No abstract

No abstract

No abstract

Temperature-induced changes of the states of beta-lactoglobulin have been studied calorimetrically. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. Its heat denaturation is approximated very closely by a two-state transition, while the cold denaturation deviates considerably from the two-state transition and this deviation increases as the temperature decreases. The heat effect of cold denaturation is opposite in sign to that of heat denaturation and is noticeably larger in magnitude. This difference in magnitude is caused by the temperature-dependent negative heat effect of additional binding of urea to the polypeptide chain of the protein upon its unfolding, which decreases the positive enthalpy of heat denaturation and increases the negative enthalpy of cold denaturation. The binding of urea considerably increases the partial heat capacity of the protein, especially in the denatured state. However, when corrected for the heat capacity effect of urea binding, the partial heat capacity of the denatured protein is close in magnitude to that expected for the unfolded polypeptide chain in aqueous solution without urea but only for temperatures below 10 degrees C. At higher temperatures, the heat capacity of the denatured protein is lower than that expected for the unfolded polypeptide chain. It appears that at temperatures above 10 degrees C not all the surface of the beta-lactoglobulin polypeptide chain is exposed to the solvent, even in the presence of 6 M urea; i.e., the denatured protein is not completely unfolded and unfolds only at temperatures lower than 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Denaturation of staphylococcal nuclease was studied in a temperature range from -7 to 70'C by scanning microcalorimetry and spectropolarimetry. It was found that the native protein is maximally stable at about 20'C and is denatured upon heating and cooling from this temperature. The heat and cold denaturation processes are approximated rather well by a two-state transition showing that the molecule is composed of a single cooperative system. The main difference between these two processes is in the sign of the enthalpy and entropy of denaturation: whereas the heat denaturation proceeds with increases in the enthalpy and entropy, the cold denaturation proceeds with decreases in both quantities. The inversion of the enthalpy sign occurs at about 15'C in an acetate buffer, but this temperature can be raised by addition of urea to the solvent.It is known that the denaturation of a protein is always accompanied by a significant increase in its enthalpy (for reviews, see refs. 1 and 2). The usually observed positive denaturational increment of heat capacity means that the enthalpy of protein denaturation is a temperature-dependent function. Thus, one can expect that the enthalpy of denaturation can, in principle, become zero and then even invert its sign at some low enough temperature, 7liV, changing from the factor stabilizing the native protein structure into a factor destabilizing this structure. Therefore, one can imagine that protein denaturation can occur not only upon heating but also upon cooling. In contrast with heat denaturation, which proceeds with heat absorption-i.e., an increase of enthalpy and entropy-cold denaturation should proceed with a release of heat-i.e., a decrease of enthalpy and entropy (3).The decrease of entropy upon the breakdown of the ordered native structure of protein molecules sounded like a paradox when it was first predicted more than 20 years ago by Brandts (4,5). As this prediction of cold denaturation was based on a long extrapolation of indirect data, there have been many attempts since then to confirm it by direct experiment. The first successful demonstration of cold denaturation was accomplished with myoglobin and apomyoglobin (3, 6).However, to show that cold denaturation is a general phenomenon specific for all globular proteins and not only for globins, it is highly desirable to demonstrate it on some other proteins. One of them was found to be lactate dehydrogenase on which cold denaturation was recently observed by Hatley and Franks (7). The other is staphylococcal nuclease (Nase), the denaturation of which is considered in this paper.Heat denaturation of Nase was studied calorimetrically by Calderon et al. (8) and it was shown that it is accompanied by a rather large increase of heat capacity, which indicated that cold denaturation of this protein might be observable under experimentally realizable conditions. MATERIALS AND METHODSThe Nase was prepared as described in detail by Calderon et al. (8) and was kindly supplied by John Gerlt (University of Maryland)...

Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the ␣-helical content increases from 60-64% to 80-90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3-to 4-fold increase in the average area subtended per molecule to 4,200 Å 2 on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled ␣-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.

It has been shown that the denaturation of phosphoglycerate kinase (PGK) can be observed not only when the solution is heated above 3O"C, but also when it is cooled below this temperature. The disruption of the native PGK structure upon cooling and the subsequent formation of this structure upon heating both proceed in two distinct stages which correspond to the independent disruption or reformation of each of its domains. In contrast, the heat denaturation of PGK proceeds in one stage, showing that the two domains of the molecule are associated into a single complex which figures in the denaturation process as a cooperative unit. It follows that, at elevated temperature, there is a positive interaction between the domains, which disappears at lower temperatures. This might be due to hydrophobic interactions, which are known to be temperature dependent. The temperature decrease leads to a decrease in inter-and intradomain interactions, which results in an increase of the independence of the domains and a decrease in their stability.

Thermodynamic properties of a mutant lambda Cro repressor with Cys replacing Val55 were studied calorimetrically. Formation of the S-S cross-link between neighboring Cys55 residues in this dimeric molecule leads to stabilization of a structure formed by the C-terminal parts of the two polypeptide chains, which behave as a single cooperative domain upon protein denaturation by heating. This composite domain is very stable at neutral pH and disrupts at 110 degrees C. The S-S-cross-linked tryptic fragment (residues 22-66), which includes this C-terminal domain, has similar stability. The N-terminal parts of the polypeptide chains do not form any stable structure when isolated, but in S-S-cross-linked dimer, they form a single cooperative block which melts in an all-or-none way 9 degrees C higher than the un-cross-linked protein. The observed cooperation of the distant N-terminal parts in dimer raises questions regarding lambda Cro repressor structure in solution.