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S M van der Vies

Publications and source records attributed to S M van der Vies.

At least 19 recordsLinked to original sources
PubMed2001

The chaperonins of Synechocystis PCC 6803 differ in heat inducibility and chaperone activity.

The chaperonins GroEL and Cpn60 were isolated from the cyanobacterium Synechocystis PCC 6803 and characterized. In cells grown under optimal conditions their ratio was about one to one. However, the amount of GroEL increased considerably more than that of Cpn60 in response to heat stress. The labile chaperonin oligomer required stabilization by MgATP or glycerol during isolation. Use of the E. coli mutant strain, groEL44 revealed that the functional properties of the two cyanobacterial chaperonins are strikingly different. Overexpression of cyanobacterial GroEL in the E. coli mutant strain allowed growth at elevated temperature, the formation of mature bacteriophage T4, and active Rubisco enzyme assembly. In contrast, Cpn60 partially complemented the temperature-sensitive phenotype, the Rubisco assembly defect and did not promote the growth of the bacteriophage T4. The difference in chaperone activity of the two cyanobacterial chaperonins very probably reflects the unique chaperonin properties required during the life of Synechocystis PCC 6803.

Bacteriophage T4↗
PubMed1999

Compensatory changes in GroEL/Gp31 affinity as a mechanism for allele-specific genetic interaction.

Previous work has shown that the GroEL-GroES interaction is primarily mediated by the GroES mobile loop. In bacteriophage T4 infection, GroES is substituted by the gene 31-encoded cochaperonin, Gp31. Using a genetic selection scheme, we have identified a new set of mutations in gene 31 that affect interaction with GroEL; all mutations result in changes in the mobile loop of Gp31. Biochemical analyses reveal that the mobile loop mutations alter the affinity between Gp31 and GroEL, most likely by modulating the stability of the GroEL-bound hairpin conformation of the mobile loop. Surprisingly, mutations in groEL that display allele-specific interactions with mutations in gene 31 alter residues in the GroEL intermediate domain, distantly located from the mobile loop binding site. The observed patterns of genetic and biochemical interaction between GroES or Gp31 and GroEL point to a mechanism of genetic allele specificity based on compensatory changes in affinity of the protein-protein interaction. Mutations studied in this work indirectly alter affinity by modulating a folding transition in the Gp31 mobile loop or by modulating a hinged conformational change in GroEL.

Alleles↗
PubMed1997

Structural adaptations in the specialized bacteriophage T4 co-chaperonin Gp31 expand the size of the Anfinsen cage.

The Gp31 protein from bacteriophage T4 functionally substitutes for the bacterial co-chaperonin GroES in assisted protein folding reactions both in vitro and in vivo. But Gp31 is required for the folding and/or assembly of the T4 major capsid protein Gp23, and this requirement cannot be satisfied by GroES. The 2.3 A crystal structure of Gp31 shows that its tertiary and quaternary structures are similar to those of GroES despite the existence of only 14% sequence identity between the two proteins. However, Gp31 shows a series of structural adaptations which will increase the size and the hydrophilicity of the "Anfinsen cage," the enclosed cavity within the GroEL/GroES complex that is the location of the chaperonin-assisted protein folding reaction.

Amino Acid Sequence↗
PubMed1996

Interplay of structure and disorder in cochaperonin mobile loops.

Protein-protein interactions typically are characterized by highly specific interfaces that mediate binding with precisely tuned affinities. Binding of the Escherichia coli cochaperonin GroES to chaperonin GroEL is mediated, at least in part, by a mobile polypeptide loop in GroES that becomes immobilized in the GroEL/GroES/nucleotide complex. The bacteriophage T4 cochaperonin Gp31 possesses a similar highly flexible polypeptide loop in a region of the protein that shows low, but significant, amino acid similarity with GroES and other cochaperonins. When bound to GroEL, a synthetic peptide representing the mobile loop of either GroES or Gp31 adopts a characteristic bulged hairpin conformation as determined by transferred nuclear Overhauser effects in NMR spectra. Thermodynamic considerations suggest that flexible disorder in the cochaperonin mobile loops moderates their affinity for GroEL to facilitate cycles of chaperonin-mediated protein folding.

