Tryptophan207 is involved in the GTP-dependent conformational switch in the α subunit of the G protein transducin: Chymotryptic digestion patterns of the GTPγS and GDP-bound forms

The limited proteolytic pattern of transducin,G _t , and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the α _t subunit were identified. The α _t subunit in the GTPγS bound form was cleaved into a major 38 kD fragment, whereas α _t -GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The βγ _t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The γ _t subunit was not cleaved and only a small portion of β _t was digested into several fragments. In order to determine which proteolytic fragment of α _t still contained the carboxyl terminal region, chymotrypsinization was carried out usingG _t previously³²P-labeled at Cys³⁴⁷ by petrussis toxin-catalyzed ADP-ribosylation. The³²P-label was mainly associated with the α _t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not³²P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of α _t , at Leu¹⁵ and Leu¹⁹. Chymotrypsin removed 15–19 amino acid residues from the amino terminus of α _t , generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp²⁰⁷ in a conformation-dependent manner. Trp²⁰⁷ of α _t -GTPγS was resistant to proteolysis but α _t -GDP and the 38 kD fragments of α _t -GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus. This proves that the environment of Trp²⁰⁷ changes when GTP or GTPγS is bound, leading to its inaccessibility to chymotrypsin.     

Translation of beta-tubulin mRNA in vitro generates multiple molecular forms

We describe the in vitro expression and characterization of the isolated beta-tubulin subunit in rabbit reticulocyte lysates and compare its assembly and chromatographic properties with that of the isolated alpha-subunit and the tubulin heterodimer. The beta-tubulin polypeptides, derived from a single chicken beta-tubulin cDNA, were found in three distinct molecular forms: a multimeric or lysate-associated form, beta I (Mr approximately 180,000); the free beta-subunit beta II (Mr approximately 55,000); and the hybrid heterodimer alpha(rabbit) beta(chick), beta III (Mr approximately 80,000-100,000). The hybrid heterodimers were 100% assembly competent, whereas beta-tubulin in the "associated" beta I and the monomeric beta II forms displayed only approximately 70 +/- 15 and 25 +/- 10% competence, respectively, in coassembly assays with bovine brain tubulin. This reduced functionality was not a consequence of diminished beta-subunit stability or protein denaturation. By comparing the elution positions of the three beta forms, the monomeric alpha-subunit, and tubulin dimer purified from bovine brain, we demonstrate that anion-exchange columns (Mono-Q) interact preferentially with the alpha-subunit and chromatograph tubulin dimer on the basis of alpha-subunit isotype. The rate of exchange of the free beta-subunit into bovine tubulin dimer was followed chromatographically. The exchange was slow at 4 degrees C and rapid at 37 degrees C where it is essentially complete in 40 min in the presence of 2.5 mg/ml bovine microtubule protein. Exogenous GTP, a potent effector of microtubule assembly, binds exchangeably to beta II and enhances the recovery of this form from the Mono-Q column, suggesting that GTP binding may occur at identical sites in the isolated beta-subunit and in the tubulin heterodimer.     

A model of the nucleotide-binding site in tubulin

Tubulin uses GTP to regulate microtubule assembly and is thought to be a member of a class of GDP/GTP-binding proteins (G-proteins) as defined by Hughes [(1983) Febs Lett. 164, 1-8]. How tubulin is structurally related to G-proteins is not known. We use a synthesis of sequence comparisons between tubulin, other G-proteins, and ADP/ATP-binding proteins and topological arguments to identify potential regions involved in nucleotide binding. We propose that the nucleotide-binding domain in the beta-subunit of tubulin is an alpha/beta structure derived from amino acid residues approximately 60-300. Five peptide sequences are identified which we suggest exist as 'loops' that extend from beta-strands and connect alpha-helices in this structure. We argue that GDP binds to four of the five loops in an Mg2+-independent manner while GTP binds in an Mg2+-dependent manner to a different combination of four loops. We propose that this switch between loops upon GTP binding induces a conformational change essential for microtubule assembly.     

