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A Shrake

Publications and source records attributed to A Shrake.

9 recordsLinked to original sources
PubMed1992

Origins and consequences of ligand-induced multiphasic thermal protein denaturation.

The presence of subsaturating levels of a high-affinity ligand has been demonstrated both by experiment and calculation to have far-reaching consequences on thermally induced protein denaturation due to the coupling between the protein denaturation and ligand-binding equilibria. Under such circumstances, a protein may undergo biphasic denaturation even though in the absence of ligand it exhibits a thermogram comprised of a single essentially symmetric endotherm. Up to now, the presence of just 2 maxima in the thermogram has been presented merely as an experimental observation or as the result of equilibrium computations. Here we develop a thermodynamic description of the linkage between these equilibria in which the number of cusps present in the thermogram correlates with the number of resolved steps in the plot of saturation level of remaining native protein vs temperature (i.e., the thermal binding curve). During thermally induced denaturation, the concentration of native protein decreases; thus, the native protein in effect is titrated with ligand at constant total ligand concentration. The free ligand concentration The free ligand concentration increases substantially through the release of bound ligand by unfolding protein thereby increasing the saturation level of the remaining native protein. The form of this thermal binding curve is a function of the number of ligand-binding sites on the protein, the magnitudes of the association constants, and the total ligand and total protein concentrations. As a result, the model predicts multiphasic denaturation of a single cooperative unit when the thermal binding curve consists of discrete multiple steps. The presence of only 2 maxima (i.e., a single cusp) in a thermogram for a protein with multiple sites on the native species derives from the form of the thermal binding curve, which in this case is a single-step sigmoidal plot, and not from the predominant denaturation of unliganded and fully liganded native species. In addition, it is shown that, in general, the contributions from the denaturation of individual native protein species are decidedly non-two-state in character; thus, simple deconvolution should not be carried out. The effects of nonzero values of delta Cp and d delta Cp/dT for denaturation and of changes in enthalpy and in heat capacity for ligand binding, as well as the interaction of ligand with the denatured protein, are explored also.

Binding Sites↗
PubMed1990

Ligand-induced biphasic protein denaturation.

The results of a thermodynamic calculation of the excess heat capacity that is based on experimental observations and that incorporates the effects of ligand binding on the two-state, thermal denaturation of a protein are presented. For a protein with a single-binding site on the native species and at subsaturating concentrations of ligand, bimodal or unimodal thermograms were computed merely by assuming a larger or smaller ligand association constant, respectively. The calculated thermograms for this simplified case show the salient features of those observed by differential scanning calorimetry for defatted human albumin monomer in the absence and presence of three ligands for which the protein has higher, intermediate, and lower affinity (Shrake, A., and Ross, P. D. (1988) J. Biol. Chem. 263, 15392-15399). The computation demonstrates that biphasic unfolding can result from a significant increase in the free energy of denaturation (and the transition temperature) during the course of unfolding due to a substantial increase in free ligand concentration caused by the release of bound ligand by denaturing protein. Such ligand-induced biphasic denaturation does not relate to macromolecular substructure but derives from a perturbation, during unfolding, of the ligand binding equilibrium, which is coupled to the equilibrium between the folded and unfolded protein species. Thus, this bimodality is not limited to thermally induced unfolding but is operative independent of the means used to effect denaturation and therefore must be considered when studying any macromolecular folding/unfolding reaction in the presence of ligand.

Kinetics↗
PubMed1989

Partial unfolding of dodecameric glutamine synthetase from Escherichia coli: temperature-induced, reversible transitions of two domains.

Glutamine synthetase (GS), Mr 622,000, from Escherichia coli contains 12 active sites formed at heterologous interfaces between subunits [Almassy, R. J., Janson, C. A., Hamlin, R., Xuong, N.-H., & Eisenberg, D. (1986) Nature (London) 323, 304-309]. Temperature-induced changes in UV spectra from 3 to 68 degrees C were reversible with the Mn2+- or Mg2+-enzyme at pH 7.0 (50 degrees C) in 100 mM KCl. No dissociation or aggregation of dodecamer occurred at high temperatures. The thermal transition involves the exposure of approximately 0.7 of the 2 Trp residues/subunit (by UV difference spectroscopy) and 2 of the 17 Tyr residues/subunit (change in exposure from 4.7 to 6.7 Tyr/subunit by second-derivative spectral analysis). Monitoring changes in Trp and Tyr exposure independently gives data that conform to a two-state model for partial unfolding with Tm values (where delta G unfolding = 0) differing by 2-3 degrees C at each level of [Mn2+] studied and with average delta HvH values of 80 and 94 kcal/mol, respectively. These observations suggest that two regions of the oligomeric structure unfold separately as independent transitions (random model). However, the data can be fit equally with a sequential model in which the Trp transition occurs first upon heating. By fitting with either model, Tm values increase from approximately 47 to approximately 54 degrees C with increasing free [Mn2+] from 3.6 to 49 microM but decrease from approximately 54 to approximately 43 degrees C by further increasing free [Mn2+] from 0.05 to 10 mM; such behavior indicates that the high-temperature form of the enzyme binds Mn2+ more weakly but has more binding sites than the native enzyme. The high-temperature Mn-enzyme form is somewhat less unfolded than is the catalytically inactive apoenzyme, which undergoes no further Trp or Tyr exposure on heating and therefore is assumed to be the high-temperature form of divalent cation-free GS. Adding substrates [ADP, L-Met-(SR)-sulfoximine, Gln, Gln + NH2OH, or Gln + ADP] to Mn.GS increased Tm to varying extents by preferential binding to the folded form. Indeed, the transition-state analogue complex GS.(Mn2.ADP.L-Met-(S)-sulfoximine phosphate)12 was stable in the folded form to at least 72 degrees C. Moreover, an Arrhenius plot for gamma-glutamyl transfer activity was linear from 4 to 72 degrees C with Ea = 18.3 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1988

