Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E-coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms. (c) 2005 Elsevier Ltd. All rights reserved.

The cyclotide family of circular miniproteins: Nature's combinatorial peptide template

The cyclotides are a recently discovered family of miniproteins that contain a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. They are approximately 30 amino acids in size and are present in high abundance in plants from the Violaceae, Rubiaceae, and Cucurbitaceae families, with individual plants containing a suite of up to 100 cyclotides. They have a diverse range of biological activities, including uterotonic, anti-HIV, antitumor, and antimicrobial activities, although their natural function is likely that of defending their host plants from pathogens and pests. This review focuses on the structural aspects of cyclotides, which may be thought of as a natural combinatorial peptide template in which a wide range of amino acids is displayed on a compact molecular core made up of the cyclic cystine knot structural motif. Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The struclural features that contribute to their remarkable stability are described ill this review. (c) 2006 Wiley Periodicals, Inc.

Dilution versus dialysis - A quantitative study of the oxidative refolding of recombinant human alpha-fetoprotein

Alpha-fetoprotein (AFP) is a commercially important polypeptide with important diagnostic. physiological and immunomodulatory functions. Previous studies into the refolding of this macromolecule are contradictory. and variously suggest that AFP denaturation may be irreversible or that refolding may be achieved by reducing denaturant concentration through dilution but not dialysis. Importantly, these same previous studies do not provide quantitative metrics by which the Success of refolding, and the potential for bioprocess development. can be assessed. Moreover, these same studies do not optimize and control refolding redox potential - an important factor considering that AFP contains 32 cysteines which form 16 disulfide bonds. In this current study, a quantitative comparison of recombinant human AFP (rhAFP) refolding by dilution and dialysis is conducted under optimized redox conditions. rhAFP refolding yields were > 35% (dialysis refolding) and > 75% (dilution refolding) as assessed by RP-HPLC and ELISA, with structural Similarity to the native state confirmed by UV spectroscopy. Dialysis refolding yield was believed to be lower because the gradual reduction in denaturant concentration allowed extended conformational searching. enabling more time for undesirable interaction with other protein molecules and/or the dialysis membrane, leading to a Sub-optimal process outcome. Significant yield sensitivity to redox environment was also observed, emphasizing the importance of physicochemical optimization. This study demonstrates that very high refolding yields can be obtained, for a physiologically relevant protein, with optimized dilution refolding. The study also highlights the quantitative metrics and macromolecular physical spectroscopic 'fingerprints' required to facilitate transition from laboratory to process scale.

Functional split and crosslinking of the membrane domain of the β subunit of proton-translocating transhydrogenase from Escherichia coli

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α subunit with the NAD(H)-binding domain I and a β subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the α and β subunits. The interface in domain II between the α and the β subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the α subunit and loops connecting the nine transmembrane helices in the β subunit. However, to investigate the organization of the nine transmembrane helices in the β subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type α subunit and the two new peptides β1 and β2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD + by NADPH, the cyclic reduction of 3-acetylpyridine-NAD + by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the α subunit was normally folded, followed by a concerted folding of β1 + β2. Cross-linking of a βS105C-βS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same β subunit has been demonstrated.

Mutations of beta-arrestin 2 that limit self-association also interfere with interactions with the beta2-adrenoceptor and the ERK1/2 MAPKs:implications for beta2-adrenoceptor signalling via the ERK1/2 MAPKs

FRET (fluorescence resonance energy transfer) and co-immunoprecipitation studies confirmed the capacity of beta-arrestin 2 to self-associate. Amino acids potentially involved in direct protein-protein interaction were identified via combinations of spot-immobilized peptide arrays and mapping of surface exposure. Among potential key amino acids, Lys(285), Arg(286) and Lys(295) are part of a continuous surface epitope located in the polar core between the N- and C-terminal domains. Introduction of K285A/R286A mutations into beta-arrestin 2-eCFP (where eCFP is enhanced cyan fluorescent protein) and beta-arrestin 2-eYFP (where eYFP is enhanced yellow fluorescent protein) constructs substantially reduced FRET, whereas introduction of a K295A mutation had a more limited effect. Neither of these mutants was able to promote beta2-adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both beta-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys(285), Arg(286) and particularly Lys(295) to be important for this interaction. Direct interactions between beta-arrestin 2 and the beta2-adrenoceptor were also compromised by both K285A/R286A and K295A mutations of beta-arrestin 2. These were not non-specific effects linked to improper folding of beta-arrestin 2 as limited proteolysis was unable to distinguish the K285A/R286A or K295A mutants from wild-type beta-arrestin 2, and the interaction of beta-arrestin 2 with JNK3 (c-Jun N-terminal kinase 3) was unaffected by the K285A/R286A or L295A mutations. These results suggest that amino acids important for self-association of beta-arrestin 2 also play an important role in the interaction with both the beta2-adrenoceptor and the ERK1/2 MAPKs. Regulation of beta-arrestin 2 self-association may therefore control beta-arrestin 2-mediated beta2-adrenoceptor-ERK1/2 MAPK signalling.

