The alpha-, beta- and gamma-crystallins are the major protein components of the vertebrate eye lens, alpha-crystallin as a molecular chaperone as well as a structural protein, beta- and gamma-crystallins as structural proteins. For the lens to be able to retain life-long transparency in the absence of protein turnover, the crystallins must meet not only the requirement of solubility associated with high cellular concentration but that of longevity as well. For proteins, longevity is commonly assumed to be correlated with long-term retention of native structure, which in turn can be due to inherent thermodynamic stability, efficient capture and refolding of non-native protein by chaperones, or a combination of both. Understanding how the specific interactions that confer intrinsic stability of the protein fold are combined with the stabilizing effect of protein assembly, and how the non-specific interactions and associations of the assemblies enable the generation of highly concentrated solutions, is thus of importance to understand the loss of transparency of the lens with age. Post-translational modification can have a major effect on protein stability but an emerging theme of the few studies of the effect of post-translational modification of the crystallins is one of solubility and assembly. Here we review the structure, assembly, interactions, stability and post-translational modifications of the crystallins, not only in isolation but also as part of a multi-component system. The available data are discussed in the context of the establishment, the maintenance and finally, with age, the loss of transparency of the lens. Understanding the structural basis of protein stability and interactions in the healthy eye lens is the route to solve the enormous medical and economical problem of cataract.

A new method based on neural network theory is presented to analyze and quantify the information content of far UV circular dichroism spectra. Using a backpropagation network model with a single hidden layer between input and output, it was possible to deduce five different secondary structure fractions (helix, parallel and antiparallel beta-sheet, beta-turn and random coil) with satisfactory correlations between calculated and measured secondary structure data. We demonstrate that for each wavelength interval a specific network is suitable. The remaining discrepancy between the secondary structure data from neural network prediction and crystallography may be attributed to errors in the determination of protein concentration and random noise in the CD signal, as indicated by simulations.

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Abstract. The NEM-sensitive fusion protein, NSF, together with SNAPs (soluble NSF attachment proteins) and the SNAREs (SNAP receptors), is thought to be generally used for the fusion of transport vesicles to their target membranes. NSF is a homotrimer whose polypeptide subunits are made up of three distinct domains: an amino-terminal domain (N) and two homologous ATP-binding domains (D1 and D2). Mutants of NSF were produced in which either the order or composition of the three domains were altered. These mutants could not support intra-Golgi transport, but they indicated that the D2 domain was required for trimerization of the NSF subunits. Mutations of the first ATP-binding site that affected either the binding (K266A) or hydrolysis (E329Q) of ATP completely eliminated NSF activity. The hydrolysis mutant was an effective, reversible inhibitor of Golgi transport with an IC50 of 125 ng/50 #1 assay. Mutants in the second ATP-binding site (binding, K549A; hydrolysis, D604Q) had either 14 or 42 % the specific activity of the wild-type protein, respectively. Using coexpression of an inactive mutant with wild-type subunits, it was possible to produce a recombinant form of trimeric NSF that contained a mixture of subunits. The mixed NSF trimers were inactive, even when only one mutant subunit was present, suggesting that NSF action requires each of the three subunits in a concerted mechanism. These studies demonstrate that the ability of the D1 domain to hydrolyze ATP is required for NSF activity and, therefore is required for membrane fusion. The D2 domain is required for trimerization, but its ability to hydrolyze ATP is not absolutely required for NSF function.

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Oceans not only cover the major part of the earth's surface but also reach into depths exceeding the height of the Mt Everest. They are populated down to the deepest levels (=11800 m), which means that a significant proportion of the global biosphere is exposed to pressures of up to 120 MPa. Although this fact has been known for more than a century, the ecology of the 'abyss' is still in its infancy. Only recently, barophilic adaptation, i.e. the requirement of elevated pressure for viability, has been firmly established. In non-adapted organisms, increased pressure leads to morphological anomalies or growth inhibition, and ultimately to cell death. The detailed molecular mechanism of the underlying 'metabolic dislocation' is unresolved.Effects of pressure as a variable in microbiology, biochemistry and biotechnology allow the structure/function relationship of proteins and protein conjugates to be analyzed. In this context, stabilization by cofactors or accessory proteins has been observed. High-pressure equipment available today allows the comprehensive characterization of the behaviour of proteins under pressure. Single-chain proteins undergo pressure-induced denaturation in the 100-MPa range, which, in the case of oligomeric proteins or protein assemblies, is preceded by dissociation at lower pressure. The effects may be ascribed to the positive reaction volumes connected with the formation of hydrophobic and ionic interactions. In addition, the possibility of conformational effects exerted by moderate, non-denaturing pressures, and related to the intrinsic compressibility of proteins, is discussed. Crystallization may serve as a model reaction of protein self-organization. Kinetic aspects of its pressureinduced inhibition can be described by a model based on the Oosawa theory of molecular association. Barosensitivity is known to be correlated with the pressure-induced inhibition of protein biosynthesis. Attempts to track down the ultimate cause in the dissociation of ribosomes have revealed remarkable stabilization of functional complexes under pseudo-physiological conditions, with the post-translational complex as the most pressure-sensitive species. Apart from the key issue of barosensitivity and barophilic adaptation, high-pressure biochemistry may provide means to develop new approaches to nonthermic industrial processes, especially in the field of food technology.Life in the deep sea faces a whole set of unfavourable conditions, including pressures up to 120 MPa (1200 bar), temperatures around 2°C or, in volcanic regions, above 1OO"C, absence of sunlight, and scarce supply of organic nutrients. When 'deep' means deeper than 1000 m, these biotopes include more than half (~6 2 % ) of the volume of the global biosphere (Jannasch and Taylor, 1984). The average pressure on the ocean floor is of the order of 38 MPa, indicatCorrespondence to R. Jaenicke,

The molecular chaperone GroE facilitates correct protein folding in vivo and in vitro. The mode of action of GroE was investigated by using refolding of citrate synthase as a model system. In vitro denaturation of this dimeric protein is almost irreversible, since the refolding polypeptide chains aggregate rapidly, as shown directly by a strong, concentration-dependent increase in light scattering. The yields of reactivated citrate synthase were strongly increased upon addition of GroE and MgATP. GroE inhibits aggregation reactions that compete with correct protein folding, as indicated by specific suppression of light scattering. GroEL rapidly forms a complex with unfolded or partially folded citrate synthase molecules. In this complex the refolding protein is protected from aggregation. Addition of GroES and ATP hydrolysis is required to release the polypeptide chain bound to GroEL and to allow further folding to its final, active state.

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