The mechanism of ligand binding coupled to conformational changes in macromolecules has recently attracted considerable interest. The 2 limiting cases are the ''induced fit'' mechanism (binding first) or ''conformational selection'' (conformational change first). Described here are the criteria by which the sequence of events can be determined quantitatively. The relative importance of the 2 pathways is determined not by comparing rate constants (a common misconception) but instead by comparing the flux through each pathway. The simple rules for calculating flux in multistep mechanisms are described and then applied to 2 examples from the literature, neither of which has previously been analyzed using the concept of flux. The first example is the mechanism of conformational change in the binding of NADPH to dihydrofolate reductase. The second example is the mechanism of flavodoxin folding coupled to binding of its cofactor, flavin mononucleotide. In both cases, the mechanism switches from being dominated by the conformational selection pathway at low ligand concentration to induced fit at high ligand concentration. Over a wide range of conditions, a significant fraction of the flux occurs through both pathways. Such a mixed mechanism likely will be discovered for many cases of coupled conformational change and ligand binding when kinetic data are analyzed by using a fluxbased approach.kinetics ͉ binding ͉ folding ͉ coupled equilibria ͉ mechanism T he binding of ligands by macromolecules is crucial to a multitude of physiological processes. These include the cascade of reactions accompanying hormone binding to receptors (1, 2), enzyme reactions initiated by the binding of substrates to the enzyme (3, 4), gene regulation by the binding of molecules to DNA (5) and RNA (6), and the folding of unstructured proteins to produce biologically active molecules (7,8). In virtually all cases, the binding of ligands and conformational changes go hand in hand. Consequently, considerable effort has been expended in assessing the detailed mechanism by which ligand binding and conformational changes are coupled (3,4,9). Two limiting mechanisms are generally considered: (i) ''conformational selection,'' whereby the ligand selectively binds to a form of the macromolecule that is present only in small amounts, eventually converting the macromolecule to the ligand-bound conformation; and (ii) ''induced fit,'' (9) whereby ligand binds to the predominant free conformation followed by a conformational change in the macromolecule to give the preferred ligandbound conformation.The 2 limiting mechanisms can be written as:Conformational Selection: P weak º P tightInduced Fit: P weak ϩ L º P weak ⅐LIn these equations, P weak and P tight represent the tightly and weakly binding conformations of the protein (or other macromolecule), and L is the ligand. In point of fact, these 2 limiting mechanisms can be distinguished by kinetic measurements of the rate of the conformational change. In the simplest case, which often prevails, the ligand-bind...

Coupled ligand binding and conformational change plays a central role in biological regulation. Ligands often regulate protein function by modulating conformational dynamics, yet the order in which binding and conformational change occurs are often hotly debated. Here we show that the “conformational selection versus induced fit” on which this debate is based is a false dichotomy because the mechanism depends on ligand concentration. Using the binding of pyrophosphate (PPi) to B. subtilis RNase P protein as a model, we show that coupled reactions are best understood as a change in flux between competing pathways with distinct orders of binding and conformational change. The degree of partitioning through each pathway depends strongly on PPi concentration, with ligand binding redistributing the conformational ensemble toward the folded state by both increasing folding rates and decreasing unfolding rates. These results indicate that ligand binding induces marked and varied changes in protein conformational dynamics, and that the order of binding and conformational change is ligand concentration dependent.

The thermodynamic stability of proteins is typically measured at high denaturant concentrations and then extrapolated back to zero denaturant conditions to obtain unfolding free energies under native conditions. For membrane proteins, the extrapolations are fraught with considerable uncertainty as the denaturants may have complex effects on the membrane or micellar structure. We therefore sought to measure stability under native conditions, using a method that does not perturb the properties of the membrane or membrane mimetics. We use a technique called steric trapping to measure the thermodynamic stability of bacteriorhodopsin in bicelles and micelles. We find that bacteriorhodopsin has a high thermodynamic stability, with an unfolding free energy of ∼11 kcal/mol in dimyristoyl phosphatidylcholine bicelles. Nevertheless, the stability is much lower than predicted by extrapolation of measurements made at high denaturant concentrations. We investigated the discrepancy and found that unfolding free energy is not linear with denaturant concentration. Apparently, long extrapolations of helical membrane protein unfolding free energies must be treated with caution. Steric trapping, however, provides a method for making these measurements.membrane protein folding | steric trap M ethods to measure the thermodynamic stability of membrane proteins have largely followed methods developed for soluble protein folding (1). The fraction unfolded is first measured as a function of denaturant concentration (urea, guanidine-HCl, etc.), which in turn provides the unfolding free energy (ΔG U ) as a function of denaturant. The fraction unfolded, however, can be accurately measured only at high denaturant concentration, where the amount of unfolded protein is large enough-the so-called transition zone. Thus, obtaining a measure of the unfolding free energy in the absence of denaturant requires extrapolation from the transition zone. For chemical denaturation, the unfolding free energy is typically linearly dependent on the denaturant concentration in the transition zone, allowing a linear extrapolation back to zero denaturant. However, although there is now considerable experimental and theoretical validation of this approach for soluble proteins (2-4), the validity of these extrapolations is not clear for measuring stability of membrane proteins.Since the observation of Braiman et al. that bacteriorhodopsin (bR) can be refolded from an SDS-denatured state (5), SDS has been commonly used to study the folding of helical membrane proteins. The Booth laboratory has pioneered and extensively studied the refolding kinetics of bR from an SDS-denatured state (6-8). We introduced SDS unfolding to measure the thermodynamic stability of the membrane enzyme diacylglycerol kinase (9) and a similar approach can be used to measure bR thermodynamic stability (10, 11). bR contains a covalently bound retinal chromophore that complicates unfolding analysis because it can slowly hydrolyze off in the SDS unfolded protein (12). We and others originally ...

Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in the present study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine-N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations showed multiphasic kinetics. Equilibrium “cotitration” experiments were performed using both TMAO and urea during the titration to obtain a TMAO-urea titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated and the folding rate constants involved in the reaction are relatively slow compared to intrinsically folded proteins of similar size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.