Biomedical subjects

R Golbik

Publications and source records attributed to R Golbik.

32 records · Page 2Linked to original sources

PubMed1997

Refolding chromatography with immobilized mini-chaperones.

Mini-chaperones (e.g., a peptide consisting of residues 191-345 of GroEL) that are immobilized on agarose have very efficient chaperoning activity with several proteins that are otherwise recalcitrant to renaturation by conventional methods. We have used immobilized mini-chaperones both in column chromatography and batchwise to renature an insoluble protein from an inclusion body, to refold apparently irreversibly denatured proteins, and to recondition enzymes that have lost activity on storage. Refolding chromatography offers an efficient and simple means to renature proteins in high yield and with biological activity.

Chaperonin 60↗

PubMed1997

Fluorescence properties of a tryptophan residue in an aromatic core of the protein subunit of ribonuclease P from Escherichia coli.

Escherichia coli ribonuclease P (RNase P), a ribonucleoprotein complex which primarily functions in tRNA biosynthesis, is composed of a catalytic RNA subunit, M1 RNA, and a protein cofactor, C5 protein. The fluorescence emission spectrum of the single tryptophan residue-containing C5 protein exhibits maxima at 318 nm and 332 nm. Based on a comparison of the emission spectra of wild-type C5 protein and some of its mutant derivatives, we have determined that the 318 nm maximum could be the result of a complex formed in the excited state as a result of hydrophobic interactions between Trp109, Phe18 and Phe73. The analogous tryptophan fluorescence emission spectra of wild-type C5 protein and the barstar mutant W38F/W44F, taken together with the detailed structural information available for barstar, provide a possible explanation for the unusual emission spectrum of C5 protein.

Bacterial Proteins↗

PubMed1997

The folding pathway of a protein at high resolution from microseconds to seconds.

We have documented the folding pathway of the 10-kDa protein barstar from the first few microseconds at the resolution of individual residues from its well characterized denatured state. The denatured state had been shown from NMR to have flickering native-like structure in the first two of its four alpha-helices. phi-value analysis shows that the first helix becomes substantially consolidated as the intermediate is formed in a few hundred microseconds, as does the second to a lesser extent. A native-like structure then is formed in a few hundred milliseconds as the whole structure consolidates. Peptide fragments corresponding to sequences containing the first two helices separately and together as a helix-loop-helix motif have little helical structure under conditions that favor folding. The early stages of folding fit the nucleation-condensation model that was proposed for the smaller chymotrypsin inhibitor 2, which is a single module of structure and folds by two-state kinetics. The early stages of the multistate folding of the larger, multimodular, barnase have proved experimentally inaccessible. The folding pathway of barstar links those of CI2 and barnase to give a unified scheme for folding.

Bacterial Proteins↗

PubMed1996

Chaperone activity and structure of monomeric polypeptide binding domains of GroEL.

The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions.

Allosteric Regulation↗

PubMed1996

Mutations that suppress the thermosensitivity of green fluorescent protein.

BACKGROUND: The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently attracted great interest as the first example of a cloned reporter protein that is intrinsically fluorescent. Although successful in some organisms, heterologous expression of GFP has not always been straight forward. In particular, expression of GFP in cells that require incubation temperatures around 37 degrees C has been problematic. RESULTS: We have carried out a screen for mutant forms of GFP that fluoresce more intensely than the wild-type protein when expressed in E. coli at 37 degrees C. We have characterized a bright mutant (GFPA) with reduced sensitivity to temperature in both bacteria and yeast, and have shown that the amino acids substituted in GFPA act by preventing temperature-dependent misfolding of the GFP apoprotein. We have shown that the excitation and emission spectra of GFPA can be manipulated by site-directed mutagenesis without disturbing its improved folding characteristics, and have produced a thermostable folding mutant (GFP5) that can be efficiently excited using either long-wavelength ultraviolet or blue light. Expression of GFP5 results in greatly improved levels of fluorescence in both microbial and mammalian cells cultured at 37 degrees C. CONCLUSIONS: The thermotolerant mutants of GFP greatly improve the sensitivity of the protein as a visible reporter molecule in bacterial, yeast and mammalian cells. The fluorescence spectra of these mutants can be manipulated by further mutagenesis without deleteriously affecting their improved folding characteristics, so it may be possible to engineer a range of spectral variants with improved tolerance to temperature. Such a range of sensitive reporter proteins will greatly improve the prospects for GFP-based applications in cells that require relatively high incubation temperatures.

