Conformational properties of a 12-residue tryptophan zipper (trpzip) -hairpin peptide (AWAWENGKWAWK-NH 2 , a modification of the original trpzip2 sequence) are analyzed under equilibrium conditions using ECD and IR spectra of a series of variants, singly and doubly C 1 -labeled with 13 C on the amide CdO. The characteristic features of the 13 CdO component of the amide I′ IR band and their sensitivity to the local structure of the peptide are used to differentiate stabilities for parts of the hairpin structure. Doubly labeled peptide spectra indicate that the ends of the -strands are frayed and that the center part is more stable as would be expected from formation of a stable hydrophobic core consisting of four tryptophan residues, and supported by MD simulations. NMR analyses were used to determine a best fit solution structure that is in close agreement with that of trpzip2, except for a small variation in the turn geometry. The distinct vibrational coupling patterns of the labeled sites based on this structure are also well matched by ab initio DFT-level calculations of their IR spectral patterns. Thermal unfolding of the peptides as studied with CD spectra could be fit with an apparent two-state transition model. ECD senses only the tryptophan interactions (tertiary-like) and their overall environment, as shown by TD-DFT modeling of the Trp-Trp π-π* ECD. However, variation of the amide I IR spectra of 13 C-isotopomers showed that the thermal unfolding process is not cooperative in terms of the peptide backbone (secondary structure), since the transition temperatures sensed for labeled modes differ from those for the whole peptide. The thermal data also evidence dependence on concentration and pH but these cause little spectral variation. This study illustrates the consequences of multistate conformational change at the residue-or sequence-specific level in a system whose structure is dominated by hydrophobic collapse.

In this work, time dependent density functional theory (TD-DFT) is used to provide a reliable basis for interpretation of the electronic spectra of coupled tryptophan (Trp) residues, particularly those in a model Trpzip b-hairpin peptide. Pairs of isolated indoles form chiral coupled chromophores whose computed electronic ultraviolet circular dichroism (CD) is in excellent agreement with observed transition wavelengths and intensities. The calculations were compared to experimental data for pairwise coupling in mutant Trpzip peptides that are recently available. A study of variation of the basis set, geometry optimization, and the solvent environment on the spectra showed limited impact on bandshapes. An alternative simplified computational scheme, dependent on the transition dipole coupling (TDC) mechanism, is shown to give a representation of qualitative aspects of the intense CD for the 1 B bands at 228 and 213 nm. The results confirm the origin of the Trpzip diagnostic CD as primarily a dipolar interaction between Trp sidechains, and show that quantum computations of electronic CD can provide a reliable basis for interpretation of these chirally coupled aromatic spectral phenomena.

Coupling between the amide linkages in a peptide or protein is the key physical property that gives vibrational spectra and circular dichroism sensitivity to secondary structures. By use of (13)C isotopic labeling on individual and pairs of amide C═O groups, the amide I band for selected residues was effectively isolated in designed hexa- and octapeptides having dominant 3(10)-helical conformations. The resultant frequency and intensity responses were measured with IR absorption, vibrational circular dichroism (VCD), and Raman spectroscopies and simulated with density functional theory (DFT) based computations. Band fitting the spectral components and correlating the results to the computed coupling between selected labeled positions were used to determine coupling constant signs and to estimate their magnitudes for specific sequences. The observed frequency and intensity patterns, and their variation between IR and VCD with label position in the sequence, follow the theoretical predictions to a large degree, but are complicated by end effects that alter the local force field (FF) for some residues in these short peptides. These FF variations were overestimated in the theoretical models which may be evidence of structural variations not included in the model. By analyzing the simulations with different coupling models, the coupling constants were determined to lie in a range (positive) +3-5 cm(-1) for sequential residues (i,i+1) and with (negative) -3 cm(-1) as an upper bound for alternate ones (i,i+2). The sequential amide coupling for 3(10)-helices is weaker than for α-helices but has the same sign and is larger than and oppositely signed as compared to 3(1)-, or poly-(Pro)(n) type-II, helices.

Laser induced temperature jump (T-jump) relaxation kinetics were measured with infrared absorbance (IR) detection for a set of beta-hairpin peptides, related to the Trpzip2 hairpin, but containing single isotopic labels, (13)C on the amide C horizontal lineO of selected residues both in the center of the strands and at the terminal regions of the hairpin. Variations in the behavior of single labeled peptides are compared to those previously reported for double labeled variants. Although single labels do not result in spectral intensity enhancement, as seen for cross-strand labeling, the IR frequency shifts are still diagnostic of hairpin unfolding. If C horizontal lineO's in the beta-strand portion of the hairpin (between the Trp residues) are labeled, the dynamic behavior of the local modes is similar to the results obtained with double labels in terms of relaxation time and activation energy and closely tracks the kinetics of the beta-strand components. This implies that either property, local secondary structure (change of varphi,psi), or cross-strand coupling enabled by strand formation and H-bonding relaxes with the same kinetic mechanism. Single labeled residues on the terminal positions have a different behavior and are less able to be detected due to overlap with the (12)C components, in contrast to double labels involving these positions, which are enhanced due to coupling. DFT-based spectral simulations that use the NMR structure of Trpzip2C indicate that the single labeled peptides should have roughly equivalent (12)C bands but the (13)C mode frequencies will vary with sequence position. Effective solvent corrections using COSMO yield significant changes in the frequencies but not in the relative isotope shifts obtained in our calculated spectra. Sequence positional dependence of labels is shown to be more discriminatory for kinetics changes than for thermodynamic variations.