The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes of several bacilli by binding single-stranded RNA. The binding sequence is composed of eleven triplet repeats, predominantly GAG, separated by two or three non-conserved nucleotides. Here we present the crystal structure of a complex of TRAP and a 53-base single-stranded RNA containing eleven GAG triplets, revealing that each triplet is accommodated in a binding pocket formed by beta-strands. In the complex, the RNA has an extended structure without any base-pairing and binds to the protein mostly by specific protein-base interactions. Eleven binding pockets on the circular TRAP 11-mer form a belt with a diameter of about 80 A. This simple but elegant mechanism of arresting the RNA segment by encircling it around a protein disk is applicable to both transcription, when TRAP binds the nascent RNA, and to translation, when TRAP binds the same sequence within a non-coding leader region of the messenger RNA.

Background: The database of protein structures contains representatives from organisms with a range of growth temperatures. Various properties have been studied in a search for the molecular basis of protein adaptation to higher growth temperature. Charged groups have emerged as key distinguishing factors for proteins from thermophiles and mesophiles.

Fibrinogen B␤Arg448Lys is a common polymorphism, positioned within the carboxyl terminus of the B␤-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. IntroductionAn arginine to lysine substitution in the coding region of the ␤-chain of fibrinogen, B␤Arg448Lys, is a relatively common polymorphism, of which the Lys448 allele has a frequency of 15% to 20% in whites. 1,2 A previous study has shown an association between B␤Arg448Lys polymorphism and the severity of coronary artery disease as assessed by angiography, 3 with an increased frequency of the Lys448 allele in patients with triple-vessel disease compared with patients having single-vessel or double-vessel disease. The polymorphism has also been implicated in predisposition to stroke in female patients, an effect that was independent of fibrinogen levels, suggesting a functional role for this polymorphism. 2 We have previously shown that genetic factors contribute to the ultrastructure of the fibrin clot and that more than one-third of the variation in fibrin structure is determined by genes. 4 Clot structure has a role in determining the predisposition to atherothrombotic disease, as clots composed of tightly packed, thin fibers and small pores are associated with increased risk of cardiovascular disease. 5 Furthermore, changes in clot structure have been reported to affect interactions with endothelial cells and fibroblasts, whereby dense fibrin structures with small pores impaired reorganization of these cells into microtubules, 6 implicating effects on angiogenesis, wound healing, and atherosclerosis. 7 Fibrin clot formation initiates with cleavage of fibrinopeptide (Fp)A from the fibrinogen A␣-chain by thrombin, exposing a binding site in the E-region that interacts with a complementary binding site on the ␥-chain in the D-region, allowing formation of protofibrils. Thrombin cleavage of fibrinopeptide (Fp)B from the B␤-chain occurs at a slower rate and is thought to also expose a binding site, this time for a complementary region on the ␤-chain, again located in the D-region. The precise function of this latter interaction is still a matter for debate. Interactions of the ␤-chain have been proposed ...

Predicting efficacy and optimal drug delivery strategies for small molecule and biological therapeutics is challenging due to the complex interactions between diverse cell types in different tissues that determine disease outcome. Here we present a new methodology to simulate inflammatory disease manifestation and test potential intervention strategies in silico using agent-based computational models. Simulations created using this methodology have explicit spatial and temporal representations, and capture the heterogeneous and stochastic cellular behaviours that lead to emergence of pathology or disease resolution. To demonstrate this methodology we have simulated the prototypic murine T cell-mediated autoimmune disease experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. In the simulation immune cell dynamics, neuronal damage and tissue specific pathology emerge, closely resembling behaviour found in the murine model. Using the calibrated simulation we have analysed how changes in the timing and efficacy of T cell receptor signalling inhibition leads to either disease exacerbation or resolution. The technology described is a powerful new method to understand cellular behaviours in complex inflammatory disease, permits rational design of drug interventional strategies and has provided new insights into the role of TCR signalling in autoimmune disease progression.

The acylation and deacylation stages of the hydrolysis of N -acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential p K (a) values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed. It is concluded that Asp(161) is the source of a key electrostatic modulator (p K (a) 5.0+/-0.1) in actinidin, analogous to Asp(158) in papain, whose influence is not detected kinetically; it is always in the 'on' state because of its low p K (a) value (2.8+/-0.06).