Amino Acid Sequence↗

PubMed1995

Submillisecond events in protein folding.

The pathway of protein folding is now being analyzed at the resolution of individual residues by kinetic measurements on suitably engineered mutants. The kinetic methods generally employed for studying folding are typically limited to the time range of > or = 1 ms because the folding of denatured proteins is usually initiated by mixing them with buffers that favor folding, and the dead time of rapid mixing experiments is about a millisecond. We now show that the study of protein folding may be extended to the microsecond time region by using temperature-jump measurements on the cold-unfolded state of a suitable protein. We are able to detect early events in the folding of mutants of barstar, the polypeptide inhibitor of barnase. A preliminary characterization of the fast phase from spectroscopic and phi-value analysis indicates that it is a transition between two relatively solvent-exposed states with little consolidation of structure.

Bacterial Proteins↗

PubMed1995

Structural requirement for cell adhesion to kalinin (laminin-5).

Laminin-5 (kalinin) was purified from spent cell culture media (SCC25 cells) by affinity chromatography on monoclonal antibody BM165. The protein was recovered as a mixture of the typical polypeptides of 165-155, 140, and 105 kDa as judged b SDS-polyacrylamide gel electrophoresis analysis under reducing conditions. The amino acid composition of purified laminin-5 was in agreement with that compiled from the recently published cDNA sequences of the alpha 3-, beta 3-, and gamma 2-laminin chains. Moreover, the content of half-cystine residues in laminin-5 was about two-thirds that in laminin-1, which confirms the prediction of a smaller number of epidermal growth factor-like repeats in the amino-terminal portion of the three chains. The content of coiled-coil alpha-helices (27%) determined by CD spectroscopy was comparable to that reported for laminin-1, which indicates that the long arm portion of laminin-5 is equivalent to that of other laminin isoforms. The melting temperature was recorded at 72 degrees C by CD monitoring of unfolding and refolding of the coiled-coil structures during thermal denaturation and renaturation, respectively. The thermal stability of laminin-5 is therefore significantly higher than that of laminin-1 or alpha 2-chain-containing laminins, which suggests higher ionic interactions between the three polypeptide chains of laminin-5. Cell adhesion-promoting activity of laminin-5 was found to be strictly and entirely dependent on the presence of coiled-coil structures. It decreased gradually after heat denaturation of the protein above 65 degrees C and was totally abrogated at 75 degrees C. This is in contrast to laminin-1, which contains both conformation-dependent and -independent cell-binding sites on the long and short arm domains, respectively.

Binding Sites↗

PubMed1995

Tetrabrachion: a filamentous archaebacterial surface protein assembly of unusual structure and extreme stability.