酵母tRNA结合蛋白(43kD蛋白)羧端cDNA的克隆及其序列测定

为了研究酵母分子量为 4 3kD的tRNA结合蛋白的基因来源 ,通过溴化氰部分化学裂解此蛋白 ,产生的 18kD肽段经过蛋白质氨端序列测定 ,测得 18kD肽段氨端部分序列为AFTFKK .针对序列AFTFKK设计简并引物 ,利用简并PCR方法和cDNA的 3′末端的快速扩增方法 (3′RACE) ,成功克隆 4 3kD蛋白mRNA的 3′端序列 .DNA序列测定结果表明 ,该序列位于 3 磷酸甘油酸激酶 (PGK1)基因 10 0 3位— 170 0位 ,长度为 6 98bp ,编码 3 磷酸甘油酸激酶羧基端的 180氨基酸残基以及 3′端非翻译区 .结果证实 ,4 3kD蛋白的基因就是PGK1酶的基因     

Relating nucleotide‐dependent conformational changes in free tubulin dimers to tubulin assembly

The complex dynamic behavior of microtubules (MTs) is believed to be primarily due to the αβ‐tubulin dimer architecture and its intrinsic GTPase activity. Hence, a detailed knowledge of the conformational variations of isolated α‐GTP‐β‐GTP‐ and α‐GTP‐β‐GDP‐tubulin dimers in solution and their implications to interdimer interactions and stability is directly relevant to understand the MT dynamics. An attempt has been made here by combining molecular dynamics (MD) simulations and protein–protein docking studies that unravels key structural features of tubulin dimer in different nucleotide states and correlates their association to tubulin assembly. Results from simulations suggest that tubulin dimers and oligomers attain curved conformations in both GTP and GDP states. Results also indicate that the tubulin C‐terminal domain and the nucleotide state are closely linked. Protein–protein docking in combination with MD simulations suggest that the GTP‐tubulin dimers engage in relatively stronger interdimer interactions even though the interdimer interfaces are bent in both GTP and GDP tubulin complexes, providing valuable insights on in vitro finding that GTP‐tubulin is a better assembly candidate than GDP‐tubulin during the MT nucleation and elongation processes. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 282–291, 2013.     

A photoactive phosphonamide derivative of GTP for the identification of the GTP-binding domain in beta-tubulin

A GTP photoaffinity probe (125I-APTG) was developed that incorporated an [125I]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the gamma-position of GTP through a phosphonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 microM GTP with an apparent Kd of 5 microM) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an 125I radiolabel external to the GTP moiety, in contrast to the previously reported [gamma 32P]-8-N3GTP that possessed an internal 32P radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [125I]APTG was used to identify a peptide (beta:65-79) in the exchangeable GTP-binding domain of the beta-subunit of tubulin.     

Influence of guanine nucleotides on vincristine binding in tumor cytosols and purified tubulin: evidence for an inhibitor of vincristine binding

In cytosols from human rhabdomyosarcoma xenografts, the formation of vincristine (VCR)-tubulin complex and its stability were increased by GTP (Bowman et al.: Biochem. Biophys. Res. Commun., 135:695-700, 1986). We have further examined this modulation to determine whether a) GTP was protecting the VCR binding site from denaturation, b) the enhancement of complex formation was guanosine specific, and c) whether this influence was a direct interaction between GTP, VCR, and tubulin, or was mediated through another factor. In GTP-depleted cytosols from tumor xenografts HxRH18 and HxRh12, VCR binding activity was stable for at least 2 hours at 37 degrees C, indicating that the enhancement of complex formation and stability was not due to protection of tubulin integrity as measured by VCR binding; 10 nM GTP increased complex formation slightly, with complex formation increasing as GTP concentrations were increased to 5 microM, where maximum effect was observed. GTP and GDP (0.1 mM) both increased complex formation three-fold, while GMP, GMP-PNP, and ITP increased formation 1.5-fold. IMP, CTP, and ATP had no significant effect. Therefore, the modulation of VCR binding was relatively specific for the guanine nucleotides GDP and GTP. Microtubule protein, purified from Rh18 and Rh12 tumors by cycles of polymerization-depolymerization, bound VCR rapidly and binding was not influenced by GTP. This suggested that GTP modulation of VCR binding in cytosols was through a soluble factor lost in tubulin purification. In experiments with cytosol fractionated by molecular weight, there was inhibition of VCR binding activity by fractions with an mw range 20-50 kD. This inhibition was decreased by 25% by the addition of GTP. These data suggest that in tumor cytosols there may be competition between VCR and a natural ligand that is modulated by GTP. Two potential models for VCR binding are proposed.     

