Conformational stability of dimeric proteins: Quantitative studies by equilibrium denaturation

Abstract

The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, AG,(H,O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing AG,(H20) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.Keywords: conformational stability; dimeric proteins; equilibrium denaturation Equilibrium denaturation studies have been very useful in understanding the structure, stabilization, and folding of small, monomeric proteins (Tanford, 1968;Pace, 1986Pace, , 1990Timasheff, 1992). The methods for analyzing the energetics of reversible unfolding of proteins by thermal or chemical denaturant techniques (Privalov, 1979;Pace, 1986;Privalov & Gill, 1988) and the conclusions that can be drawn from thorough analysis of a few proteins (Pace, 1986(Pace, , 1990Pace et al., 1990) have been presented. Many of these studies have been done with proteins whose disulfide bonds are intact, but entropic effects of the disulfide crosslinks have also been estimated (Pace, 1990). The range of stabilities calculated for monomers is between 6 and 14 kcal/mol and represents the small difference between multiple noncovalent interactions favoring the folded protein structure and unfavorable entropic terms. Such studies have been particularly useful for analysis of packing forces in protein interiors (Alber & Matthews, 1987;Matsumura et al., 1988), the testing of the globular folding of mutant proteins (Fersht et al., 1992), and the functional interaction of residues (Carter et al., Reprint requests to: Kenneth E. Neet, Department of Biological Chemistry, UHWChicago Medical School, 3333 Green Bay Road, North Chicago, Illinois 60064; e-mail: neetk@mis.fuhscms.edu. 1984). However, application of similar techniques to oligomeric proteins has, until recently, been less common, despite the potential for providing additional information on subunit interactions. Classically, the kinetics of folding pathways have been used to determine the relationship of folding to activity of oligomeric pro...

Structural Features Responsible for the Biological Stability of Histoplasma’s Virulence Factor CBP

“…The conformational stabilities, ΔG°, determined for dimeric proteins that denature with a single transition of native dimer to denatured monomer range from 10 to 27 kcal/mol (37). The unfolding free energy for CBP determined from equilibrium urea denaturation studies would suggest that the protein is not thermodynamically stable.…”

Conformational stability of apoflavodoxin

“…10) indicates that the molten globule is not a monomeric species and provides an explanation for these anomalous results. The increase in apparent stability at increasing concentrations of protein implies that the unfolding transition is coupled to dissociation of associated monomers (Neet & Timm, 1994). This could also explain the relatively high value of m .…”

Thermodynamic Dissection of a Low Affinity Protein-Protein Interface Involved in Human Immunodeficiency Virus Assembly

“…2 and Table I). This allowed determination of the association constant K a and the free energy difference ⌬G a between dimer and monomer, and thus, the contribution of quaternary interactions to the dissociation process (37,42). The difference between ⌬G a for nonmutated CA-C and ⌬G a for each mutant (⌬⌬G a ) was taken as a measure of the relative energetic contribution of the truncated side chain (beyond C␤) (Fig.…”

“…For T188A and N193A the native baseline could not be experimentally determined, and the ⌬G ud values were obtained assuming the baseline values determined for nonmutated CA-C, which were also very similar to those obtained for all other dimeric mutants. ⌬G ur (the free energy difference corresponding to the tertiary rearrangement of each monomeric intermediate during dissociation of the CA-C dimer) was calculated from ⌬G ur ϭ (⌬G ud Ϫ ⌬G dis )/2 (37,42).…”