Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.

The folding mechanism of many proteins involves the population of partially organized structures en route to the native state. Identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. Following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding mechanism of the thermostable cytochrome c 552 from Hydrogenobacter thermophilus does involve the presence of an elusive, yet compact, on-pathway intermediate. Characterization of the folding mechanism of this cytochrome c is particularly interesting for the purpose of comparative folding studies, because H. thermophilus cytochrome c 552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c 551 , which refolds through a broad energy barrier without the accumulation of intermediates. Analysis of the folding kinetics and correlation with the three-dimensional structure add new evidence for the validity of a consensus folding mechanism in the cytochrome c family.

(1) to account for the efficient fluorescence quenching of internal tryptophans by dioxygen. In the crystal structure of enzymes processing large substrates, a channel involved in the capture and optimal presentation to the catalytic site is seen (2). Cavities and connecting channels identified in the interior space of a protein (3) confer flexibility and alternative packing arrangements that allow rapid transitions between structurally distinct states; in small globular proteins, however, it is not known whether these packing defects are important in controlling function by defining alternative pathways and hosting stations in the diffusion of a ligand to the active site. The ultra-low temperature x-ray diffraction experiment on the sperm whale myoglobin (Mb) mutant presented in this paper supports the viewpoint that pre-existing internal cavities play a major role in controlling the dynamics of ligand binding and can be modified by protein engineering.The role of protein relaxation in Mb has been extensively investigated by laser photolysis because the photosensitivity of the complex of ferrous Mb with CO, O 2 , and NO makes it possible to populate intermediate states and to follow the structural dynamics involved in the relaxation of the protein and the migration of the ligand through the matrix. These diatomic molecules are small enough to migrate rapidly away from the metal and large enough to probe accessibility of atomic-sized cavities that are sparse in the structure and interconnected through fluctuating channels (3-5). The high photosensitivity of the MbCO adduct, with unitary quantum yield (6), makes this derivative an ideal model for time-resolved crystallography (7) using intense synchrotron x-ray sources.Previous crystallographic studies on the photolyzed state of wild-type (wt) sperm whale Mb showed the photodissociated CO* to reside in the heme pocket near the metal but at nonbonding distance, both at room temperature in the first time-resolved nsec diffraction experiment (7) and at ultra-low temperatures (8-10). Differences in the location of CO* observed in the cryogenic x-ray diffraction experiments were attributed (partially or totally) to a variable degree of photolysis related to the experimental protocol and͞or the temperature. The results indicate (see ref. 10 for a discussion) that the photolyzed CO* is located largely 3.6-3.7 Å from the Fe (which moves out of the heme plane by Ϸ 0.3 Å), in agreement with optical absorption and polarized IR time-resolved studies, as well as molecular dynamics calculations (11)(12)(13)(14). A study of the structural dynamics of Mb by x-ray diffraction may provide an answer to some interesting questions, such as: (i) How far could the niche hosting the photolyzed CO* be considered a docking site? (ii) To what extent could this niche be controlled by the nature of the side chains in the distal heme pocket? (iii) Could protein engineering allow one to modify the pathways for ligand migration through cavities and channels within the protein matrix, to and fr...

PDZ domains are protein adapter modules present in a few hundred human proteins. They play important roles in scaffolding and signal transduction. PDZ domains usually bind to the C termini of their target proteins. To assess the binding mechanism of this interaction we have performed the first in-solution kinetic study for PDZ domains and peptides corresponding to target ligands. Both PDZ3 from postsynaptic density protein 95 and PDZ2 from protein tyrosine phosphatase L1 bind their respective target peptides through an apparent A ؉ B 3 A⅐B mechanism without rate-limiting conformational changes. But a mutant with a fluorescent probe (Trp) outside of the binding pocket suggests that slight changes in the structure take place upon binding in protein tyrosine phosphatase-L1 PDZ2. For PDZ3 from postsynaptic density protein 95 the pH dependence of the binding reaction is consistent with a one-step mechanism with one titratable group. The salt dependence of the interaction shows that the formation of electrostatic interactions is rate-limiting for the association reaction but not for dissociation of the complex. PDZ4 domains are found in a few hundred human proteins, either as a single domain or in arrays. These domains mediate binding to other proteins and in this way play important roles in scaffolding and signal transduction (1, 2). Structural studies have shown that the PDZ domains usually bind to the C terminus of their target proteins. A number of crystal and NMR structures of PDZ domains have been solved both with and without bound peptide (for example, Refs 3-6) ( Fig. 1). A wealth of data on different peptides binding to different PDZ domains has been obtained by screening (for example, Refs. 7 and 8) and selection (for example, Refs. 9 and 10). Such studies and those using the yeast twohybrid technique (for example, Ref. 11) provide important information on possible cellular targets for distinct PDZ domains as well as the specificity of the interaction. Moreover, theory and NMR experiments have suggested that the dynamics of PDZ domains and the residues outside of the binding pocket influence their interaction with ligands (12, 13). Despite considerable effort to clarify the structural basis for the PDZ-ligand interaction, only a handful of studies have assessed the binding energetics and specificity of PDZ-peptide interactions using proper equilibrium assays in solution (3, 11, 14 -24). Kinetics of chemical reactions not only provide "end point data" such as equilibrium constants but also yield microscopic rate constants and, more importantly, the possibility of elucidating the mechanisms of binding and probing the binding dynamics as well as the properties of the transition state of the reaction. To assess the binding mechanism, we have performed the first kinetic study of PDZ domains in solution using stopped-flow fluorimetry. The PDZ domains chosen were PDZ3 from human PSD-95, one of the most well studied PDZ domains, and the second PDZ domain from mouse protein tyrosine phosphatase-L1 (PTP-BL; also known...