The surface (S-) layer of the hyperthermophilic archaebacterium Staphylothermus marinus was isolated, dissected into separate domains by chemical and proteolytic methods, and analyzed by spectroscopic, electron microscopic and biochemical techniques. The S-layer is formed by a poorly ordered meshwork of branched, filiform morphological subunits resembling dandelion seed-heads. A morphological subunit (christened by us tetrabrachion) consists of a 70 nm long, almost perfectly straight stalk ending in four straight arms of 24 nm length that provide lateral connectivity by end-to-end contacts. At 32 nm from the branching point, tetrabrachion carries two globular particles of 10 nm diameter that have both tryptic and chymotryptic protease activity. Tetrabrachion is built by a tetramer of M(r) 92,000 polypeptides that form a parallel, four-stranded alpha-helical rod and separate at one end into four strands. These strands interact in a 1:1 stoichiometry with polypeptides of M(r) 85,000 to form the arms. The arms are composed entirely of beta-sheets. All S-layer components contain bound carbohydrates (glucose, mannose, and glucosamine) at a ratio of 38 g/100 g protein for the complete tetrabrachion-protease complex. The unique structure of tetrabrachion is reflected in an extreme thermal stability in the presence of strong denaturants (1% (w/v) SDS of 6M guanidine): the arms, which are stabilized by intramolecular disulphide bridges, melt around 115 degrees C under non-reducing conditions, whereas the stalk sustains heating up to about 130 degrees C. Complete denaturation of the stalk domain requires treatment with 70% (v/v) sulfuric acid or with fuming trifluoromethanesulfonic acid. The globular protease can be heated to 90 degrees C in 6M guanidine and to 120 degrees C in 1% SDS and represents one of the most stable proteases characterized to date.

Archaea↗

PubMed1994

Recombinant expression and structural and binding properties of alpha 1(VI) and alpha 2(VI) chains of human collagen type VI.

Full-length alpha 1(VI) and alpha 2(VI) cDNAs in an eukaryotic expression vector were used to obtain stably transfected human kidney cell clones and to purify these collagen-VI chains in substantial quantities from the culture medium. Both chains appeared mainly as monomers together with some dimers that were disulfide linked through their C-terminal globular domains. Despite sufficient hydroxylation of proline and lysine residues, the chains did not form a triple-helix, as shown by electronmicroscopy, CD spectra and pepsin sensitivity. Digestion of the chains with bacterial collagenase released the N-terminal and C-terminal globular domains, which were identified by their size and partial sequences. They showed a substantial content of alpha-helical conformation and a distinct globular structure after rotary shadowing. Antibodies could be raised that distinguished between the two chains and reacted with the globular domains. The alpha 2(VI) but not the alpha 1(VI) chain showed binding to a heparan sulfate proteoglycan (perlecan), fibronectin and pepsin-solubilized collagen VI. Purified globular domains did not bind these ligands indicating the localization of binding sites within the triple-helical domain. Both chains showed a distinct affinity for heparin but failed to bind to various collagen types.

Amino Acid Sequence↗

PubMed1994

Structure and stability of the triple-helical domains of human collagen XIV.

Two triple-helical domains, Col 1 and Col 2, were obtained from a pepsin digest of human placental collagen XIV and separated from each other under nondenaturing conditions. Edman degradation demonstrated 106 amino acids residues in the Col 1 and 149 residues in the Col 2 domain. All except one of the 37 prolines in the Yaa position of the Gly-Xaa-Yaa triplets were completely hydroxylated to 4-hydroxyproline, and there were three imperfections in the triplet repeat. Partial or complete hydroxylation and glycosylation were found for all seven lysines in the Yaa position. Domain Col 1 was joined by disulfide bonds into a trimer, while Col 2 appeared as a mixture of monomers and disulfide-linked dimers. Circular dichroic spectra were typical for the collagen triple helix and revealed relatively high melting temperatures for Col 1 (38 degrees C) and Col 2 (43 degrees C). An almost perfect refolding of the triple helix was observed for Col 1 but not for Col 2, emphasizing the importance of disulfide bonds for the folding kinetics and in part the stability of the triple helix. Circular dichroic spectra of the large nontriple helical domain, NC3, of collagen XIV indicated 11% alpha helix and 63% beta structure. Comparative melting profiles of NC3 and intact collagen XIV indicated that the triple helices in intact collagen XIV have a melting temperature of 44 degrees C.

Amino Acid Sequence↗

PubMed1993

Interaction of type IV collagen with the isolated integrins alpha 1 beta 1 and alpha 2 beta 1.