Role of GTP hydrolysis in microtubule polymerization: evidence for a coupled hydrolysis mechanism

The relationship between GTP hydrolysis and microtubule assembly has been investigated by using a rapid filtration method. Microtubules assembled from phosphocellulose-purified tubulin, double-labeled with [gamma-32P]- and [3H]GTP, were trapped and washed free of unbound nucleotide on glass fiber filters. The transient accumulation of microtubule-bound GTP predicted by uncoupled GTP hydrolysis models [Carlier & Pantaloni (1981) Biochemistry 20, 1918-1924; Carlier et al. (1987) Biochemistry 26, 4428-4437] during the rapid assembly of microtubules was not detectable under our experimental conditions. By calculating hypothetical time courses for the transient accumulation of microtubule-bound GTP, we demonstrate that microtubule-bound GTP would have been detectable even if the first-order rate constant for GTP hydrolysis were 4-5 times greater than the pseudo-first-order rate constant for tubulin subunit addition to microtubules. In a similar manner, we demonstrate that if GTP hydrolysis were uncoupled from microtubule assembly but were limited to the interface between GTP subunits and GDP subunits (uncoupled vectorial hydrolysis), then microtubule-bound GTP would have been detectable if GTP hydrolysis became uncoupled from microtubule assembly at less than 50 microM free tubulin, 5 times the steady-state tubulin concentration of our experimental conditions. In addition, during rapid microtubule assembly, we have not detected any microtubule-bound Pi, which has been proposed to form a stabilizing cap at the ends of microtubules [Carlier et al. (1988) Biochemistry 27, 3555-3559]. Also, several conditions that could be expected to increase the degree of potential uncoupling between GTP hydrolysis and microtubule assembly were examined, and no evidence of uncoupling was found. Our results are consistent with models that propose cooperative mechanisms that limit GTP hydrolysis to the terminal ring of tubulin subunits [e.g., O'Brien et al. (1987) Biochemistry 26, 4148-4156]. The results are also consistent with the hypothesis that a slow conformational change in tubulin subunits after GTP hydrolysis and Pi release occurs that results in destabilized microtubule ends when such subunits become exposed at the ends.     

Microtubule-binding domain of tau proteins

Limited chymotryptic digestion of whole tau proteins produced a fragment of Mr 14,000 (CT14), which was able to bind to microtubules reconstituted from tubulin alone in the presence of taxol. This fragment was also found to persist in microtubules when microtubules consisting of tau proteins and tubulin were digested by chymotrypsin. Analysis of amino acid composition revealed that CT14 was rich in lysine and proline residues, suggesting unique structure of microtubule-binding domain of tau proteins. Amino-terminal sequence of CT14 was determined to be Ser-Ser-Pro-Gly-Ser-Pro-Gly-Thr-Pro-Gly-Ser-Arg-Ser-Arg-X-Pro-Ser-Leu-Pr o. No heterogeneity was detected in this amino-terminal sequence of 19 residues. Five species of polypeptides consisting of tau proteins were separated from each other by gel electrophoresis and subjected to chymotryptic digestion. CT14 was produced from each of the tau polypeptides by chymotryptic digestion, indicating that all tau polypeptides have a common microtubule-binding domain.     

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     

Identification of an 80kD Protein Associated with the α3β1 Integrin as a Proteolytic Fragment of the α3 Subunit: Studies with Human Keratinocytes

We have characterised a protein of approximately 80kD previously observed to co-immunoprecipitate with the α₃β₁ integrin in lysates of surface labelled human epiderrnalkerati-nocytes. The 80kD protein only appeared when keratinocytes were harvested with trypsin/EDTA prior to lysis and a protein of similar molecular mass could be immunoprecipitated from human dermal fibroblasts following treatment of the cells with trypsin/EDTA. N terminal sequencing established that the 80kD protein had homology with the as integrin subunit. Peptide-mass fingerprinting was used to confirm that the protein comprised the amino terminus of α₃ and established that the site of cleavage was after amino acid 629. The 80kD fragment could be coimmunoprecipitated with α₃β₁ using an antibody to the cytoplasmic domain of the α₃ subunit, showing that the fragment remained complexed with intact α₃β₁. When antibodies to the cytoplasmic and extracellular domains of α₃ were used to label human epidermis by immunofluorescence, the staining patterns were indistinguishable and there is therefore no evidence that proteolysis of α₃ plays a role in keratinocyte detachment from the basement membrane during terminal differentiation. Whether the 80kD fragment has any effects, positive or negative, on α₃β₁-mediated adhesion remains to be determined.     

