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This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. 1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. 2. α-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxytic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of α-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 °C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date. [ABSTRACT FROM AUTHOR]

Partially purified enzymatic fractions from extracts of Escherichia coli B/r catalyse transfer of the isotope label from [adenine-2,8-3H]NAD+ to some bacterial proteins, as well as to hen egg-white lysozyme. The radio-active group in the modified lysozyme was identified as mono(ADP-ribose). Several bacterial proteins were labelled in vivo with 32P; the presence of the label in the form of an ADP-ribosyl group was shown in one of them. [ABSTRACT FROM AUTHOR]

Deciphering the mechanism of activation of heterotrimeric G proteins by their cognate receptors continues to be an intriguing area of research. The recently solved crystal structure of the ternary complex captured the receptor-bound α-subunit in an open conformation, without bound nucleotide has improved our understanding of the activation process. Despite these advancements, the mechanism by which the receptor causes GDP release from the α-subunit remains elusive. To elucidate the mechanism of activation, we studied guanine nucleotide-induced structural stability of the α-subunit (in response to thermal/chaotrope-mediated stress). Inherent stabilities of the inactive ( GDP-bound) and active ( GTP-bound) forms contribute antagonistically to the difference in conformational stability whereas the GDP-bound protein is able to switch to a stable intermediate state, GTP-bound protein loses this ability. Partial perturbation of the protein fold reveals the underlying influence of the bound nucleotide providing an insight into the mechanism of activation. An extra stable, pretransition intermediate, 'empty pocket' state (conformationally active-state like) in the unfolding pathway of GDP-bound protein mimics a gating system - the activation process having to overcome this stable intermediate state. We demonstrate that a relatively more complex conformational fold of the GDP-bound protein is at the core of the gating system. We report capturing this threshold, 'metastable empty pocket' conformation (the gate) of α-subunit of G protein and hypothesize that the receptor activates the G protein by enabling it to achieve this structure through mild structural perturbation. [ABSTRACT FROM AUTHOR]

At the time when the first membrane-protein crystal structure was determined, crystallization of these molecules was widely perceived as extremely arduous. Today, that perception has changed drastically, and the process is regarded as routine (or nearly so). On the occasion of the International Year of Crystallography 2014, this review presents a snapshot of the current state of the art, with an emphasis on the role of detergents in this process. A survey of membrane-protein crystal structures published since 2012 reveals that the direct crystallization of protein-detergent complexes remains the dominant methodology; in addition, lipidic mesophases have proven immensely useful, particularly in specific niches, and bicelles, while perhaps undervalued, have provided important contributions as well. Evolving trends include the addition of lipids to protein-detergent complexes and the gradual incorporation of new detergents into the standard repertoire. Stability has emerged as a critical parameter controlling how a membrane protein behaves in the presence of detergent, and efforts to enhance stability are discussed. Finally, although discovery-based screening approaches continue to dwarf mechanistic efforts to unravel crystallization, recent technical advances offer hope that future experiments might incorporate the rational manipulation of crystallization behaviors. [ABSTRACT FROM AUTHOR]

New technologies enable acquisition of large data-sets containing genomic, proteomic and metabolic information that describe the state of a cell. These data-sets call for systematic methods enabling relevant information about the inner workings of the cell to be extracted. One important issue at hand is the understanding of the functional interactions between genes, proteins and metabolites. We here present a method for identifying the dynamic interactions between biochemical components within the cell, in the vicinity of a steady-state. Key features of the proposed method are that it can deal with data obtained under perturbations of any system parameter, not only concentrations of specific components, and that the direct effect of the perturbations does not need to be known. This is important as concentration perturbations are often difficult to perform in biochemical systems and the specific effects of general type perturbations are usually highly uncertain, or unknown. The basis of the method is a linear least-squares estimation, using time-series measurements of concentrations and expression profiles, in which system states and parameter perturbations are estimated simultaneously. An important side-effect of also employing estimation of the parameter perturbations is that knowledge of the system's steady-state concentrations, or activities, is not required and that deviations from steady-state prior to the perturbation can be dealt with. Time derivatives are computed using a zero-order hold discretization, shown to yield significant improvements over the widely used Euler approximation. We also show how network interactions with dynamics that are too fast to be captured within the available sampling time can be determined and excluded from the network identification. Known and unknown moiety conservation relationships can be processed in the same manner. The method requires that the number of samples equals at least the number of network components and, hence, is at present restricted to relatively small-scale networks. We demonstrate herein the performance of the method on two small-scalein silicogenetic networks. [ABSTRACT FROM AUTHOR]

