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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Tryptophan Interactions that Stabilize Folding Motifs:A Guide to Placement, Dynamics Applications, andOptimizing Fold StabilizationIrene Shu, Michele Scian, Brandon L. Kier, D. Victoria Williams, andNiels H. AndersenDepartment of Chemistry, University of Washington, Seattle, WA, 98195, U.S.A.IntroductionTrp/Trp interactions in proteins have been observed by CD since at least 1994 [1], with anexciton couplet centered at 222 nm. The melting of such a CD couplet in a hairpin with aW-turn-W motif is illustrated in Figure 1. This feature is the result of a chiral edge-to-face(EtF) interaction geometry between the indole rings of the Trp residues. This feature hasbeen used in designing hairpin folds since 1999 [2 and references cited therein]. Morerecently a capping unit for hairpin structures that also contains an EtF W/W interaction hasbeen developed [3]. In the present report, we detail how to place W/W pairs in hairpin so asto enhance fold stability. In addition, the EtF W/W interaction produces large chemicalshift deviations (CSDs) in the folded state. The protons with very large chemical shiftchanges between the folded and unfolded state also serve as probes for NMR dynamicsstudies based on exchange broadening data. This allows access to hairpin folding andunfolding rates in the sec timescale [6]. We illustrate unexpected changes in hairpinfolding rates, with a dependence on both loop sequence and the presence or absence ofCoulombic interactions at the hairpin termini being observed.Results and DiscussionIn our continuing studies, four distinct EtF W/W interaction geometries have beenobserved; these are illustrated in Figure 2. Geometry A is observed when P12 of theTrp-cage is replaced by an additional Trp residue [4 and unpublished studies at UW byWilliams and Kier]. The other geometries areobserved for cross-strand W/W pairs in hairpins[2,3,5]. Panel B represents two geometries as the“edge Trp” can either be at the N- or C-terminus of asequence. The geometry in panel C has beenobserved in hairpins only in the presence offluoroalcohol co-solvents. All of these geometriesproduce a far upfield indole ring-H in the NMRspectrum. In the case of geometry B, H 3(indole H4) on the edge-indole is upfield by2-2.5 ppm. In less stable versions of hairpins, theH 3 CSD provides the extent of folding.Fig.1. The melting of a W/Wexciton couplet in a hairpin.EtF W/W interaction only appear when theW/W pair is placed at non-H-bonded hairpin strandpositions: the positions which also produce foldFig. 2. Trp-Trp geometries observed in miniprotein and hairpin constructs.614