鸡含锰超氧化物歧化酶cDNA克隆及序列分析

 为弄清鸡含锰超氧化物歧化酶 (manganese containingsuperoxidedismutase ,MnSOD)的cDNA序列 ,以开展动物锰营养学的深入研究 ,根据已知鸡MnSOD的N端氨基酸序列设计简并引物 ,应用 3′RACE(rapidamplificationofcDNAends)技术 ,扩增克隆了鸡心肌MnSOD 990bp的 3′cDNA片段 .再根据 3′RACE片段测序结果设计引物进行 5′RACE ,结果获取了一个与 3′RACE片段相互重叠的鸡心肌MnSOD 52 1bp的 5′RACE片段 ,并对其进行了克隆测序 .最后根据 3′RACE片段和 5′RACE片段序列信息进行拼接 ,从而获取鸡MnSODcDNA的全序列信息 .研究结果表明 :鸡MnSODcDNA全长为 110 8个核苷酸 ,其中 5′非翻译区 2 5个核苷酸 ,编码区 675个核苷酸 ,3′非翻译区 4 0 8个核苷酸 ,编码一个长 2 2 4个氨基酸残基的蛋白质前体 .其中信号肽长 2 6个氨基酸残基 ,成熟肽长 198个氨基酸残基 ,分子量为 2 2kD .与人、大鼠、线虫、果蝇等真核生物MnSOD氨基酸序列的同源性分别为82 4 %、84 .7%、62 .4 %、59.3% .     

人内皮抑素在毕赤酵母中的表达、纯化与生物功能研究

内皮抑素（Endostatin）是近年来新发现的一种内源性新生血管生成（Angiogenesis）抑制因子,通过抑制新血管生成而抑制肿瘤的形成和转移且不会引起耐药性,具有极高的临床应用前景。巴斯德毕赤酵母（Pichia pastoris)具有表达率高、产物可分泌、可对高等真核生物蛋白正确进行翻译后加工、遗传稳定、发酵工艺成熟等优点被用来进行重组人Endostatin的表达。本研究用PCR的方法从人胎肝cDNA文库中扩增出人Endostatin的cDNA,测序正确后转入毕赤巴斯德甲醇酵母,并获得了高效可溶型表达,用肝素亲和层析的方法进行纯化,纯化后产物经SDSPAGE薄层扫描分析纯度达987％以上,质谱测定分子量为2043kD与理论值一致,蛋白质N端序列测定结果为SPPAHTHRDFQPVLH与天然序列一致。生物活性检测证明可抑制鸡胚尿囊绒毛膜（CAM）的新生血管生成（Angiogenesis),并可抑制血管内皮细胞的增殖。因此用酵母表达系统可以得到具有生物活性的内皮抑素,经纯化后可用于进一步的生物功能和作用机理试验。     

Stimulation of Tubulin Synthesis by Lactate in Isolated Spermatogenic Cells

The effect of lactate on synthesis of new proteins in isolated spermatids and spermatocytes of rats was examined. Lactate stimulated[³⁵S]methionine ([³⁵S]met) incorporation into both spermatids and spermatocytes. The rate of protein synthesis was positively correlated with the intracellular level of ATP. The [³⁵S]met-labeled proteins in the two types of cells were compared by one and two dimensional polyacrylamide gel electrophoresis (1D and 2D-PAGE) and autoradiography. The syntheses of several stagespecific and non-specific proteins were observed. When spermatids and spermatocytes were cultured in medium without lactate, two major proteins of molecular weight (Mr) 43 kD and 55 kD were detected in the water-soluble fraction (105,000 g supernatant), and one major protein of Mr 24 kD was observed in the membrane-rich fraction. Addition of lactate to the incubation medium dramatically increased the synthesis of six proteins (Mr 14 kD, 16 kD, 43 kD, 55 kD, 84 kD and 135 kD) in the water-soluble fractions of spermatids and spermatocytes, but did not stimulate the synthesis of the Mr 24 kD protein in the membrane-rich fraction. In addition, after 1D and 2D-PAGE and electrophoretic transfer to nitrocellulose, two proteins of Mr 43 kD and 55 kD were identified as actin and tubulin, respectively, on the basis of their reactivities with specific antisera. Tubulin was also produced by in vitro translation using a spermatid lysate. These results suggest that lactate may play an important role in changing the cell structure and shape during spermatogenesis by regulating the syntheses of actin and tubulin.     

