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G H Lorimer

Publications and source records attributed to G H Lorimer.

At least 19 recordsLinked to original sources
PubMed1996

A thermodynamic coupling mechanism for GroEL-mediated unfolding.

Chaperonins prevent the aggregation of partially folded or misfolded forms of a protein and, thus, keep it competent for productive folding. It was suggested that GroEL, the chaperonin of Escherichia coli, exerts this function 1 unfolding such intermediates, presumably in a catalytic fashion. We investigated the kinetic mechanism of GroEL-induced protein unfolding by using a reduced and carbamidomethylated variant of RNase T1, RCAM-T1, as a substrate. RCAM-T1 cannot fold to completion, because the two disulfide bonds are missing, and it is, thus, a good model for long-lived folding intermediates. RCAM-T1 unfolds when GroEL is added, but GroEL does not change the microscopic rate constant of unfolding, ruling out that it catalyzes unfolding. GroEL unfolds RCAM-T1 because it binds with high affinity to the unfolded form of the protein and thereby shifts the overall equilibrium toward the unfolded state. GroEL can unfold a partially folded or misfolded intermediate by this thermodynamic coupling mechanism when the Gibbs free energy of the binding to GroEL is larger than the conformational stability of the intermediate and when the rate of its unfolding is high.

Adenosine Diphosphate↗
PubMed1996

Chaperonin-facilitated protein folding: optimization of rate and yield by an iterative annealing mechanism.

We develop a heuristic model for chaperonin-facilitated protein folding, the iterative annealing mechanism, based on theoretical descriptions of "rugged" conformational free energy landscapes for protein folding, and on experimental evidence that (i) folding proceeds by a nucleation mechanism whereby correct and incorrect nucleation lead to fast and slow folding kinetics, respectively, and (ii) chaperonins optimize the rate and yield of protein folding by an active ATP-dependent process. The chaperonins GroEL and GroES catalyze the folding of ribulose bisphosphate carboxylase at a rate proportional to the GroEL concentration. Kinetically trapped folding-incompetent conformers of ribulose bisphosphate carboxylase are converted to the native state in a reaction involving multiple rounds of quantized ATP hydrolysis by GroEL. We propose that chaperonins optimize protein folding by an iterative annealing mechanism; they repeatedly bind kinetically trapped conformers, randomly disrupt their structure, and release them in less folded states, allowing substrate proteins multiple opportunities to find pathways leading to the most thermodynamically stable state. By this mechanism, chaperonins greatly expand the range of environmental conditions in which folding to the native state is possible. We suggest that the development of this device for optimizing protein folding was an early and significant evolutionary event.

Adenosine Triphosphate↗
PubMed1996

A quantitative assessment of the role of the chaperonin proteins in protein folding in vivo.

In vitro the chaperonin proteins, GroEL and GroES, facilitate the folding of some other proteins under conditions where that process does not occur spontaneously. Using values drawn from a number of such in vitro studies, together with the known rates of in vivo protein synthesis by Escherichia coli and the known quantities of GroEL and GroES in E. coli, an assessment of the general role of these proteins in protein folding in vivo has been made. Three specific cases are examined, where compelling evidence points to the involvement of the chaperonins; the in vivo folding of the bacteriophage coat protein during the burst phase of phage morphogenesis and of Rubisco during chloroplast development and during expression of recombinant Rubisco in E. coli. In each case the maximum in vitro rates are nearly sufficient to account for the observed in vivo rates of formation of the native protein. However, in general, there appears to be sufficient GroEL and GroES to facilitate the folding of no more than 5% of all of the proteins within E. coli.

Capsid↗
PubMed1995

Dethiobiotin synthetase: the carbonylation of 7,8-diaminonanoic acid proceeds regiospecifically via the N7-carbamate.

