Biomedical subjects

K L Reid

Publications and source records attributed to K L Reid.

10 recordsLinked to original sources

PubMed2001

Examination of the folding of E. coli CspA through tryptophan substitutions.

Escherichia coli cold shock protein, CspA, folds very rapidly (time constant, tau = 4 msec) by an apparent two-state mechanism. However, recent time-resolved infrared (IR) temperature-jump experiments indicate that the folding trajectory of CspA may be more complicated. The sole tryptophan of wild-type CspA (Trp11), which is used to monitor the folding process by fluorescence spectroscopy, is located in an unusual aromatic cluster on the surface of CspA within the nucleic acid binding site. To gain a more global picture of the folding kinetics of CspA and to determine if there are any previously undetected intermediates, we have introduced a second tryptophan at three different surface locations in the protein. The three mutations did not significantly alter the tertiary structure of CspA, although two of the substitutions were found to be slightly stabilizing. Two-state folding, as detected by stopped-flow fluorescence spectroscopy, is preserved in all three mutants. These results indicate that the fast folding of CspA is driven by a concerted mechanism.

Amino Acid Substitution↗

PubMed1998

Neural precursor differentiation into astrocytes requires signaling through the leukemia inhibitory factor receptor.

The differentiation of precursor cells into neurons or astrocytes in the developing brain has been thought to be regulated in part by growth factors. We show here that neural precursors isolated from the developing forebrain of mice that are deficient in the gene for the low-affinity leukemia inhibitory factor receptor (LIFR-/-) fail to generate astrocytes expressing glial fibrillary acidic protein (GFAP) when cultured in vitro. Precursors from mice heterozygous for the null allele show normal levels of GFAP expression. These findings support the in vivo findings that show extremely low levels of GFAP mRNA in brains of embryonic day 19 LIFR-/- mice. In addition, monolayers of neural cells from LIFR-/- mice are far less able to support the neuronal differentiation of normal neural precursors than are monolayers from heterozygous or wild-type animals, indicating that endogenous signaling through the LIFR is required for the expression of both functional and phenotypic markers of astrocyte differentiation. LIFR-/- precursors are not irreversibly blocked from differentiating into astrocytes: they express GFAP after long-term passaging or stimulation with bone morphogenetic protein-2. These findings strongly implicate the LIF family of cytokines in the regulation of astrocyte differentiation and indeed the LIF-deficient animals show a significant reduction in the number of GFAP cells in the hippocampus. However, because this reduction is only partial it suggests that LIF may not be the predominant endogenous ligand signaling through the LIFR.

Animals↗

PubMed1998

Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA.

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.

Bacterial Proteins↗

PubMed1996

Physical interactions between members of the DnaK chaperone machinery: characterization of the DnaK.GrpE complex.

Many of the functions of the Escherichia coli Hsp70, Dnak, require two cofactors, DnaJ and GrpE. GrpE acts as a nucleotide exchange factor in the DnaK reaction cycle but the details of its mechanism remain unclear. GrpE has high affinity for monomeric native DnaK, with a Kd estimated at < or = 50 nM. GrpE is a very asymmetric molecule and exists as either a dimer or trimer in its native state. The stoichiometry of GrpE to DnaK in the isolated complex was 3:1, suggesting a trimer. Formation of the complex is quite fast (kan > 1 s-1), whereas the off-rate is very slow on the HPLC timescale (kant < or = 10(-4) s-1). GrpE has no affinity for ATP or ADP, nor the oligomeric and molten globule states of DnaK. The complex is much more thermally stable than either GrpE or DnaK alone, and prevents the formation of the molten globule-like state of DnaK at physiologically relevant temperatures. Formation of the complex does not cause any change in secondary structure, as determined by the lack of change in the circular dichroism spectrum. However, binding of GrpE induces a similar tertiary structural change in DnaK to that induced by binding of ATP, based on the blue shift in lambda max from the fluorescence of the single tryptophan in DnaK. The nucleotide exchange properties of GrpE can be explained by the conformational change which may represent the opening of the nucleotide cleft on DnaK, subsequently inducing a low affinity state for ADP.

Adenosine Diphosphate↗

PubMed1994

hsp70-protein complexes. Complex stability and conformation of bound substrate protein.

The presence of bound substrate protein increases the thermostability of hsp70 molecular chaperones (heat shock proteins of molecular mass 70 kDa). Complexes between hsp70 and unfolded substrate proteins were isolated by size-exclusion high performance liquid chromatography. The isolated complexes were observed to dissociate at a significant rate even in the absence of ATP. The presence of ADP caused a substantial increase in the stability of the complex. Both ADP and inorganic phosphate were found to inhibit the ATP-induced dissociation of complex. ADP was also observed to increase both the rate of complex formation and its stability as a function of temperature, suggesting an important regulatory role for nucleotides during heat shock. Circular dichroism and fluorescence studies of the complex between DnaK and a thermally unstable mutant of staphylococcal nuclease indicate that the bound substrate protein is significantly unfolded. A model for hsp70 cycle of complex formation and dissociation, which accounts for the regulatory role of nucleotides, is proposed.