Undue influence and the family home

This book provides a comprehensive analysis of the doctrine of undue influence in the context of the family home and fully incorporates the recent House of Lords ruling in Royal Bank of Scotland v Etridge (No 2) (2001). It is aimed predominantly at the legal practitioner, but will also act as a useful source of reference for academics and students of contract, land law and equity and trusts. Emphasis is placed on claims brought by spouses (usually the wife) seeking to set aside a charge over the matrimonial home made in favour of a lending institution. The role of lenders in this context is also examined in depth, as is the part played by the solicitor acting on behalf of the parties. Apart from providing an exposition of the doctrine and its key elements, the book also gives a broader outlook by examining the Commonwealth experience (notably in Australia, Canada and New Zealand) and suggesting an underlying concept of unconscionability as governing undue influence claims. There is also a separate chapter on remedies, as well as an appendix containing a number of draft pleadings for use by the legal practitioner. In the foreword, the Honourable Mr Justice Neuberger writes: 'Pawlowski and Brown are to be congratulated for having produced a book ...as comprehensive and user-friendly as this volume. Not only have they considered the effect of the authorities in a clear and logical way, but they have also highlighted problems which have yet to be resolved and questions which have yet to be answered ...one of the hallmarks of a good legal book.'

Mapping the yeast host cell response to recombinant membrane protein production:relieving the biological bottlenecks

Understanding the structures and functions of membrane proteins is an active area of research within bioscience. Membrane proteins are key players in essential cellular processes such as the uptake of nutrients, the export of waste products, and the way in which cells communicate with their environment. It is therefore not surprising that membrane proteins are targeted by over half of all prescription drugs. Since most membrane proteins are not abundant in their native membranes, it is necessary to produce them in recombinant host cells to enable further structural and functional studies. Unfortunately, achieving the required yields of functional recombinant membrane proteins is still a bottleneck in contemporary bioscience. This has highlighted the need for defined and rational optimization strategies based upon experimental observation rather than relying on trial and error. We have published a transcriptome and subsequent genetic analysis that has identified genes implicated in high-yielding yeast cells. These results have highlighted a role for alterations to a cell's protein synthetic capacity in the production of high yields of recombinant membrane protein: paradoxically, reduced protein synthesis favors higher yields. These results highlight a potential bottleneck at the protein folding or translocation stage of protein production.

Multiple roles of charged amino acids in cytoplasmic loop 7 for expression and function of the multidrug and organic anion transporter MRP1 (ABCC1)

Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, an amino acid with the same charge, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu(1169), Glu(1170), and Glu(1172) were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, (1169)AAQA). Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and (1169)AAQA were accompanied by changes in orthovanadate-induced trapping of [alpha-(32)P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1.

Self-contained encrypted image folding

The recently introduced approach for Encrypted Image Folding is generalized to make it self-contained. The goal is achieved by enlarging the folded image so as to embed all the necessary information for the image recovery. The need for extra size is somewhat compensated by considering a transformation with higher folding capacity. Numerical examples show that the size of the resulting cipher image may be significantly smaller than the plain text one. The implementation of the approach is further extended to deal also with color images.

Sparsity and "something else": an approach to encrypted image folding

A property of sparse representations in relation to their capacity for information storage is discussed. It is shown that this feature can be used for an application that we term Encrypted Image Folding. The proposed procedure is realizable through any suitable transformation. In particular, in this paper we illustrate the approach by recourse to the Discrete Cosine Transform and a combination of redundant Cosine and Dirac dictionaries. The main advantage of the proposed technique is that both storage and encryption can be achieved simultaneously using simple processing steps.

Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor

The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.

Hijacked then lost in translation:the plight of the recombinant host cell in membrane protein structural biology projects

Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells.

Integer Programming Approach to HP Folding

One of the most widely studied protein structure prediction models is the hydrophobic-hydrophilic (HP) model, which explains the hydrophobic interaction and tries to maximize the number of contacts among hydrophobic amino-acids. In order to find a lower bound for the number of contacts, a number of heuristics have been proposed, but finding the optimal solution is still a challenge. In this research, we focus on creating a new integer programming model which is capable to provide tractable input for mixed-integer programming solvers, is general enough and allows relaxation with provable good upper bounds. Computational experiments using benchmark problems show that our formulation achieves these goals.