Biphasic denaturation of human albumin due to ligand redistribution during unfolding.

Denaturation of defatted human albumin monomer, monitored by differential scanning calorimetry, is monophasic as reflected by the single, resulting endotherm. With low levels of various ligands, biphasic or monophasic unfolding processes are manifested as bimodal or unimodal thermograms, respectively. The greater the affinity of native protein for ligand, the greater is the tendency for biphasic denaturation. We propose that such a biphasic unfolding process arises from a substantial increase in stability (transition temperature) of remaining native protein during denaturation. This increase in stability derives from the free energy of ligand binding becoming more negative due to the release of high affinity ligand by unfolding protein. The tendency for biphasic denaturation is greatest at low (subsaturating) levels of ligand where greatest increases in stability occur. Biphasic unfolding arising from such ligand redistribution results from denaturation of different kinds of protein molecules, ligand-poor and ligand-rich species, and not from sequential unfolding of domains within the same molecule. Differentiating between these two mechanisms is necessary for the correct interpretation of biphasic denaturation data. Furthermore, biphasic unfolding due to ligand redistribution occurs independently of the means used to effect denaturation. The maximum increase in stability due to ligand binding relative to the stability of defatted albumin monomer alone occurs with the intermediate affinity ligand octanoate (22 degrees C) and not with the high affinity ligand hexadecanoate (15 degrees C). This indicates a much greater affinity of denatured albumin for hexadecanoate since increase in stability derives from the difference between free energy of ligand binding to folded and unfolded protein forms.

Hot Temperature↗
PubMed1988

Decrease in stability of human albumin with increase in protein concentration.

The stability (reflected in denaturation temperature, Td) of defatted human albumin monomer, monitored by differential scanning calorimetry, decreases with increasing protein concentration. This is shown to be compatible with a simple model in which reversible polymerization of denatured monomer promotes unfolding. This model also predicts an increase in transition cooperativity with decreasing protein concentration whereas experimentally cooperativity decreases because the rate of thermally induced polymerization of unfolded monomer is slow relative to the scan rate of the calorimeter. The denaturation of undefatted human albumin monomer, subsaturated with high affinity endogenous long-chain fatty acid (LCFA), was previously observed by differential scanning calorimetry to be a biphasic process. Td for the first endotherm, associated with the denaturation of LCFA-poor species, decreases with increasing protein concentration similar to that for defatted monomer whereas Td for the second endotherm, associated with denaturation of LCFA-rich species, is independent of concentration. The magnitude of the concentration dependence of Td relates directly to the extent of polymerization of denatured monomer, which decreases with increasing level of bound ligand. The bimodal thermogram observed for undefatted monomer persists upon simultaneous extrapolation of Td values to low concentration and low scan rate thereby demonstrating that this biphasic denaturation arising from ligand redistribution during denaturation is a true thermodynamic phenomenon and not an artifact of specific experimental conditions or the method used to induce denaturation.

Algorithms↗
PubMed1975

Optical properties of lysozyme. pH and saccharide binding difference spectra.

Difference spectra associated with changes in pH and with binding of saccharides have been recorded for hen egg white (HEW) lysozyme, turkey egg white (TEW) lysozyme, and for the derivatives of the hen protein in which Tre-62 or Trp-108 had been oxidized specifically to oxindolealanine to give the Oxa-62 or Oxa-108-proteins. Identical pH difference spectra were obtained for HEW, TEW, and Oxa-62-lysozymes. Oxidation of Trp-108 is reflected in both the high and low pH (pH 7 versus 5 and pH 2 versus 5) difference spectra. The magnitude of the low pH difference spectrum is enhanced by binding of saccharide for HEW and Oxa-62-lysozymes but not for TEW lysozyme. The shapes and magnitudes of saccharide binding difference spectra are affected by oxidation of residues 62 or 108. These results can be interpreted in terms of the perturbations responsible for the lysozyme difference spectra. The pH 7 versus 5 difference spectrum results from perturbation by Glu-35 of Trp-108 and another tryptophan, probably Trp-63. Perturbation of Trp-108 and one or more other tryptophan residues by several carboxylate groups is responsible for the low pH difference spectra of the unliganded HEW and TEW lysozyme molecules. Perturbation of Trp-108 makes a principal contribution to the saccharide-binding difference spectrum. Perturbation of the Oxa-108 chromophore by ionization of Glu-35 or by saccharide binding produces absorbance changes in the 250 to 265 nm region.

Acetylglucosamine↗