The analytical toolkit developed for investigations into water-soluble protein folding has yet to be applied in earnest to membrane proteins. A major problem is the difficulty in collecting kinetic data, which are crucial to understanding any reaction. Here, we combine kinetic and thermodynamic studies of the reversible unfolding of an α-helical membrane protein to provide a definitive value for the reaction free energy and a means to probe the transition state. Our analyses show that the major unfolding step in the SDS-induced denaturation of bacteriorhodopsin involves a reduction in α-helical structure and proceeds with a large free-energy change; both our equilibrium and kinetic measurements predict that the free energy of unfolding in the absence of denaturant is +20 kcal·mol ⁻¹ , with an associated m -value of 25 kcal·mol ⁻¹ . The rate of unfolding in the absence of denaturant, k _u ^{H ₂ O} , is surprisingly very slow (≈10 ⁻¹⁵ s ⁻¹ ). The kinetics also give information on the transition state for this major unfolding step, with a value for β ( m _f /[ m _f + m _u ]) of ≈0.1, indicating that the transition state is close to the unfolded state. We thus present a basis for mapping the structural and energetic properties of membrane protein folding by mutagenesis and classical kinetics.

Investigations into protein folding have concentrated on experimentally tractable proteins with the result that membrane protein folding remains unsolved. New evidence is providing insight into the nature of the interactions stabilising the folded state of alpha-helical membrane proteins as well as giving hints on the character of the folding transition state. These developments show that classical methods used for water-soluble proteins can be successfully adapted for membrane proteins. The advances, coupled with increasing numbers of solved crystal structures, augur well for future research into the mechanisms of membrane protein folding.

The 19-mer synthetic peptide known as R5 has been used widely in studies of peptide-driven silica condensation. Despite this, the structure and function of R5 have not yet been fully characterized. Here, we present a systematic study of R5 silicification focusing on three key variables: the concentration of the peptide, the concentration of the silica precursor silicic acid, and the solution pH. Additionally, we present the first study of R5 secondary structure in the presence and absence of silicic acid and introduce one-dimensional and two-dimensional solution NMR to probe both structure and higher-order peptide aggregation. We find that R5-directed silicification is linear with regard to silicic acid and H⁺ but, unexpectedly, that silicification appears to be cooperative with respect to peptide concentration. We also find that R5 is a random coil ensemble at subsaturating silicic acid concentrations and does not spontaneously self-assemble to form discrete aggregates in solution. These data contradict a model that invokes the functional micellization of R5 and provide a framework for future studies with the R5 peptide.