Dethiobiotin synthetase (DTBS) catalyzes the penultimate step in biotin biosynthesis, the formation of the ureido ring of dethiobiotin from (7R,8S)-7,8-diaminononanoic acid (7,8-diaminopelargonic acid, DAPA), CO2, and ATP. Solutions of DAPA at neutral pH readily formed a mixture of the N7- and N8-carbamates in the presence of CO2. However, four lines of evidence together indicated that only the N7-carbamate of DAPA was an intermediate in the reaction catalyzed by DTBS. (1) Addition of diazomethane to mixtures of DAPA and [14C]CO2 yielded a mixture of the N7- and N8-methyl carbamate esters, consistent with carbamate formation in free solution. In the presence of excess DTBS (over DAPA), the ratio of N7:N8-methyl carbamate esters recovered was roughly doubled, suggesting that the enzyme preferentially bound the N7-DAPA-carbamate. (2) Both N7- and N8-DAPA-carbamates were observed directly by 1H and 13C NMR in solutions containing DAPA and [13C]CO2. In the presence of excess DTBS (over DAPA) only one carbamate was observed, showing that carbamate binding to the enzyme was regiospecific. 13C NMR of mixtures containing enzyme, [7-15N]DAPA, and [13C]CO2 showed that the enzyme-bound carbamate was at N7 of DAPA. In addition, pulse-chase experiments showed that the binary complex of DTBS and N7-DAPA-carbamate became kinetically committed upon addition of MgATP. (3) The N7-DAPA-carbamate mimic, 3-(1-aminoethyl)nonanedioic acid, in which the carbamate nitrogen was replaced with a methylene group, cyclized to the corresponding lactam in the presence of DTBS and ATP; ADP and P(i) were also formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids, Diamino↗
PubMed1995

Functional characterization of the higher plant chloroplast chaperonins.

The higher plant chloroplast chaperonins (ch-cpn60 and ch-cpn10) have been purified and their structural/functional properties examined. In all plants surveyed, both proteins were constitutively expressed, and only modest increases in their levels were detected upon heat shock. Like GroEL and GroES of Escherichia coli, the chloroplast chaperonins can physically interact with each other. The asymmetric complexes that form in the presence of ADP are "bullet-shaped" particles that likely consist of 1 mol each of ch-cpn60 and ch-cpn10. The purified ch-cpn60 is a functional molecular chaperone. Under "nonpermissive" conditions, where spontaneous folding was not observed, it was able to assist in the refolding of two different target proteins. In both cases, successful partitioning to the native state also required ATP hydrolysis and chaperonin 10. Surprisingly, however, the "double-domain" ch-cpn10, comprised of unique 21-kDa subunits, was not an obligatory co-chaperonin. Both GroES and a mammalian mitochondrial homolog were equally compatible with the ch-cpn60. Finally, the assisted-folding reaction mediated by the chloroplast chaperonins does not require K+ ions. Thus, the K(+)-dependent ATPase activity that is observed with other known groEL homologs is not a universal property of all chaperonin 60s.

Adenosine Diphosphate↗
PubMed1995

On the distribution of ligands within the asymmetric chaperonin complex, GroEL14.ADP7.GroES7.

In the presence of MgATP or MgADP the E. coli chaperonin proteins, GroEL and GroES, form a stable asymmetric complex with a stoichiometry of two GroEL7:one GroES7: seven MgADP. The distribution of the ligands between the two heptameric GroEL rings is crucial to our understanding of the mechanism of chaperonin-assisted folding, being either cis (i.e. [GroEL7.MgADP7.GroES7]-[GroEL7]) or trans (i.e. [GroEL7.MgADP7]-[GroEL7.GroES7]. On the basis of cross-linking experiments with 8-azido-ATP and the heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), it was suggested that GroES and MgADP are bound to the same GroEL ring which resists proteinase K digestion [Nature 366 (1993) 228-233]. However, we find that the SPDP-promoted cross linking of GroES and GroEL occurs in the absence of Mg2+, ADP or ATP, which are required for the formation of the asymmetric complex. Cross-linking is shown to occur only when the SPDP-modified GroES is co-precipitated with GroEL by trichloracetic acid. Furthermore, there are structural grounds for questioning whether SPDP can crosslink, in a physiologically relevant manner, an amino group of GroES with any of the cysteinyl groups of GroEL.

Adenosine Diphosphate↗
PubMed1995

Stability of the asymmetric Escherichia coli chaperonin complex. Guanidine chloride causes rapid dissociation.

