The ion permeation process, driven by a membrane potential through an outer membrane protein, OmpF porin of Escherichia coli, was simulated by molecular dynamics. A Na+ ion, initially placed in the solvent region at the outer side of the porin channel, moved along the electric field passing through the porin channel in a 1.3 nsec simulation; the permeation rate was consistent with the experimentally estimated channel activity (10(8)-10(9)/sec). It this simulation, it was indicated that the ion permeation through the porin channel proceeds by a "push-out" mechanism, and that Asp113 is an important residue for the channel activity.

We report the crystal structure of an enolase from Enterococcus hirae, which is the first report of a structure determination among gram-positive bacteria. We isolated the enolase gene and determined the base sequence. The amino acid sequence deduced from the DNA sequence suggests that this enolase is composed of 431 amino acids. The amino acid sequence is very similar to those of enolases from eukaryotic and prokaryotic organisms, being 65% and 50% identical to enolases from Escherichia coli and yeast, respectively. The enolase prepared from E. hirae lysate yielded crystals containing one dimer per asymmetric unit. X-ray diffraction patterns were obtained at 2.8 A resolution on a SPring-8 synchrotron radiation source. Crystals belong to space group I4 with unit cell dimensions of a = b = 153.5 A, c = 90.7 A. The E. hirae, yeast, E. coli and lobster enolase structures are very similar. The E. hirae enolase takes an "Open" conformation. The regions in the structure that differ most from other enolases are loops L4 (132-140) and L3 (244-265). Considering the positions of these loops relative to the active site, they seem to have no direct involvement in function. Our findings show that the three dimensional structure of an important enzyme in the glycolytic pathway is evolutionarily conserved among eukaryotes and prokaryotes, including gram-positive bacteria.

No abstract

The low-humidity IB crystal form of aspartame (L-alphaaspartyl-L-phenylalanine methyl ester) is prepared via humidity-induced transition from the highly hydrated IA crystal form and is used widely as a sweetener. The crystal structure of the low-humidity IB form is determined at 1.05 A resolution (0.476 A(-1) in maximum sintheta/lambda) from an extremely fine fibrous crystal using synchrotron radiation. There are three aspartame molecules and two water molecules in the asymmetric unit of the monoclinic space group P2(1). Each aspartame molecule adopts an almost identical extended conformation which is commonly observed in other crystal forms of aspartame. Three aspartame molecules are assembled into a triangular trimer, and trimer units are stacked along the b-axis via hydrogen-bonding and electrostatic interactions in the main chains and also via hydrophobic contacts in the phenyl side-chains. Six trimer units are related by pseudo 6(1)-screw axis symmetry and form a hydrophilic channel at their center. The hydrophilic channel in the IB form contains only four water molecules in the unit cell, compared with 16 in the IA form. Although the IB form exhibits a trimer structure similar to that of the IA form, one aspartame molecule is rotated by approximately equals 20 degrees from the orientation in the IA form. This arrangement of the molecule implies that the humidity-induced transition is accompanied by a flapping motion of its methyl ester group. These structural differences may imply the stepwise transition from the IA to the IB forms.

Three-dimensional local structures located near active sites are responsible for protein functions. To predict protein functions, we developed a method to extract three-dimensional structural characteristics of functions. We chose EF-hand calcium binding proteins as the model target. We defined a local structure as the structure within 12Å from an arbitrary Asp(C α). Analysis of the amino acid propensities in the active sites showed that there were clear preferences for certain amino acids to locate at the binding sites. Furthermore, the analysis of the distributions of the distances between Asp(C α) and any other amino acids(C α) showed that there were preferred distances for respective amino acids. Using such structural data, we derived a DB (distance based) score and a DPB (distance and propensity based) score for the prediction of functional sites. A search for EF-hand calcium binding sites was performed to test the method. EF-hand calcium binding sites were successfully predicted.