No abstract

Enhanced structural insights into the folding energy landscape of the N-terminal dimerization domain of E. coli tryptophan repressor, 2 TR, were obtained from a combined experimental and theoretical analysis of its equilibrium folding reaction. Previous studies have shown that the three intertwined helices in [2-66] 2 TR are sufficient to drive the formation of a stable dimer for the fulllength protein, [2-107] 2 TR. The monomeric and dimeric folding intermediates that appear during the folding reactions of [2-66] 2 TR have counterparts in the folding mechanism of the full-length protein. The equilibrium unfolding energy surface on which the folding and dimerization reactions occur for [2-66] 2 TR was examined with a combination of native-state hydrogen exchange analysis, pepsin digestion and MALDI mass spectrometry performed at several protein and denaturant concentrations. Peptides corresponding to all three helices in [2-66] 2 TR show multi-layered protection patterns consistent with the relative stabilities of the dimeric and monomeric folding intermediates. The observation of protection exceeding that offered by the dimeric intermediate in segments from all three helices implies that a segment-swapping mechanism may be operative in the monomeric intermediate. Protection greater than that expected from the global stability for a single amide hydrogen in a peptide from the A-helix and another from the C-helix may reflect non-random structure, possibly a pre-cursor for segment swapping, in the urea-denatured state. Native topologybased model simulations that correspond to a funnel energy landscape capture both the monomeric and dimeric intermediates suggested by the HX-MS data and provide a rationale for the progressive acquisition of secondary structure in their conformational ensembles.

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Carbonic anhydrase from the archeon Methanosarcina thermophila (Cam) is a homo-trimeric enzyme, the left-handed beta-helical subunits of which bind three catalytic Zn(2+) ions at symmetry-related subunit interfaces. The observation of activity for holo-Cam at nanomolar concentrations provides a minimal estimated free energy of folding and assembly of the trimeric holo-complex of approximately 70 kcal (mol trimer)(-1) at standard state. Although the direct measurement of stability by chemical denaturation was precluded by the irreversible unfolding of the holo-enzyme, the reversible unfolding of metal-free apo-Cam is well described by a three-state model involving the folded apo-trimer, the folded monomer and the unfolded monomer. The monomer is estimated to have a stability of 4.0 +/- 0.3 kcal (mol monomer)(-1). The association to form apo-trimer contributes 13.2 +/- 0.4 kcal (mol trimer)(-1), a value confirmed by analytical ultracentrifugation measurements. Far- and near-UV circular dichroism data show a progressive increase in secondary and tertiary structure as the apo-monomer is converted to holo-trimer. The literature value for the free energy of binding of one Zn(2+) ion to a canonical active site, 16.4 kcal mol(-1), is consistent with the presumption that the >45 kcal (mol trimer)(-1) generated by the binding of three ions represents the major contribution to the stability of the holo-trimeric Cam.