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.

Phosphorylation is ubiquitous in control of protein activity, yet its effects on protein structure are poorly understood. Here we investigate the effect of serine phosphorylation in the interior of an α-helix when a salt bridge is present between the phosphate group and a positively charged side chain (in this case lysine) at i,i + 4 spacing. The stabilization of the helix is considerable and can overcome the intrinsically low preference of phosphoserine for the interior of the helix. The effect is pH dependent, as both the lysine and phosphate groups are titratable, and so calculations are given for several charge combinations. These results, with our previous work, highlight the different, context-dependent effects of phosphorylation in the α-helix. The interaction between the phosphate 2− group and the lysine side chain is the strongest yet recorded in helix-coil studies. The results are of interest both in de novo design of peptides and in understanding the structural modes of control by phosphorylation.Phosphorylation and dephosphorylation have long been known as control mechanisms for various processes in biochemistry, and roughly one-third of all proteins in eukaryotes are estimated to undergo reversible phosphorylation (1). Since the middle of the last century it has been known that protein activity can be controlled by the addition or removal of a phosphate group (2-5). The control of gene expression, macromolecule production, and cellular proliferation have since been shown to be orchestrated through multiple intracellular signal transduction pathways. These pathways, or protein kinase cascades, propagate signals received at the plasma membrane to the interior of the cell through a series of phosphorylation-controlled events.The mechanisms of action of phosphorylation are, however, still relatively poorly understood at the structural level, though many studies of individual systems now show some conformational differences between proteins and synthetic peptides before and after phosphorylation (see, e.g., refs 6-13). Previous work from our own group (14) and others (15-17) has shown that phosphorylation at the N-terminus stabilizes α-helices but also that the effect is position dependent. When interior serine residues are phosphorylated, the effect is greatly destabilizing, but at the three N-terminal positions it is greatly stabilizing (phosphoserine at the N2 position in a helix is the most stabilizing interaction yet found at this position). More recent work has suggested that protein phosphorylation sites, especially those for serine and threonine, are predominantly disordered prior to phosphorylation and that phosphorylation sites resemble natively unstructured proteins in terms of their charge, hydrophobicity, and amino acid composition. Some of the exceptions to these sites may be crystallization artifacts, and in others it may be that the substrate undergoes an order- † This work was funded by The Wellcome Trust (award reference 065106). Europe PMC Funders Author ManuscriptsEurope PMC ...

The alpha-helix is the most abundant secondary structure in proteins. We now have an excellent understanding of the rules for helix formation because of experimental studies of helices in isolated peptides and within proteins, examination of helices in crystal structures, computer modeling and simulations, and theoretical work. Here we discuss structural features that are important for designing peptide helices, including amino acid preferences for interior and terminal positions, side chain interactions, disulfide bonding, metal binding, and phosphorylation. The solubility and stability of a potential design can be predicted with helical wheels and helix/coil theory, respectively. The helical content of a peptide is most often quantified by circular dichroism, so its use is discussed in detail.

N3 is the third position from the N terminus in the ␣-helix with helical backbone dihedral angles. All 20 amino acids have been placed in the N3 position of a synthetic helical peptide (CH 3 CO-[AAX AAAAKAAAAKAGY]-NH 2 ) and the helix content measured by circular dichroism spectroscopy at 273 K. The dependence of peptide helicity on N3 residue identity has been used to determine a free energy scale by analysis with a modified Lifson-Roig helix coil theory that includes a parameter for the N3 energy (n3). The most stabilizing residues at N3 in rank order are Ala, Glu, Met/Ile, Leu, Lys, Ser, Gln, Thr, Tyr, Phe, Asp, His, and Trp. Free energies for the most destabilizing residues (Cys, Gly, Asn, Arg, and Pro) could not be fitted. The results correlate with N1, N2, and helix interior energies and not at all with N-cap preferences. This completes our work on studying the structural and energetic preferences of the amino acids for the N-terminal positions of the ␣-helix. These results can be used to rationally modify protein stability, help design helices, and improve prediction of helix location and stability.Keywords: ␣-helix; N3 position; circular dichroism; protein folding; protein stability; helix propensities; helix-coil theory; macrodipoleThe ␣-helix is the most frequently observed secondary structure in proteins. N1, N2, and N3 are the first three amino acids with helical , backbone dihedral angles at the helix N terminus. They differ from interior positions as their amide NH groups do not participate in backbonebackbone i,i+4 hydrogen bonds within the helix. The presence of these otherwise unsatisfied hydrogen bond donors has profound structural effects, most often satisfied by hydrogen bonds to side chains local in sequence, such as to preceding N-cap side chains.In native proteins, Ala, Asp, Gln, and Glu have the highest propensity for the N3 position. We have made a thorough study of the structures adopted by side chains at the N-cap, N1, N2, and N3 positions in proteins Penel et al. 1999). Asn, Asp, Gln, Glu, and Thr occasionally form i,i hydrogen bonds to the N3 backbone NH group, although these bonds may be weak, as they are nonlinear, particularly for the shorter side chains (Penel et al. 1999;Wan and Milner-White 1999). A second important feature of the N3 position is the capping box, where the N3 side chain accepts a hydrogen bond from the N-cap backbone NH group (Harper and Rose 1993). This is most important for Gln or Glu at N3. Helix termini are highly solvent exposed, and tertiary interactions are rare, so the environment of peptide and protein helices are very similar. The unique structural trends for N3 imply that the free energies for substituting amino acids at this position differ from all other positions in the helix. In this work, we measure these free energies using helical peptides.␣-Helices in aqueous solution adopt a large number of structures, with fully helical, fully coil, and partly helical conformations all populated. For a complete understanding of helix formation and st...

The hydrophobic Arg-Phe and Phe-Met side chain interactions stabilize the alpha-helix by -0.29 and -0.59 kcal/mol, respectively, when placed i, i + 4 in an alanine-based peptide. When both interactions are present simultaneously, however, they stabilize the helix by an additional -0.75 kcal/mol, nearly as much as the sum of its parts. We attribute this coupling to a shared rotamer preference, as the central Phe is t in both bonds. The energetic cost of restricting the Phe residue into a t conformation is only paid once in the triplet, rather than twice when the interactions are separate. Coupling is thus demonstrated to have large effects on protein stability.