Amino Acid Sequence↗
PubMed1995

Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin 60.

Chloroplasts contain a 21-kDa co-chaperonin polypeptide (cpn21) formed by two GroES-like domains fused together in tandem. Expression of a double-domain spinach cpn21 in Escherichia coli groES mutant strains supports growth of bacteriophages lambda and T5, and will also suppress a temperature-sensitive growth phenotype of a groES619 strain. Each domain of cpn21 expressed separately can function independently to support bacteriophage lambda growth, and the N-terminal domain will additionally suppress the temperature-sensitive growth phenotype. These results indicate that chloroplast cpn21 has two functional domains, either of which can interact with GroEL in vivo to facilitate bacteriophage morphogenesis. Purified spinach cpn21 has a ring-like toroidal structure and forms a stable complex with E. coli GroEL in the presence of ADP and is functionally interchangeable with bacterial GroES in the chaperonin-facilitated refolding of denatured ribulose-1,5-bisphosphate carboxylase. Cpn21 also inhibits the ATPase activity of GroEL. Cpn21 binds with similar efficiency to both the alpha and beta subunits of spinach cpn60 in the presence of adenine nucleotides, with ATP being more effective than ADP. The tandemly fused domains of cpn21 evolved early and are present in a wide range of photosynthetic eukaryotes examined, indicating a high degree of conservation of this structure in chloroplasts.

Adenosine Triphosphatases↗
PubMed1994

Bacteriophage T4 encodes a co-chaperonin that can substitute for Escherichia coli GroES in protein folding.

Several bacteriophages use the Escherichia coli GroES and GroEL chaperonins for folding and assembly of their morphogenetic structures. Bacteriophage T4 is unusual in that it encodes a specialized protein (Gp31) that is thought to interact with the host GroEL and to be absolutely required for the correct assembly of the major capsid protein (Gp23) in vivo. Here we show that despite the absence of amino-acid sequence similarity between Gp31 and GroES, Gp31 can functionally substitute for the GroES co-chaperonin in the morphogenesis of bacteriophages lambda and T5, the in vivo and in vitro chaperonin-dependent assembly of ribulose bisphosphate carboxylase (Rubisco), as well as overall bacterial growth at the non-permissive temperature. Like GroES, the bacteriophage Gp31 protein forms a stable complex with the E. coli GroEL protein in the presence of Mg-ATP and inhibits the ATPase activity of GroEL in vitro.

Bacterial Proteins↗
PubMed1993

Chaperonin-mediated reconstitution of the phytochrome photoreceptor.

The native phytochrome photoreceptor was purified to homogeneity from etiolated seedlings of oat (Avena sativa L.) and used for renaturation experiments. Light scattering measurements showed that the GroEL molecular chaperone interacts with non-native phytochrome to suppress aggregation of the refolding polypeptide, following its dilution from a chaotrope. The binary complex formed between non-native phytochrome and GroEL was stable and could be isolated by size exclusion chromatography. Discharge of the photoreceptor from GroEL was obtained with 2 mM MgATP, although 6 mM adenosine 5'-O-(3-thiotriphosphate) was also effective. Phytochrome released from GroEL with MgATP was found primarily in the form of 124-kDa monomers, as judged by size exclusion chromatography and nondenaturing gel electrophoresis, although dimers and other oligomeric forms were also observed. The reconstituted dimers, and other oligomeric forms, preferentially cross-reacted with a monoclonal antibody that recognizes native-like epitopes. In vitro folding reactions, using chemically denatured phytochrome, revealed that successful reconstitution of the photoreceptor required the presence of the GroEL chaperonin and MgATP under the conditions tested. Reconstitution in the presence of GroEL yielded phytochrome that could exhibit photoreversibility between the red-light absorbing and far-red absorbing forms.

Adenosine Triphosphate↗
PubMed1992

Conformational states of ribulosebisphosphate carboxylase and their interaction with chaperonin 60.