The chaperonin proteins, GroEL14 and GroES7, inhibit protein aggregation and assist in protein folding in a potassium/ATP-dependent manner. In vitro, assays for chaperonin activity typically involve adding a denatured substrate protein to the chaperonins and measuring the appearance of correctly folded substrate protein. The influence of denaturant is generally ignored. Low concentrations of guanidinium chloride (< 100 mM) had a profound effect on the activity/structure of the chaperonins. Guanidinium decreased the ATPase activity of GroEL and attenuated the inhibition of GroEL ATP hydrolysis by GroES. The stable, asymmetric chaperonin complex which forms in the presence of GroES and ADP (GroES7.ADP7.GroEL7-GroEL7) rapidly dissociated upon addition of 80 mM guandinium chloride. Dissociation was enhanced at high ionic strength, but rapid dissociation was guanidinium-specific. Accelerated release of the GroES from the complex was also demonstrated. Unfolded proteins alone had no effect on complex stability. Residual guanidinium depressed the rate of Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase (Rubisco) folding; an increased aggregation rate also decreased the yield of folded Rubisco. Chaperonin-assisted folding is therefore best studied using proteins denatured by means other than guanidinium chloride.

Adenosine Diphosphate↗
PubMed1995

GroES and the chaperonin-assisted protein folding cycle: GroES has no affinity for nucleotides.

The E. coli chaperonin proteins, GroEL and GroES, assist in folding newly synthesized proteins. GroES is necessary for GroEL-assisted folding under conditions where the substrate protein cannot spontaneously fold. On the basis of photolabelling of GroES with 8-azido-ATP, a role for nucleotide binding to GroES in chaperonin function was suggested [Martin, et al., Nature, 366 (1993) 279-282]. We confirm the photolabeling of GroES with 8-azido-ATP. However, other proteins not known to contain nucleotide binding sites also became photolabeled suggesting that labeling is non-specific. Using rigorous physical methods, isothermal calorimetry and equilibrium binding, no interaction between GroES and nucleotides could be detected. We conclude that GroES has no nucleotide binding site.

Adenosine Triphosphate↗
PubMed1994

Dynamics of the chaperonin ATPase cycle: implications for facilitated protein folding.

The Escherichia coli chaperonins GroEL and GroES facilitate protein folding in an adenosine triphosphate (ATP)-dependent manner. After a single cycle of ATP hydrolysis by the adenosine triphosphatase (ATPase) activity of GroEL, the bi-toroidal GroEL formed a stable asymmetric ternary complex with GroES and nucleotide (bulletlike structures). With each subsequent turnover, ATP was hydrolyzed by one ring of GroEL in a quantized manner, completely releasing the adenosine diphosphate and GroES that were tightly bound to the other ring as a result of the previous turnover. The catalytic cycle involved formation of a symmetric complex (football-like structures) as an intermediate that accumulated before the rate-determining hydrolytic step. After one to two cycles, most of the substrate protein dissociated still in a nonnative state, which is consistent with intermolecular transfer of the substrate protein between toroids of high and low affinity. A unifying model for chaperonin-facilitated protein folding based on successive rounds of binding and release, and partitioning between committed and kinetically trapped intermediates, is proposed.

Adenosine Triphosphatases↗
PubMed1994

On the role of groES in the chaperonin-assisted folding reaction. Three case studies.

The mechanism by which correctly folded proteins are recovered from stable complexes with groEL is not well understood. Certain target proteins require ATP and groES, while others seemingly dispense with the cochaperonin. Here, we examine the chaperonin-assisted folding of ribulose-1,5-bisphosphate carboxylase, malate dehydrogenase, and citrate synthase, three proteins that are believed to require both chaperonin components for successful reactivation. Surprisingly, in all cases, the need for groES depended on the folding environment. Under "non-permissive" conditions, where unassisted spontaneous folding could not occur, reactivation to the native state required the complete chaperonin system (e.g. groEL, groES, and MgATP). However, under "permissive" conditions where spontaneous folding could occur groES was no longer mandatory. Instead, upon the addition of ATP alone, all three target proteins could be released from groEL, in a form that was capable of reaching the native state. In the permissive setting, groES merely accelerated the rate of the ATP-dependent release process. The results suggest that the incompletely folded protein species that are released from groEL, in the absence of groES, are not necessarily committed to the native state. Similar to the unassisted folding reaction, they still partition between productive and unproductive folding pathways in an environment-dependent manner. It follows that the mechanistic contribution of the co-chaperonin, groES, and its physiological significance in cellular protein folding, could be entirely missed in a permissive in vitro environment.

Animals↗
PubMed1993

Plurality of protein conformations of ribulose-1,5-bisphosphate carboxylase/oxygenase monomers probed by high pressure electrophoresis.

