Biomedical subjects

D R Palleros

Publications and source records attributed to D R Palleros.

8 recordsLinked to original sources

PubMed1994

Classification of acid denaturation of proteins: intermediates and unfolded states.

A systematic investigation of the effect of acid on the denaturation of some 20 monomeric proteins indicates that several different types of conformational behavior occur, depending on the protein, the acid, the presence of salts or denaturant, and the temperature. Three major types of effects were observed. Type I proteins, when titrated with HCl in the absence of salts, show two transitions, initially unfolding in the vicinity of pH 3-4 and then refolding to a molten globule-like conformation, the A state, at lower pH. Two variations in this behavior were noted: some type I proteins, when titrated with HCl in the absence of salts, show only partial unfolding at pH 2 before the transition to the molten globule state; others of this class form an A state that is a less compact from of the molten globule state. In the presence of salts, these proteins transform directly from the native state to the molten globule conformation. Type II proteins, upon acid titration, do not fully unfold but directly transform to the molten globule state, typically in the vicinity of pH 3. Type III proteins show no significant unfolding to pH as low as 1, but may be caused to behave similarly to type I in the presence of urea. Thus, the exact behavior of a given protein at low pH is a complex interplay between a variety of stabilizing and destabilizing forces, some of which are very sensitive to the environment. In particular, the protein conformation is quite sensitive to salts (anions) that affect the electrostatic interactions, denaturants, and temperature, which cause additional global destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Acids↗

PubMed1994

Protein conformational changes induced by 1,1'-bis(4-anilino-5-naphthalenesulfonic acid): preferential binding to the molten globule of DnaK.

1,1'-Bis(4-anilino-5-naphthalenesulfonic acid) (bis-ANS), a hydrophobic fluorescent molecular probe which has been shown to bind to compact intermediate states of proteins (molten globules) and also to many nucleotide binding sites, induces a conformational change in DnaK by preferentially binding to its partially folded intermediate state (I) and thus shifting the equilibrium from favoring the native state (N) to favoring the I state. The conformational change was detected by CD, fluorescence emission, size exclusion chromatography, and small-angle X-ray scattering. The presence of bis-ANS significantly decreases the midpoint, Tm, of the initial transition (N-->I) in the thermal unfolding of DnaK, resulting in the apparent destabilization of the native state of DnaK. There is a linear correlation between the apparent free energy (reflected by Tm) of this transition and the concentration of bis-ANS. Bis-ANS does not affect the midpoint of the transition for DnaK from the intermediate to the unfolded state (U). An additional small transition from I to I*, a more expanded intermediate state, was observed, suggesting that the thermal denaturation of DnaK proceeds via a four-state (N-->I-->I*-->U) unfolding process. The addition of nucleotides, ADP or ATP, to the DnaK-bis-ANS complex causes a decrease in bis-ANS fluorescence emission due to the release of bound bis-ANS from the intermediate state of DnaK. This is due to preferential binding of the nucleotide to the native state of DnaK, resulting in a shift in the equilibrium from the intermediate toward the native state rather than the direct displacement of bis-ANS bound in the nucleotide binding site. Denaturation of DnaK induced by bis-ANS can be minimized by working at a temperature much lower than the Tm of the protein, at low dye concentration, and in the presence of nucleotide. Under these conditions, bis-ANS binds to the native state of DnaK.

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↗

PubMed1991

Interaction of hsp70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding.

Circular dichroism and HPLC gel filtration were used to show that cytosolic hsp70 is thermally stable but undergoes a conformational transition (midpoint, 43 degrees C; 57 degrees C in the presence of ATP or ADP) leading to oligomerization. hsp70 binds to unfolded, but not to folded, proteins in a temperature-dependent manner; complex formation is significant only at physiologically relevant temperatures. hsp70 binds ADP more tightly than ATP to form a binary complex, which binds to the unfolded protein more rapidly than free hsp70. ADP also inhibits the ATP-induced dissociation of the hsp70-protein complex. A regulatory role for the hsp70-nucleotide binary complexes is proposed.

Adenosine Diphosphate↗