Conformational states of ribulosebisphosphate carboxylase (Rubisco) from Rhodospirillum rubrum were examined by far-UV circular dichroism (CD), tryptophan fluorescence, and 1-anilino-naphthalenesulfonate (ANS) binding. At pH 2 and low ionic strength (I = 0.01), Rubisco adopts an unfolded, monomeric conformation (UA1 state) as judged by far-UV CD and tryptophan fluorescence. As with other acid-unfolded proteins [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577], an intermediate conformation (A1 state) is observed at pH 2 and high ionic strength. The A1 state has an alpha-helical content equivalent to 64% of that present in the native dimer (N2 state). However, fluorescence measurements indicate that the tertiary structure of the A1 state is largely disordered. A site-directed mutant, K168E, which exists as a stable monomer [Mural, R. J., Soper, T. S., Larimer, F. W., & Hartman, F. C. (1990) J. Biol. Chem. 265, 6501-6505] was used to characterize the "native" monomer (N1 state). The far-UV CD spectra of the N1 and N2 states are almost identical, indicating a similar secondary structure content. However, the tertiary structure of the N1 state is less ordered than that of the N2 state. Nevertheless, when appropriately complemented in vitro, K168E forms an active heterodimer. Upon neutralization of acid-denatured Rubisco or dilution of guanidine hydrochloride-denatured Rubisco, unstable folding intermediates (I1 state) are rapidly formed. At concentrations at or below the "critical aggregation concentration" (CAC), the I1 state reverts spontaneously but slowly to the native states with high yield (greater than 65%). The CAC is temperature-dependent. At concentrations above the CAC, the I1 and the A1 states undergo irreversible aggregation. The commitment to aggregation is rapid [ef. Goldberg, M. E., Rudolph, R., & Jaenicke, R. (1991) Biochemistry 30, 2790-2797] and proceeds until the concentration of folding intermediate(s) has fallen to the CAC. In the presence of a molar excess of chaperonin 60 oligomers, the I1 state forms a stable binary complex. No stable binary complex between chaperonin 60 and the N1 state could be detected. Formation of the chaperonin 60-I1 binary complex arrests the spontaneous folding process. The I1 state becomes resistant to interaction with chaperonin 60 with kinetics indistinguishable from those associated with the appearance of the native states. In vitro complementation analysis indicated that the product of the chaperonin-facilitated process is monomeric.(ABSTRACT TRUNCATED AT 400 WORDS)

Anilino Naphthalenesulfonates↗
PubMed1989

The molecular chaperone concept.

Molecular chaperones are a ubiquitous family of proteins whose proposed role is to mediate the folding and assembly of other proteins into oligomeric structures. The essential function of molecular chaperones is to prevent the formation of incorrect structures which may result from the transient exposure of charged or hydrophobic surfaces normally involved in interactions between or within polypeptide chains. Such transient exposure may occur during the synthesis of polypeptides, the unfolding and refolding that occurs during their transport across membranes, the association of polypeptides made in one subcellular compartment with those made in another, changes in protein-protein interactions during the normal functioning of a complex, and recovery from stresses such as heat shock. Three classes of molecular chaperone are discussed: the nucleoplasmins, the BiP group, and the chaperonins.

Bacterial Proteins↗
PubMed1988

Homologous plant and bacterial proteins chaperone oligomeric protein assembly.

An abundant chloroplast protein is implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants. The product of the Escherichia coli groEL gene is essential for cell viability and is required for the assembly of bacteriophage capsids. Sequencing of the groEL gene and the complementary cDNA encoding the chloroplast protein has revealed that these proteins are evolutionary homologues which we term 'chaperonins'. Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes. Assisted post-translational assembly of oligomeric protein structures is emerging as a general cellular phenomenon.

Adenosine Triphosphatases↗
PubMed1987

Co-expression of both the maize large and wheat small subunit genes of ribulose-bisphosphate carboxylase in Escherichia coli.

A cDNA clone for the precursor form of the small subunit of wheat ribulose-bisphosphate carboxylase has been modified to allow the expression in Escherichia coli of a mature form of small subunit that lacks the transit peptide. Synthesis of the protein is controlled by a lac promoter, and translation is initiated from a lacZ ribosome binding site, giving rise to a small subunit with several beta-galactosidase amino acids fused to its N-terminus. A plasmid has been constructed that enables both wheat small subunits and maize large subunits to be synthesized in the bacterial cell, but using different promoters to allow independent expression of the rbcS and rbcL genes. When the small subunit is synthesized in the absence of the large subunit, it is found in the soluble fraction but the polypeptide is unstable and has a half-life of less than 15 min. Its size on sucrose gradients indicates a monomeric or dimeric form. When large subunit synthesis is induced in cells containing the small subunit, both subunits are found predominantly in the insoluble fraction and are fully stable for more than 120 min, suggesting that aggregation of the subunits may occur. The two subunits do not assemble together to form an active holoenzyme in vivo, even when nascent large subunits ware synthesized in a pool of mature small subunits. This indicates that other factors may be required to mediate the assembly of the higher plant enzyme.

