We introduce the CC+ Database, a detailed, searchable repository of coiled-coil assignments, which is freely available at http://coiledcoils.chm.bris.ac.uk/ccplus. Coiled coils were identified using the program SOCKET, which locates coiled coils based on knobs-into-holes packing of side chains between α-helices. A method for determining the overall sequence identity of coiled-coil sequences was introduced to reduce statistical bias inherent in coiled-coil data sets. There are two points of entry into the CC+ Database: the ‘Periodic Table of Coiled-coil Structures’, which presents a graphical path through coiled-coil space based on manually validated data, and the ‘Dynamic Interface’, which allows queries of the database at different levels of complexity and detail. The latter entry level, which is the focus of this article, enables the efficient and rapid compilation of subsets of coiled-coil structures. These can be created and interrogated with increasingly sophisticated pull-down, keyword and sequence-based searches to return detailed structural and sequence information. Also provided are means for outputting the retrieved coiled-coil data in various formats, including PyMOL and RasMol scripts, and Position-Specific Scoring Matrices (or amino-acid profiles), which may be used, for example, in protein-structure prediction.

There are several approaches to creating synthetic-biological systems. Here, we describe a molecular-design approach. First, we lay out a possible synthetic-biology space, which we define with a plot of complexity of components versus divergence from nature. In this scheme, there are basic units, which range from natural amino acids to totally synthetic small molecules. These are linked together to form programmable tectons, for example, amphipathic alpha-helices. In turn, tectons can interact to give self-assembled units, which can combine and organize further to produce functional assemblies and systems. To illustrate one path through this vast landscape, we focus on protein engineering and design. We describe how, for certain protein-folding motifs, polypeptide chains can be instructed to fold. These folds can be combined to give structured complexes, and function can be incorporated through computational design. Finally, we describe how protein-based systems may be encapsulated to control and investigate their functions.

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Electrostatic interactions play important roles in diverse biological phenomena controlling the function of many proteins. Polar molecules can be studied with the FDPB method solving the Poisson-Boltzmann equation on a finite difference grid. A method for the prediction of pK a s and redox potentials in the thioredoxin superfamily is introduced. The results are compared with experimental pK a data where available, and predictions are made for members lacking such data. Studying CxxC motif variation in the context of different background structures permits analysis of contributions to cysteine ⌬pK a s. The motif itself and the overall framework regulate pK a variation. The reported method includes generation of multiple sidechain rotamers for the CxxC motif and is an effective predictive tool for functional pK a variation across the superfamily. Redox potential follows the trend in cysteine pK a variation, but some residual discrepancy indicates that a pH-independent factor plays a role in determining redox potentials for at least some members of the superfamily. A possible molecular basis for this feature is discussed.Keywords: electrostatics calculations; thioredoxin superfamily; ionizable groups; redox potential Proteins in the thioredoxin superfamily characteristically include a thioredoxin fold, consisting of a mixed ␤-sheet of five strands surrounded by four ␣-helices, and a CxxC sequence motif in which x denotes an unspecified amino acid, in their active site (Martin 1995;Åslund and Beckwith 1999). This sequence is CPHC starting at residue C30 in Escherichia coli DsbA, CGPC starting at C32 in human thioredoxin, CGYC starting at C98 in E. coli DsbC, and CVYC starting at C14 in T4 bacteriophage glutaredoxin. Different members of the superfamily can have opposite functions depending on the redox potential and the environment, either reducing disulfide bonds or oxidizing cysteine thiols (Debarbieux and Beckwith 2000). The CxxC motif has been extensively studied in terms of redox potential and function (Grauschopf et al. 1995; Huber-Wunderlich and Glockshuber 1998) and has been characterized as a rheostat in the active site (Chivers et al. 1997). The function of each member of the thioredoxin superfamily is determined by their redox potential and by the direction of the electron transport pathway within which each participates. Thioredoxin 1 of E. coli, with a redox potential of −270 mV, is a major reductant in the cytoplasm, whereas DsbA, a periplasmic protein with a redox potential of −122 mV, is highly oxidizing and required for the disulfide bond formation in the cell envelope (Åslund and Beckwith 1999). The active sites of members of the thioredoxin superfamily have remarkably similar conformation but exhibit extensive variation in redox equilibria.Electrostatic energies play a significant role in the control of protein function (Honig and Nicholls 1995), and accurate determination of these energies is therefore required to aid functional prediction. The magnitudes of electrostatic interactions of ...

An active site containing aC XXC motif is always foundi nt he thiol-disulphide oxidoreductase superfamily. As urvey of crystals tructuresr evealed that the CXXC motif had av eryh ighl ocal propensity (26.3 6 6.2) for theNtermini of a -helices. Ahelical peptide with the sequence CAAC at the Nt erminus was synthesized to examine the helix-stabilizing capacity of the CXXC motif. Circular dichroism was used to confirm the helicalnature of thepeptide andstudy behavior under titration with various species. With DTT,aredox potentialo fE o ¼À 230m Vw as measured, indicating that the isolated peptide is reducingi nn ature ands imilar to native human thioredoxin. The pK a values of the individual Cys residues could notb es eparated in thet itration of ther educed state, giving as ingle transition with an apparent pK a of 6.74 ( 6 0.06). In the oxidized state, theN -terminal pK a is 5.96 ( 6 0.05). Analysisofresults with themodifiedhelix-coiltheoryindica tedthatthe disulfidebondstabilized the a -helical structureb y0 .5 kcal/mol.R educingt he disulfided estabilizest he helixb y0 .9 kcal/mol.Keywords: a -helix;N -cap; N3; circular dichroism; proteinf olding; protein stability; CXXC; helix-coil theory An active site containing aC XXC motif is always found in the thiol-disulphideo xidoreductase superfamily.T his superfamilyi ncludest hioltransferase, thioredoxin, glutaredoxin,a nd protein disulphidei somerase.I na ll of these proteins characterized thus far, the firsta nd the second Cysinthe motifare always at theN-cap and N3 positions of an a -helix.R esidues in these positions are in close proximitya nd can potentially interact with each other. There have been af ew studies on the CXXC motif in an isolated peptide (Ookuraetal. 1995; Nguyenetal. 2003) and no study on itse ffect on helical structure and stability.Disulphides have been showntostabilize helical peptides based on apamin, though with adifferent spacing and locationi nt he helix (Pease et al.1 990). Here we synthesized ahelical peptide containingthe CXXC motif to understand its behavior,i ndependent of thet ertiary structureand neighboring aminoacids. Itsenergetic properties were analyzed using theh elix-coil theory.