Conformational dynamics play a key role in enzyme catalysis. While protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of enzyme catalysis has not been clearly established. We generated a mutant of E. coli dihydrofolate reductase (DHFR) that abrogates millisecond time scale fluctuations in the enzyme active site without perturbing its structural and electrostatic preorganization. Remarkably, this dynamic knockout severely impairs hydride transfer. Thus we have found a link between conformational fluctuations on the millisecond timescale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for attempts to design novel protein catalysts.

Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR). With no optimization, PAS-DHFR exhibited light-dependent catalytic activity that depended on the site of connection and on known signaling mechanisms in both proteins. PAS-DHFR serves as a proof of concept for engineering regulatory activities into proteins through interface design at conserved allosteric sites.Proteins typically adopt well-packed three-dimensional structures in which amino acids are engaged in a dense network of contacts (1,2). This emphasizes the energetic importance of local interactions, but protein function also depends on nonlocal, long-range communication between amino acids. For example, information transmission between distant functional surfaces on signaling proteins (3), the distributed dynamics of amino acids involved in enzyme catalysis (4-6), and allosteric regulation in various proteins (7) all represent manifestations of nonlocal interactions between residues. To the extent that these features contribute to defining biological properties of protein lineages, we expect that the underlying mechanisms represent conserved rather than idiosyncratic features in protein families.On the basis of this conjecture, methods such as statistical coupling analysis (SCA) quantitatively examine the long-term correlated evolution of amino acids in a protein family -the statistical signature of functional constraints arising from conserved communication between positions (8,9). This approach has identified sparse but physically connected networks of coevolving amino acids in the core of proteins (8)(9)(10)(11)(12). The connectivity of these networks is remarkable, given that a small fraction of total residues are involved and that no tertiary structural information is used in their identification. Empirical observation in several protein families shows that these networks connect the main functional site with distantly † To whom correspondence should be addressed. rama.ranganathan@utsouthwestern.edu. * These authors contributed equally to this work. The finding that certain surface sites might be statistical "hotspots" for functional interaction with active sites suggests an idea for engineering new regulatory mechanisms into proteins. What if two proteins were joined at surface sites such that their statistically correlated networks were juxtaposed and could form functional interactions (Fig. 1A)? If the connection sites are functionally linked to their respective activ...

The adequate stimuli and molecular receptors for muscle metaboreceptors and nociceptors are still under investigation. We used calcium imaging of cultured primary sensory dorsal root ganglion (DRG) neurons from C57Bl/6 mice to determine candidates for metabolites that could be the adequate stimuli and receptors that could detect these stimuli. Retrograde DiI labeling determined that some of these neurons innervated skeletal muscle. We found that combinations of protons, ATP, and lactate were much more effective than individually applied compounds for activating rapid calcium increases in muscle-innervating dorsal root ganglion neurons. Antagonists for P2X, ASIC, and TRPV1 receptors suggested that these three receptors act together to detect protons, ATP, and lactate when presented together in physiologically relevant concentrations. Two populations of muscle-innervating DRG neurons were found. One responded to low metabolite levels (likely nonnoxious) and used ASIC3, P2X5, and TRPV1 as molecular receptors to detect these metabolites. The other responded to high levels of metabolites (likely noxious) and used ASIC3, P2X4, and TRPV1 as their molecular receptors. We conclude that a combination of ASIC, P2X5 and/or P2X4, and TRPV1 are the molecular receptors used to detect metabolites by muscle-innervating sensory neurons. We further conclude that the adequate stimuli for muscle metaboreceptors and nociceptors are combinations of protons, ATP, and lactate.