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...

The energy landscape theory provides a general framework for describing protein folding reactions. Because a large number of studies, however, have focused on two-state proteins with single well-defined folding pathways and without detectable intermediates, the extent to which free energy landscapes are shaped up by the native topology at the early stages of the folding process has not been fully characterized experimentally. To this end, we have investigated the folding mechanisms of two homologous threestate proteins, PTP-BL PDZ2 and PSD-95 PDZ3, and compared the early and late transition states on their folding pathways. Through a combination of ⌽ value analysis and molecular dynamics simulations we obtained atomic-level structures of the transition states of these homologous three-state proteins and found that the late transition states are much more structurally similar than the early ones. Our findings thus reveal that, while the native state topology defines essentially in a unique way the late stages of folding, it leaves significant freedom to the early events, a result that reflects the funneling of the free energy landscape toward the native state.energy landscape ͉ kinetics ͉ molecular dynamics ͉ phi analysis ͉ protein folding T he description of the folding process of a protein in terms of pathways on a free energy landscape has provided much insight into the mechanism of the folding reaction (1-4). Free energy landscapes of many proteins appear to resemble the shape of a funnel that guides the folding process toward the native state (5-7). According to this view, folding is considered a stochastic process so that a protein reaches its native conformation through folding pathways made up by an ensemble of different trajectories (6,(8)(9)(10). It has been difficult, however, to establish experimentally the extent to which these trajectories can differ from each other, in particular in the wider region of the free energy funnel corresponding to the initial stages of the folding process, where a considerable heterogeneity may be expected. Many proteins exhibit single folding pathways composed of families of closely related trajectories, but parallel folding pathways have also been observed (11), as well as significant changes of pathways and transition state structures upon circular permutation (12, 13) or solvent conditions (14-16). In addition, different transition state structures were characterized in two homologous proteins with a highly symmetric native structure (17).To obtain a glimpse of the width of the free energy landscape at the early stages of the folding reaction, we compared the folding pathways of two homologous three-state proteins. The study of homologous proteins represents a powerful approach to obtain insight into the process of protein folding (18-22), especially when combined with structural information on intermediate events (17,(23)(24)(25). The transition states of two-state proteins were compared in a series of studies (17, 23, 24, 26).Here we present a vivid illustration of...

Theoretical ab initio quantum mechanical charge field molecular dynamics (QMCF MD) formalism has been applied in conjunction to experimental large angle X-ray scattering to study the structure and dynamics of the hydrated sulfite ion in aqueous solution. The results show that there is a considerable effect of the lone electron-pair on sulfur concerning structure and dynamics in comparison with the sulfate ion with higher oxidation number and symmetry of the hydration shell. The S-O bond distance in the hydrated sulfite ion has been determined to 1.53(1) Å by both methods. The hydrogen bonds between the three water molecules bound to each sulfite oxygen are only slightly stronger than those in bulk water. The sulfite ion can therefore be regarded as a weak structure maker. The water exchange rate is somewhat slower for the sulfite ion than for the sulfate ion, τ(0.5) = 3.2 and 2.6 ps, respectively. An even more striking observation in the angular radial distribution (ARD) functions is that the for sulfite ion the water exchange takes place in close vicinity of the lone electron-pair directed at its sides, while in principle no water exchange did take place of the water molecules hydrogen bound to sulfite oxygens during the simulation time. This is also confirmed when detailed pathway analysis is conducted. The simulation showed that the water molecules hydrogen bound to the sulfite oxygens can move inside the hydration shell to the area outside the lone electron-pair and there be exchanged. On the other hand, for the hydrated sulfate ion in aqueous solution one can clearly see from the ARD that the distribution of exchange events is symmetrical around the entire hydration sphere.

Graphical Entry Textual abstractThe selenite ion has an asymmetric hydration sphere with loosely electrostatically bound water molecules outside the free electron pair. 2 AbstractStructure and hydrogen bonding of the hydrated selenite, SeO 3 2-, and selenate, SeO 4 2-, ions have been studied in aqueous solution by large angle X-ray scattering (LAXS), EXAFS and double difference infrared (DDIR) spectroscopy. The mean Se-O bond distances are 1.709(2) and 1.657(2) Å, respectively, as determined by LAXS, and 1.701(3) and 1.643(3) Å by EXAFS.These bond distances are slightly longer than the mean distances found in the solid state, 1.691 and 1.634 Å, respectively. The structures of The DDIR spectra show peaks for affected water bound to the selenite and selenate ions at 2491±2 and 2480±39 cm -1 , respectively, compared to 2509 cm -1 in pure water. This shows that the selenite and selenite ions shall be regarded as weak structure makers. 3 IntroductionThe selenium oxo acids and their salts have many similarities with the corresponding sulfur oxo acids, including similar physico-chemical parameters as e.g. the acid dissociations constants; H 2 SeO 4 : pK a2 =1.70; H 2 SO 4 : pK a2 =1.99; H 2 SeO 3 , pK a1 =2.62, pK a2 =8.32, H 2 SO 3 ; pK a1 =1.85, pK a2 =7.20 . 1 The structure and hydrogen bonding of the hydrated sulfite and sulfate ions have previously been studied in aqueous solution using large angle X-ray scattering (LAXS) and double difference infrared spectroscopy as well as simulations on QMCF MD level. 2-4 Three water molecules are hydrogen bound to each oxygen atom for both ions. Furthermore, some water molecules are clustered outside the lone electron-pair of the sulfite ion at a reasonably welldefined distance. 4 In studies of other oxoanions it has been shown that the number of hydrogen bound water molecules seems to decrease when the central atom belong to the third and fourth series. The arsenate ions, independent of degree of protonation, and arsenous and telluric acid all bind approximately two water molecules to each oxygen. 5 The sulfate and sulfite ions are both weak structure makers, thus the hydrogen bond between the sulfate/sulfite oxygens and the hydrating water molecules is slightly shorter and stronger than between water molecules in the aqueous bulk. [2][3][4] It is of fundamental interest to compare the hydration of the selenite and sulfite ions as they have asymmetric hydration shells due to the presence of a free electron pair in the fourth tetrahedron vortex. Previously reported simulations of the water exchange dynamics of the hydrated sulfate and sulfite ions in aqueous solution showed significantly different mechanisms. 4 The hydrogen bound water molecules on the sulfate oxygen exchange directly with a bulk water. On the other hand, the asymmetrically hydrated sulfite ion exchanges almost all water molecules in close vicinity of the free electron pair with a transport path from the sulfite oxygens to this region. 4 The aim of this study is to determine the structures and the hydrogen b...

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