Direct interaction between the C-terminal portion of dystrophin and dystrophin-associated proteins wax investigated. The binding of dystrophin to each protein wax reconstituted by overlaying bacterially expressed dystrophin fusion proteins onto the blot membranes to which dystrophinassociated proteins were transferred after separation by SDS/PAGE with the following results. (a) Among the components of the glycoprotein complex which links dystrophin to the sarcolemma, a 43-kDa dystrophin-associated glycoprotein binds directly to dystrophin. Although at least one of the binding sites of this protein resides within the cysteine-rich domain of dystrophin, a contribution of additional amino acid residues within the first half of the C-terminal domain was also suggested for more secure binding. (b) Two other proteins also directly bind to dystrophin. Their binding sites are suggested to reside within the last half of the C-terminal domain which is alternatively spliced depending on the tissue type. Previously, based on the enzyme digestion experiments, we showed that the binding site for the glycoprotein complex on dystrophin is present within the cysteine-rich domain and the first half of the C-terminal domain [Suzuki, A., Yoshida, M., Yamamoto, H. & Ozawa, E. (1992) FEBS Lett. 308, 154-1601. Here, we have extended this work and found that the region which is involved in interaction with the complex is widely extended to the entire length of this part of the molecule. On the basis of the present results, we propose a model of molecular architecture at the binding site for the complex on dystrophin. [ABSTRACT FROM AUTHOR]

Two derivatives of Hansenula anomala Flavocytochrome b2 have been prepared, one deprived of the flavin prosthetic group (deflavocytochrome B2), and the other consisting of the heme-h-carrying globule (b2 core). The redox potential of the heme in the two derivatives is -5 (±5) mV and -10 (±5) mV respectively, fairly similar to the value of -20 (±5) mV reported for the holoenzyme, indicating a minor effect of the flavin and of the flavodehydrogenase domain on heme potential. The kinetic of azurin and stellacyanin reduction by both derivatives have been investigated. At pH 7.0, I= 0.2 M and 20°C the second-order rate constants are k = 8 x 105M-1s-1 for azurin reduction by deflavocytochrome b2; k = 1.6 x 106 M-1s-1 for azurin reduction by b2 core; k = 1 x 107 M-1s-1 for stellacyanin reduction by deflavocytochrome b2; k = 3 x 107 M-1s-1 for stellacyanin reduction by b2 core. The change in pH markedly affects the kinetics in the case of azurin, but has no effect on stellacyanin reduction. The change in ionic strength has a significant effect when deflavocytochrome b2 is the reductant, indicating that the flavodehydrogenase domain plays a role in the stabilization of the transient kinetic complex by means of electrostatic interactions. The kinetic results are discussed in the framework of the Marcus theory. [ABSTRACT FROM AUTHOR]

The changes in the degree of phosphorylation of ribosomal protein S6 during the life cycle of the aquatic fungus Blastocladiella emersonii were analyzed by two-dimensional gel electrophoresis. Three phosphorylated derivatives of S6 are present throughout the entire life cycle. However, under certain germination condition, more highly phosphorylated derivatives of S6 appear. Nonetheless, the resumption of protein synthesis that occurs during germination is not dependent on those highly phosphorylated derivatives of S6. The pattern and sites of phosphorylation of S6 labelled in vivo with [32P]orthophosphate have been compared with those of 40S ribosomal subunit labelled in vitro by partially purified protein kinases. Three major phosphopeptides were found in S6 isolated from the zoospore, while six phosphopeptides were found after zoospore germination (in germling cells). The phosphopeptide patterns of S6 phosphorylated by the cAMP-dependent protein kinase and by casein kinases I and II were completely distinct. Only the cAMP-dependent protein kinase gives rise to a phosphopeptide found in 32P-labelled cells, indicating that one of sites phosphorylated in vivo is also phoshorylated in vitro by the cAMP-dependent protein kinase. [ABSTRACT FROM AUTHOR]