We used hydrostatic pressure in the range of 1 to 2 kbar, coupled with polyacrylamide gel electrophoresis, to investigate the properties of monomers of dimeric ribulose bisphosphate carboxylase/oxygenase. At temperatures below -5 degrees C or pressures above 1.5 kbar, only a diffuse band with low electrophoretic mobility was observed, which is assigned to a denatured monomer. In gels run at 1.0 kbar and temperatures above 0 degree C, both the wild type and a mutant in which a positively charged Lys at the dimer interface is replaced by a negatively charged glutamic acid displayed several discrete bands with retardation coefficients larger than that of the dimer. Cross-linking due to oxidation of cysteines was not the reason for the multiplicity of bands, which in addition were independent on the length of the electrophoretic run in the range of 1-3 h. Binding of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid to the pressure dissociated monomers stabilized the unfolded conformations. We propose that the dissociated monomers adopt various expanded conformations, which, under the experimental conditions, are stabilized to the extent necessary to be considered as distinct chemical species. Gel filtration high performance liquid chromatography analysis of bands eluted from nonstained gels run at 1 kbar (15 degrees C) and 1.5 kbar (-5 degrees C), respectively, was performed. In both cases the dimeric structure was fully recovered, along with the spectroscopic properties and catalytic activity characteristic of the native dimer, indicating that the unresolved unfolded conformers that appear at -5 degrees C, as well as the set of discrete conformers obtained at 15 degrees C, are able to reconstitute a single active conformation on reassociation.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

Identification and functional analysis of chaperonin 10, the groES homolog from yeast mitochondria.

Chaperonin 60 (cpn60) and chaperonin 10 (cpn10) constitute the chaperonin system in prokaryotes, mitochondria, and chloroplasts. In Escherichia coli, these two chaperonins are also termed groEL and groES. We have used a functional assay to identify the groES homolog cpn10 in yeast mitochondria. When dimeric ribulose-1,5-bisphosphate carboxylase (Rubisco) is denatured and allowed to bind to yeast cpn60, subsequent refolding of Rubisco is strictly dependent upon yeast cpn10. The heterologous combination of cpn60 from E. coli plus yeast cpn10 is also functional. In contrast, yeast cpn60 plus E. coli cpn10 do not support refolding of Rubisco. In the presence of MgATP, yeast cpn60 and yeast cpn10 form a stable complex that can be isolated by gel filtration and that facilitates refolding of denatured Rubisco. Although the potassium-dependent ATPase activity of E. coli cpn60 can be inhibited by cpn10 from either E. coli or yeast, neither of these cpn10s inhibits the ATPase activity of yeast cpn60. Amino acid sequencing of yeast cpn10 reveals substantial similarity to the corresponding cpn10 proteins from rat mitochondria and prokaryotes.

Adenosine Triphosphatases↗
PubMed1993

A role for the epsilon-amino group of lysine-334 of ribulose-1,5-bisphosphate carboxylase in the addition of carbon dioxide to the 2,3-enediol(ate) of ribulose 1,5-bisphosphate.

Earlier structural and functional studies of ribulose-1,5-bisphosphate carboxylase/oxygenase imply that K334 facilitates the addition of gaseous substrate to the 2,3-enediol(ate) derived from ribulose 1,5-bisphosphate. Crystallographic analysis of the activated spinach enzyme [Knight et al. (1990) J. Mol. Biol. 215, 113-160] shows that the lysyl side chain is appropriately positioned to stabilize the transition state for the addition of CO2 to the enediol(ate). Furthermore, despite total impairment of carboxylase and oxygenase activities, site-directed mutants of the Rhodospirillum rubrum enzyme with replacements for lysine K334 (formerly designated K329) retain the capacity to enolize ribulose bisphosphate, demonstrating that the primary catalytic lesion lies beyond this initial step [Soper et al. (1988) Protein Eng. 2, 39-44; Hartman & Lee (1989) J. Biol. Chem. 264, 11784-11789]. We now show that the K334C mutant is also competent in the latter stages of catalysis, whereby 2'-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon intermediate of the carboxylation pathway, is correctly processed to 3-phosphoglycerate. Thus, the impairment of the mutant in overall catalysis can be attributed to preferential disruption of the reaction of CO2 or O2 with the enzyme-bound enediol(ate). Chemical rescue of the K334C mutant by aminoethylation and aminopropylation shows that this disruption reflects, at least in part, a failure to adequately stabilize the relevant transition state. With several simplifying assumptions, the CO2/O2 specificity factor tau can be reduced to the ratio of the fundamental second-order rate constants for the interaction of the gaseous substrates with the enzyme-bound 2,3-enediol(ate) of ribulose bisphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1993

Hydrolysis of adenosine 5'-triphosphate by Escherichia coli GroEL: effects of GroES and potassium ion.

