Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W. C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profiles and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low-resolution threedimensional representations of the structures of gap junctions.The structural variations detailed in the preceding paper (5) establish that we are looking at not one structure of the gap junction, but at a family of structures. By observing closely related states of a molecular assembly, it is often possible to infer something about the way that transitions occur between states. These molecular rearrangements may be significant in the functional activity of the structure. Furthermore, polymorphism provides a constraint on the interpretation of the diffraction patterns. For example, the connexon structure is likely to be similar in arrays with different lattice constants, although the intensities in the X-ray patterns may be quite different.

An approach combining small-angle X-ray solution scattering (SAXS) data with coarse-grained (CG) simulations is developed to characterize the assembly states of Hck, a member of the Srcfamily kinases, under various conditions in solution. First, a basis set comprising a small number of assembly states is generated from extensive CG simulations. Second, a theoretical SAXS profile for each state in the basis set is computed by using the Fast-SAXS method. Finally, the relative population of the different assembly states is determined via a Bayesian-based Monte Carlo procedure seeking to optimize the theoretical scattering profiles against experimental SAXS data. The study establishes the concept of basis-set supported SAXS (BSS-SAXS) reconstruction combining computational and experimental techniques. Here, BSS-SAXS reconstruction is used to reveal the structural organization of Hck in solution and the different shifts in the equilibrium population of assembly states upon the binding of different signaling peptides.T yrosine kinases of the Src-family are large multidomain allosteric enzymes implicated in the signaling pathways regulating cell growth and proliferation (1). The key role that Src kinases play in the onset of many human diseases, particularly cancer, makes them important targets for therapeutic intervention (2).All Src kinases share a common structural organization comprising the SH3 and SH2 binding domains followed by a highly conserved catalytic domain connected by flexible linkers (1). Crystal structures have offered a detailed view of the down-regulated forms of Hck and c-Src (3, 4), characterized by a compact assembled form stabilized by auto-inhibitory intramolecular interactions between the N and C terminus of the catalytic domain with the SH3 and SH2 modules, respectively. The SH2 and SH3 modules of the Src-family tyrosine kinases play dual roles: both are involved in the auto-inhibitory intramolecular interactions down-regulating kinase activity, but can also serve as possible binding receptors for cellular signals leading to kinase activation.In a broadly accepted paradigm, Src inactivation/activation is pictured in terms of a simple two-state process involving an assembled (inactive) and a disassembled (active) conformation, i.e., once any of the intramolecular interaction is released, the kinase domain switches to a disassembled catalytically active state targeting down-stream cellular substrates (5-8). Crystal structures (3, 4) and mutational studies complemented by molecular dynamics (MD) simulations (9) are consistent with this view. However, one crystal structure of c-Src in a partially activated state in which the SH2-SH3 modules are reassembled in a different orientation with respect to the catalytic domain (10), suggests that the situation could be considerably more complex. In this regard, it seems likely that different signals could promote different assembly states, leading to differently configured activated kinases. While it is understood that Src increases its activity in respon...

X-ray solution scattering shows new promise for the study of protein structures, complementing crystallography and nuclear magnetic resonance. In order to realize the full potential of solution scattering, it is necessary to not only improve experimental techniques but also develop accurate and efficient computational schemes to relate atomistic models to measurements. Previous computational methods, based on continuum models of water, have been unable to calculate scattering patterns accurately, especially in the wide-angle regime which contains most of the information on the secondary, tertiary, and quaternary structures. Here we present a novel formulation based on the atomistic description of water, in which scattering patterns are calculated from atomic coordinates of protein and water. Without any empirical adjustments, this method produces scattering patterns of unprecedented accuracy in the length scale between 5 and 100 Å, as we demonstrate by comparing simulated and observed scattering patterns for myoglobin and lysozyme.

Bacterial adhesion pili are designed to bind specifically and maintain attachment of bacteria to target cells. Uropathogenic P-pili are sufficiently mechanically resilient to resist the cleansing action of urine flow that removes most other bacteria. P-pili are 68 A in diameter and approximately 1 micron long, and are composed of approximately 1,000 copies of the principal structural protein, PapA. They are attached to the outer membrane by a minor structural protein, PapH and are terminated by an approximately 20 A diameter fibrillus composed of PapK, PapE and PapF, which presents the host-binding adhesin PapG. The amino-acid sequences of PapA, PapE, and PapF are similar, with highly conserved C-termini being responsible for binding to PapD, the periplasmic chaperone. Our three-dimensional reconstruction indicates that pili are formed by the tight winding of a much thinner structure. A structural transition allows the pilus to unravel without depolymerizing, producing a thin, extended structure five times the length of the original pilus.

Two mechanisms have been proposed to drive asymmetric solvent response to a solute charge: a static potential contribution similar to the liquid-vapor potential, and a steric contribution associated with a water molecule's structure and charge distribution. In this work, we use free-energy perturbation molecular-dynamics calculations in explicit water to show that these mechanisms act in complementary regimes; the large static potential (∼44 kJ/mol/e) dominates asymmetric response for deeply buried charges, and the steric contribution dominates for charges near the solute-solvent interface. Therefore, both mechanisms must be included in order to fully account for asymmetric solvation in general. Our calculations suggest that the steric contribution leads to a remarkable deviation from the popular "linear response" model in which the reaction potential changes linearly as a function of charge. In fact, the potential varies in a piecewise-linear fashion, i.e., with different proportionality constants depending on the sign of the charge. This discrepancy is significant even when the charge is completely buried, and holds for solutes larger than single atoms. Together, these mechanisms suggest that implicit-solvent models can be improved using a combination of affine response (an offset due to the static potential) and piecewise-linear response (due to the steric contribution).

We present a coarse residue-based computational method to rapidly compute the solution scattering profile from a protein with dynamical fluctuations. The method is built upon a coarse-grained (CG) representation of the protein. This CG representation takes advantage of the intrinsic low-resolution and CG nature of solution scattering data. It allows rapid scattering determination from a large number of conformations that can be extracted from CG simulations to obtain scattering characterization of protein conformations. The method includes several important elements, effective residue structure factors derived from the Protein Data Bank, explicit treatment of water molecules in the hydration layer at the surface of the protein, and an ensemble average of scattering from a variety of appropriate conformations to account for macromolecular flexibility. This simplified method is calibrated and illustrated to accurately reproduce the experimental scattering curve of Hen egg white lysozyme. We then illustrated the applications of this CG method by computing the solution scattering patterns of several representative protein folds and multiple conformational states. The results suggest that solution scattering data, when combined with the reliable computational method that we developed, show great potential for a better structural description of multidomain complexes in different functional states, and for recognizing structural folds when sequence similarity to a protein of known structure is low.

We experimentally demonstrate a biomagnetic sensor scheme based on Brownian relaxation of magnetic nanoparticles suspended in liquids. The characteristic time scale of the Brownian relaxation can be determined directly by ac susceptibility measurements as a function of frequency. The peak in the imaginary part of the ac susceptibility shifts to lower frequencies upon binding the target molecules to the magnetic nanoparticles. The frequency shift is consistent with an increase of the hydrodynamic radius corresponding to the size of the target molecule.

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