Adenosine Diphosphate↗

PubMed1993

DnaK ATPase activity revisited.

It has recently been reported that the ATPase activity of DnaK, a 70 kDa heat shock protein from E. coli, is autostimulated by increasing protein concentration [(1993) FEBS Lett. 322, 277-279], suggesting that the DnaK dimer may be the enzymatically active species. In this paper we investigated the ATPase activity of different DnaK preparations; we found that the turnover number was very dependent on protein purification. With HPLC-purified DnaK we found a turnover number 20- to 50-fold lower than typical values previously published and no evidence of autostimulation, indicating that the monomer is the active species.

Adenosine Triphosphatases↗

PubMed1993

ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis.

The molecular chaperone proteins, particularly Hsp60 and Hsp70, have been implicated in essential cell functions under both normal and stress conditions (reviewed in refs 1-5). Members of the family of heat-shock proteins of M(r) 70K, Hsp70, bind to unfolded proteins and short peptides. Addition of Mg-ATP results in the dissociation of the substrate polypeptides from the chaperone, but as ATP-gamma S (an ATP analogue that is only slowly hydrolysable) cannot substitute for ATP in this reaction, it has been concluded that ATP hydrolysis is necessary to dissociate Hsp70-substrate protein complexes. By independently measuring the rates of ATP hydrolysis and substrate protein dissociation, we show here that Mg-ATP binding but not Mg-ATP hydrolysis is essential for substrate dissociation. We also show that there is an absolute requirement for K+ for the effect of Mg-ATP: only the combination of K+ and Mg-ATP will cause the conformational change in Hsp70 that is necessary for substrate dissociation. Moreover, in the absence of K+, Mg-ATP favours complex formation. We consider these results in terms of a G-protein-like model.

Adenosine Triphosphatases↗

PubMed1993

Three-state denaturation of DnaK induced by guanidine hydrochloride. Evidence for an expandable intermediate.

The denaturation of the heat shock protein DnaK induced by guanidine hydrochloride (Gdn-HCl) was investigated by circular dichroism, fluorescence, size-exclusion HPLC, and dynamic light scattering. DnaK unfolding takes place in two discrete steps. The midpoint (Cm) of the first transition (0.5 M) was shifted to higher denaturant concentrations (0.8 M) in the presence of Mg/ADP or Mg/ATP, whereas the second transition (Cm = 1.6 M) was unaffected by nucleotides. An intermediate state which continuously expands with increasing Gdn.HCl concentration was observed; its relation to molten globules is discussed. In addition, a direct correlation between molecular volume and ellipticity at 222 nm was found, regardless of the conformational state (native, intermediate, unfolded); the implications of these findings are discussed. The unfolding of DnaK is best explained by a hierarchical model of unfolding.

Chromatography, High Pressure Liquid↗

PubMed1992

DnaK, hsp73, and their molten globules. Two different ways heat shock proteins respond to heat.

The thermal stability of bovine brain hsp73, Escherichia coli DnaK, and its mutant T199A was studied by a combination of spectroscopic and chromatographic methods. DnaK undergoes a temperature-induced conformational change that leads to the formation of a molten globule at physiologically relevant temperatures (midpoint of the transition, tm, 41 degrees C). Native DnaK binds to a denatured form of alpha-lactalbumin in a temperature-dependent manner with maximum rate at about 40 degrees C. The molten globule of DnaK is unable to bind denatured alpha-lactalbumin but recovers native structure and activity upon cooling. The half-life of the refolding process is 10 min at 35 degrees C. Mg/ATP and Mg/ADP increase the thermal stability of DnaK; in the presence of these nucleotides the tm is shifted to 59 degrees C. Binding of Mg/ATP (but not Mg/ADP or Mg/adenosine 5'-[gamma-thio]triphosphate) causes a conformational change in DnaK as determined by the emission fluorescence spectrum. The DnaK mutant T199A which lacks the threonine residue that is essential for ATP hydrolysis and autophosphorylation activity (McCarty, J. S., and Walker, G. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9513-9517) shows nearly identical properties to the wild type in the presence or absence of nucleotides. Hsp73 undergoes similar temperature-induced transitions as determined by spectroscopic methods (Palleros, D.R., Welch, W.J., and Fink, A.L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5719-5723); however, contrary to DnaK, the molten globule of hsp73 irreversibly aggregates at temperatures higher than its tm (42 degrees C).

Animals↗

PubMed1989

Induction of the ganglioside marker A2B5 on cultured cerebellar neural cells by growth factors.

The surface ganglioside marker A2B5 was originally detected on neurons, but has subsequently been shown to be expressed on a wide range of macroglial and non-neural cells. This marker has been used in vitro to categorize subpopulations of neural cells within the central nervous system, as well as defining developmental pathways of macroglia. These categorizations are based on the assumption that this marker cannot easily be modulated. In this study of cerebellar cultures we demonstrate that A2B5 can be induced on approximately 16% of A2B5 non-expressing cells by the growth factors: basic fibroblast growth factor (b-FGF); acidic fibroblast growth factor (a-FGF); and, to a lesser degree, epidermal growth factor (EGF).

Animals↗