mean structure and the aPP crystal structure is 0.65 A, although there is only 39% identity of the residues. Furthermore, the average conformations of some (mostly from the alpha-helix) side chains of bPP in solution are closely similar to those of aPP in the crystal structure. A large number of side chains of bPP, however, show significant conformational averaging in solution. 相似文献
20.
Influence of N-terminal acetylation and C-terminal proteolysis on the analgesic activity of beta-endorphin. 总被引:6,自引:1,他引:5
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Removal of one, two and four amino-acid residues from the C-terminus of beta-endorphin ('lipotropin C-Fragment', lipotropin residues 61--91) led to the formation of peptides with progressively decreased analgesic potency; there was no change in the persistence of the analgesic effects. The four C-terminal residues are thus important for the activity of beta-endorphin, but not for the duration of action. Removal of eight amino-acid residues from the N-terminus provided a peptide that had no specific affinity for brain opiate receptors in vitro and was devoid of analgesic properties. The N-terminal sequence of beta-endorphin is therefore necessary for the production of analgesia, whereas the C-terminal residues confer potency. The N alpha-acetyl form of beta-endorphin had no specific affinity for brain opiate receptors in vitro and possessed no significant analgesic properties. Since lipotropin C'-Fragment (lipotropin residues 61--87) and the N alpha-acetyl derivative of beta-endorphin occur naturally in brain and pituitary and are only weakly active or inactive as opiates, it is suggested that proteolysis at the C-terminus and acetylation of the N-terminus of beta-endorphin may constitute physiological mechanisms for inactivation of this potent analgesic peptide. 相似文献