Bacteriorhodopsin is a transmembrane protein that uses light energy, absorbed by its chromophore retinal, to pump protons from the cytoplasm of bacteria such as Halobacterium salinarium into the extracellular space. It is made up of seven alpha-helices, and in the bacterium forms natural, two-dimensional crystals called purple membranes. We have analysed these crystals by electron cryo-microscopy to obtain images of bacteriorhodopsin at 3.0 A resolution. The structure covers nearly all 248 amino acids, including loops outside the membrane, and reveals the distribution of charged residues on both sides of the membrane surface. In addition, analysis of the electron-potential map produced by this method allows the determination of the charge status of these residues. On the extracellular side, four glutamate residues surround the entrance to the proton channel, whereas on the cytoplasmic side, four aspartic acids occur in a plane at the boundary of the hydrophobic-hydrophilic interface. The negative charges produced by these aspartate residues is encircled by areas of positive charge that may facilitate accumulation and lateral movement of protons on this surface.

A molecular dynamics (MD) method using the multicanonical algorithm, multicanonical MD, is proposed to enhance the efficiency of conformational sampling of peptides. Multicanonical MD is a constant temperature MD on a deformed potential energy surface, which gives a multicanonical ensemble characterized by a flat energy distribution. The multicanonical ensemble, in turn, is converted into a canonical ensemble by the reweighting formula of Ferrenberg and Swendsen. The multicanonical algorithm was originally developed by Berg et al. as a method of Monte Carlo simulations. The multicanonical MD is its application to MD simulations. Using a system of a simple double-well potential, it was confirmed that this method yields a correct canonical distribution. A test of the method was also performed with a five-residue peptide, Met-enkephalin, and showed that multicanonical MD samples much larger conformational space than conventional canonical MD.

A simple formula based on linear response theory is proposed to explain and predict the structural change of proteins upon ligand binding. By regarding ligand binding as an external perturbation, the structural change as a response is described by atomic fluctuations in the ligand-free form and the protein-ligand interactions. The results for three protein systems of various sizes are consistent with the observations in the crystal structures, confirming the validity of the linear relationship between the equilibrium fluctuations and the structural change upon ligand binding.

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The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.

For the third complementarity determining region of the antibody heavy chain (CDR-H3), we propose the`H3-rules', which should identify the tertiary structure from the amino acid sequence of the CDR-H3 segment. A total of 100 CDR-H3 segments from well-determined crystal structures were analyzed. Distinctive relationships between the structures and the sequences were revealed from 55 segments, and the rules were examined for the other 45 segments and were verified. In some antibodies, basic residues at specific positions were revealed to be notable signals, with their ability to form salt bridges and to assume conformations inconsistent with the rules.z 1999 Federation of European Biochemical Societies.

Large varieties in the lengths and the amino acid sequences of the third complementarity determining region of the antibody heavy chain (CDR-H3) have made it difficult to establish a relationship between the sequences and the tertiary structures, in contrast to the other CDRs, which are classified by their canonical structures. A total of 55 CDR-H3 segments from well determined crystal structures were analyzed, and we have derived several remarkable rules, which could partly govern the CDR-H3 conformation dependence on the sequence. Since the rules are physically reasonable, they are expected to be applicable to structural modeling and design of antibodies.

In line with the recent development of fragment-based drug design, a new computational method for mapping of small ligand molecules on protein surfaces is proposed. The method uses three-dimensional (3D) spatial distribution functions of the atomic sites of the ligand calculated using the molecular theory of solvation, known as the 3D reference interaction site model (3D-RISM) theory, to identify the most probable binding modes of ligand molecules. The 3D-RISM-based method is applied to the binding of several small organic molecules to thermolysin, in order to show its efficiency and accuracy in detecting binding sites. The results demonstrate that our method can reproduce the major binding modes found by X-ray crystallographic studies with sufficient precision. Moreover, the method can successfully identify some binding modes associated with a known inhibitor, which could not be detected by X-ray analysis. The dependence of ligand-binding modes on the ligand concentration, which essentially cannot be treated with other existing computational methods, is also investigated. The results indicate that some binding modes are readily affected by the ligand concentration, whereas others are not significantly altered. In the former case, it is the subtle balance in the binding affinity between the ligand and water that determines the dominant ligand-binding mode.