Cloning, Molecular↗
PubMed1986

Assembly of cyanobacterial and higher plant ribulose bisphosphate carboxylase subunits into functional homologous and heterologous enzyme molecules in Escherichia coli.

The genes for the large (rbcL) and small (rbcS) subunits of ribulose-1,5 bisphosphate carboxylase-oxygenase (RuBPCase) from the cyanobacterium Synechococcus PCC 6301, and the rbcS gene of wheat, have been expressed in Escherichia coli in order to study homologous and heterologous enzyme assembly. Synechococcus L subunits expressed in E. coli in the absence of S subunits assemble into oligomeric structures without detectable enzyme activity. Co-expression of L and S subunits, achieved after infection with an M13 recombinant phage containing the rbcS gene, restores enzyme activity, thus demonstrating the essential role of S in the formation of an active RuBPCase. The S subunit, however, is neither required for the solubility nor for the assembly of the L subunits into oligomeric forms. The specific activity of the homologous Synechococcus RuBPCase can be modulated by changing the intracellular pool size of S by phage infection. Heterologous assembly between L subunits of Synechococcus and S subunits of wheat can be demonstrated and results in a functional enzyme. The hybrid RuBPCase has approximately 10% of the activity of the homologous Synechococcus enzyme.

Journal Article↗
PubMed1984

Dissociation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach by urea.

The dissociation of D-ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach, which consists of eight large subunits (L, 53 kDa) and eight small subunits (S, 14 kDa) and thus has a quarternary structure L8S8, has been investigated using a variety of physical techniques. Gel chromatography using Sephadex G-100 indicates the quantitative dissociation of the small subunit S from the complex at 3-4 M urea (50 mM Tris/Cl pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol and 5 mM 2-mercaptoethanol). The dissociated S is monomeric. Analytical ultracentrifuge studies show that the core of large subunits, L, remaining at 3-4 M urea sediments with S20, w = 15.0 S, whereas the intact enzyme (L8S8) sediments with S20, w = 17.7S. The observed value is consistent with a quarternary structure L8. The dissociation reaction in 3-4 M urea can thus be represented by L8S8----L8 + 8S. At urea concentrations c greater than 5 M the L8 core dissociates into monomeric, unfolded large subunits. A large decrease in fluorescence emission intensity accompanies the dissociation of the small subunit S. This change is completed at 4 M urea. No changes are observed upon dissociating the L8 core. The kinetics of dissociation of the small subunit, as monitored by fluorescence spectroscopy, closely follow the kinetics of loss of carboxylase activity of the enzyme. Studies of the circular dichroism of D-ribulose-1,5-bisphosphate carboxylase in the wavelength region 200-260 nm indicate two conformational transitions. The first one ([0]220 from -8000 to -3500 deg cm2 dmol-1) is completed at 4 M urea and corresponds to the dissociation of the small subunit and coupled conformational changes. The second one ([0]220 from -3500 to -1200 deg cm2 dmol-1) is completed at 6 M urea and reflects the dissociation and unfolding of large subunits from the core. The effect of activation of the enzyme by addition of MgCl2 (10 mM) and NaHCO3 (10 mM) on these conformational transitions was investigated. The first conformational transition is then shifted to higher urea concentrations: a single transition ([0]220 from -8000 to -1200 deg cm2 dmol-1) is observed for the activated enzyme. From the urea dissociation experiments we conclude that both large (L) and small (S) subunits are important for carboxylase activity of spinach D-ribulose-1,5-bisphosphate carboxylase: the L-S subunit interactions tighten upon activation and dissociation of S leads to a coupled, proportional loss of enzyme activity.

Chemical Phenomena↗