The potassium-ion activation constant (Kact) for the ATPase activity of Escherichia coli chaperonin groEL is inversely dependent upon the ATP concentration over at least 3 orders of magnitude. The ATPase activity shows positively cooperative kinetics with respect to ATP and K+. Both the K0.5 for ATP and cooperativity (as measured by the Hill coefficient) decrease as the K+ concentration increases. Equilibrium binding studies under conditions where hydrolysis does not occur indicate that MgATP binds weakly to groEL in the absence of K+. In the absence of groES, the K(+)-dependent hydrolysis of ATP by groEL continues to completion. In the presence of groES, the time course for the hydrolysis of ATP by groEL becomes more complex. Three distinct kinetic phases can be discerned. Initially, both heptameric toroids turn over once at the same rate that they do in the absence of groES. This leads to the formation of an asymmetric binary complex, groEL14-MgADP7-groES7, in which 7 mol of ADP is trapped in a form that does not readily exchange with free ADP. In the second phase, the remaining seven sites (containing readily exchangeable ADP) turn over, or have the potential to turn over, at the same rate as they do in the absence of groES, so that the overall rate of hydrolysis is maximally 50%. These remaining sites of the asymmetric binary complex do not hydrolyze all of the available ATP. Instead, the second phase of hydrolysis gives way to a third, completely inhibited state, the onset of which is dependent upon the relative affinities of the remaining sites for MgATP and MgADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1993

Reversible dissociation and conformational stability of dimeric ribulose bisphosphate carboxylase.

Dimer-monomer dissociation of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was investigated using hydrostatic pressure in the range 1-2 kbar to promote dissociation. Intrinsic fluorescence emission and polarization, along with the polarization of the fluorescence of single-labeled AEDANS conjugates, were used to follow the dissociation. Full reversibility after dissociation was observed to depend on the presence of small ligands: glycerol, Mg2+, and NaHCO3, the last two being required to activate the enzyme. The free energy of association at 15 degrees C, -12.9 kcal mol-1, was made up of a positive change in enthalpy on association of 6.0 kcal mol-1 and an entropic contribution (T delta S) of 18.9 kcal mol-1; thus the monomer association is entropy driven. No dissociation of the quaternary complex formed by the dimer, 2-carboxy-D-arabinitol 1,5-diphosphate (CADP), Mg2+, and NaHCO3 was observed at pressures up to 2.0 kbar; the magnitude of stabilization by the inhibitor binding was estimated as 2.3 kcal mol-1. Pressurization in the presence of bis-ANS results in a time-dependent increase in fluorophore emission, indicating changes in monomer conformation with exposure of hydrophobic surfaces upon dissociation. Reactivity against the fluorescent probe 1,5-I-AEDANS was also used as a conformational probe: HPLC of a trypsin digest of rubisco labeled at atmospheric pressure revealed a single fluorescent peptide, whereas more extensive labeling was observed when the reaction was carried out at 2.0 kbar, indicative of exposure of internal cysteines.(ABSTRACT TRUNCATED AT 250 WORDS)

Bicarbonates↗
PubMed1993

Chaperonins and protein folding: unity and disunity of mechanisms.

Chaperonin-facilitated folding of proteins involves two partial reactions. The first partial reaction, the formation of stable binary complexes between chaperonin-60 and non-native states of the target protein, is common to the chaperonin-facilitated folding of all target proteins investigated to date. The structural basis for this interaction is not presently understood. The second partial reaction, the dissociation of the target protein in a form committed to the native state, appears to proceed by a variety of mechanisms, dependent upon the nature of the target protein in question. Those target proteins (e.g. rubisco, rhodanese, citrate synthase) which require the presence of chaperonin-10, also appear to require the hydrolysis of ATP to bring about the dissociation of the target protein from chaperonin-60. With one exception (pre-beta-lactamase) those target proteins which do not require the presence of chaperonin-10 to be released from chaperonin-60, also do not require the hydrolysis of ATP, since non-hydrolysable analogues of ATP support the release of the target protein in a state committed to the native state. The question of whether or not chaperonin-facilitated folding constitutes a catalysed event is addressed.

Animals↗