Detection of native-state nonadditivity in double mutant cycles via hydrogen exchange.

Proteins have evolved to exploit long-range structural and dynamic effects as a means of regulating function. Understanding communication between sites in proteins is therefore vital to our comprehension of such phenomena as allostery, catalysis, and ligand binding/ejection. Double mutant cycle analysis has long been used to determine the existence of communication between pairs of sites, proximal or distal, in proteins. Typically, nonadditivity (or "thermodynamic coupling") is measured from global transitions in concert with a single probe. Here, we have applied the atomic resolution of NMR in tandem with native-state hydrogen exchange (HX) to probe the structure/energy landscape for information transduction between a large number of distal sites in a protein. Considering the event of amide proton exchange as an energetically quantifiable structural perturbation, m n-dimensional cycles can be constructed from mutation of n-1 residues, where m is the number of residues for which HX data is available. Thus, efficient mapping of a large number of couplings is made possible. We have applied this technique to one additive and two nonadditive double mutant cycles in a model system, eglin c. We find heterogeneity of HX-monitored couplings for each cycle, yet averaging results in strong agreement with traditionally measured values. Furthermore, long-range couplings observed at locally exchanging residues indicate that the basis for communication can occur within the native state ensemble, a conclusion not apparent from traditional measurements. We propose that higher-order couplings can be obtained and show that such couplings provide a mechanistic basis for understanding lower-order couplings via "spheres of perturbation". The method is presented as an additional tool for identifying a large number of couplings with greater coverage of the protein of interest.

Dynamic coupling and allosteric behavior in a nonallosteric protein.

Long-range intraprotein interactions give rise to many important protein behaviors. Understanding how energy is transduced through protein structures to either transmit a signal or elicit conformational changes is therefore a current challenge in structural biology. In an effort to understand such linkages, multiple V --> A mutations were made in the small globular protein eglin c. The physical responses, as mapped by NMR spin relaxation, residual dipolar couplings (RDCs), and scalar couplings, illustrate that the interior of this nonallosteric protein forms a dynamic network and that local perturbations are transmitted as dynamic and structural changes to distal sites as far as 16 A away. Two basic types of propagation responses were observed: contiguous pathways of enhanced (attenuated) dynamics with no change in structure; and dispersed (noncontiguous) changes in methyl rotation rates that appear to result from subtle deformation of backbone structure. In addition, energy transmission is found to be unidirectional. In one mutant, an allosteric conformational change of a side chain is seen in the context of a pathway of propagated changes in picosecond to nanosecond dynamics. The observation of so many long-range interactions in a small, rigid system lends experimental weight to the idea that all well-folded proteins inherently possess allosteric features [Gunasekaran et al. (2004) Proteins 57, 433-443], and that dynamics are a rich source of information for mapping and gaining mechanistic insight into communication pathways in individual proteins.

Direct demonstration of structural similarity between native and denatured eglin C.

To characterize the long-range structure that persists in the unfolded form of the 70-residue protein eglin C, residual dipolar couplings (RDCs) for HN-N and HA-CA bond vectors were measured by NMR spectroscopy for both its low pH, urea denatured state and its native state. When the data sets for the two different structural states were compared, a statistically significant correlation was found, with both sets of dipolar couplings yielding a correlation coefficient of r = 0.47 to 0.51. This finding directly demonstrates that the denatured state of eglin C has a nativelike global structure, a conclusion reached indirectly for staphylococcal nuclease by combining two different types of NMR data. A simple computer simulation showed that the degree of variation in phi and psi angles that yields the RDC correlation of r = 0.5 was inversely dependent on the statistical segment length, ranging from +/-6 to +/-30 degrees at the upper limit. Stable nativelike topologies that persist on unfolding would explain the rapid refolding kinetics displayed by many proteins and might provide a natural barrier against amyloid fibril formation.