Sequence Analysis of a Proteolyzed Site in Drosophila Choline Acetyltransferase

The amino terminal sequence of the 13-kilodalton (kD) polypeptide present in purified Drosophila acetyl-CoA: choline-O-acetyltransferase (EC 2.3.1.6) was determined, and its position in the sequence of the intact enzyme was located. Enzyme polypeptides for sequencing were obtained from native enzyme protein by denaturation, followed by fractionation on reverse-phase HPLC. The 13-, 54-, and 67-kD polypeptides recovered from the separation were subjected to amino terminal sequencing. Only the 13-kD fragment yielded a sequence. The 67- and 54-kD polypeptides appeared completely blocked to gas-phase Edman sequencing. The location of the amino terminal sequence from the 13-kD polypeptide in the cDNA-deduced enzyme sequence indicated that this fragment represents the carboxyl portion of the 67-kD enzyme, with the 54-kD polypeptide providing the amino terminal portion. The proteolysis that gave rise to the 13-kD polypeptide occurred at the carboxyl side of a monobasic lysine residue. An earlier comparison of the enzyme from Drosophila and pigs indicated that the cleaved lysine may be a conserved residue in the porcine enzyme. The cleaved enzyme region characterized in this study does not coincide with the regions of high homology found in the two enzymes, but hydrophilicity profiles generated for this area showed similarities.     

Microtubule elongation and guanosine 5'-triphosphate hydrolysis. Role of guanine nucleotides in microtubule dynamics

The tubulin concentration dependence of the rates of microtubule elongation and accompanying GTP hydrolysis has been studied over a large range of tubulin concentration. GTP hydrolysis followed the elongation process closely at low tubulin concentration and became gradually uncoupled at higher concentrations, reaching a limiting rate of 35-40 s-1. The kinetic parameters for microtubule growth were different at low and high tubulin concentrations. Elongation of microtubules has also been studied in solutions containing GDP and GTP in variable proportions. Only traces of GTP present in GDP were necessary to confer a high stability (low critical concentration) to microtubules. Pure GDP-tubulin was found unable to elongate microtubules in the absence of GTP but blocked microtubule ends with an equilibrium dissociation constant of 5-6 microM. These data were accounted for by a model within which, in the presence of GTP-tubulin at high concentration, microtubules grow at a fast rate with a large GTP cap; the GTP cap may be quite short in the region of the critical concentration; microtubule stability is linked to the strong interaction between GTP and GDP subunits at the elongating site; dimeric GDP-tubulin does not have the appropriate conformation to undergo reversible polymerization. These results are discussed with regard to possible role of GDP and GTP and of GTP hydrolysis in microtubule dynamics.     

On the role of the tubulin nonexchangeable GTP site in bovine neurotubule assembly.

A procedure for radiolabeling the terminal phosphoryl group of the tubulin nonexchangeable GTP site using bacterial acetate kinase and acetyl-32P is described. Warming such samples to 37 degrees results in microtubule assembly and hydrolysis of the nonexchangeable site GTP in a parallel fashion. Removal of the microtubule-associated protein fraction from lebeled tubulin prevents hydrolysis and assembly, and recombination of these components restores both processes again in a parallel fashion. These and other experiments indicate that the nonexchangeable site GTP hydrolysis and assembly are intimately linked. The experiments also demonstrate that GRP is not required at the exchangeable nucleotide site for assembly to occur.     

Characterization of a cleavage product in the human choriogonadotropin beta-subunit

The various molecular forms of human chorionic gonadotropin present in a crude preparation of urine from pregnant women were analyzed by two-dimensional gel electrophoresis and immunoblotting with monoclonal antibodies directed to synthetic peptides corresponding to the carboxyl-terminal part of either the alpha or beta-subunit. Under reducing conditions, immunoblotting with antibodies directed to the beta-subunit revealed the presence of a low-molecular-weight material of 22 kDa. This molecular form had large heterogeneity, as analyzed by isoelectrofocusing; it was immunoreactive with antibodies directed to the 111-145 region. Using microsequencing techniques, we found that the fragment had a NH2 terminal portion corresponding to the sequence of the beta-subunit appearing from residue 48. Thus, the 22-kDa fragment comprises the 48-145 portion of the beta-subunit and is probably a cleavage product of the native protein with intrachain nicking.