The triple-helical cyanogen-bromide-derived fragment CB3[IV] of collagen IV, located 100 nm from the N-terminus of the molecule, contains the binding sites for the integrins alpha 1 beta 1 and alpha 2 beta 1. To investigate the interaction of these integrins and collagen IV, we performed solid-phase and inhibition assays using as receptor isolated alpha 1 beta 1 and alpha 2 beta 1. The ligands used were the binding-site-bearing trimeric peptide CB3[IV] and its shorter tryptic fragments F1-F4. Using titration curves, in which the binding of soluble receptors to coated ligands and the binding of soluble ligands to coated receptors were analyzed, the binding sites for alpha 1 beta 1 and alpha 2 beta 1 were in different but adjacent areas of CB3[IV]. Triple-helical conformation and distinct primary structures were required for the interaction. Dissociation constants (Kd), for the affinity of integrins for collagen IV, were determined in the 1-nM range in the presence of Mn2+ and Mg2+. In the absence of Mn2+, the Kd values indicated a 30-60-fold decrease in the affinities, which for alpha 2 beta 1 was further reduced by adding Ca2+. In the presence of Ca2+ and Mg2+ the affinity of collagen IV for alpha 1 beta 1 was four-times higher than for alpha 2 beta 1.

Amino Acid Sequence↗

PubMed1993

The alpha 1 beta 1 integrin recognition site of the basement membrane collagen molecule [alpha 1(IV)]2 alpha 2(IV).

Cells interact with type IV collagen mainly via the integrins alpha 1 beta 1 and alpha 2 beta 1. A triple helical CNBr derived fragment CB3[IV], which contains the recognition sites for both integrins, was isolated from type IV collagen. Trypsin treatment of CB3[IV] gave rise to four smaller fragments, F1-F4, of which the smallest one, F4, contained the recognition site for alpha 1 beta 1. Further fragmentation of F4 by thermolysin treatment at 50 degrees C led to fragment TL1, which represents the C-terminal half of F4, and which was no longer able to interact with alpha 1 beta 1. Therefore the recognition site of alpha 1 beta 1 had to be located within the N-terminal half of F4, a position which was verified by electron micrographs of a crosslinked F2-alpha 1 beta 1 complex. Modification of the Arg and Asp residues, which abolished the binding activity of F4, led to the identification of Arg (461) within the alpha 2(IV) and Asp (461) within the alpha 1 (IV) chain as essential residues for the alpha 1 beta 1. The array of these two residues on the surface of the triple helix is discussed.

Amino Acid Sequence↗

PubMed1993

Effects of metal ions, thiamine diphosphate analogues and subunit interactions on the reconstitution behaviour of pyruvate decarboxylase from brewer's yeast.

The reconstitution of pyruvate decarboxylase starts with reversible binding of thiamine diphosphate and Mg2(+)-ions to the apoenzyme, followed by a rate-limiting conformational change to the catalytically active holoenzyme. Investigations with diphospho-esters of 4-methyl-5-(2-hydroxyethyl)thiazolium derivatives have shown that the diphosphate residue of thiamine diphosphate is the most important part of the coenzyme responsible for the first reversible binding step. Methylation of the N1'-atom of the pyrimidine ring of thiamine diphosphate or 4'-oxythiamine diphosphate prevents the coenzyme from binding stably to the apoenzyme, so that the methylated coenzyme displays no coenzyme activity. In contrast, thiamine diphosphate analogues with bulky residues on the neighbouring C2'-atom of the pyrimidine ring form active holoenzyme complexes. This result shows the essential role of the N1'-atom of thiamine diphosphate in stable cofactor binding. The cofactor binding rate to the dimeric and tetrameric apoenzymes indicates that the cofactor is located in the contact regions of the subunits in the tetrameric enzyme.

Binding Sites↗

PubMed1992

A method for determination of transketolase activity based on the use of a pH indicator.

A new method for assaying transketolase activity is proposed. The method consists in recording the pH changes in the course of the enzymatic reaction and is based on the use of the pH-indicator p-nitrophenol. When p-nitrophenol is added to a reaction mixture containing hydroxypyruvate and glycolaldehyde as substrates the absorbance increases. The rate of the change of absorbance is proportional to the enzyme concentration.

Hydrogen-Ion Concentration↗