Anjan Roy

Experimental and theoretical spectroscopic study of 3(10)-helical peptides using isotopic labeling to evaluate vibrational coupling.

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.

TD-DFT modeling of the circular dichroism for a tryptophan zipper peptide with coupled aromatic residues†

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 beta-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.

Comparison of Isotopic Substitution Methods for Equilibrium and T-Jump Infrared Studies of β-Hairpin Peptide Conformation

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 lineOs 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.

Optical Trapping Raman Spectroscopy of Protein and Membrane Interaction

To study the interdependence of external mechanical force on membrane structure we added the capability of mechanical force measurements into a single beam optical tweezers coupled to a Raman microscope. The optical trap serves as a handle to control the interaction of the membrane to an immobilized protein and it ensures the analyte is trapped in the focal volume of the laser and hence insures that we can collect spectra of single vesicle/ single cell systems. Force measurements allow us to correlate conformational changes to binding energetics.An optical trap was constructed by incorporating a position sensitive detector (PSD) and a peizo translation stage to a home-built Raman microscope. The PSD allows for force measurements on the trapped species and trap stiffness. The peizo-stage facilitates <50 nm translation resolution. We tested this system on micron sized polystyrene beads. The bead was visually guided into the trap, while the power spectrum (force) was collected simultaneously with the Raman spectrum (structure). This was successfully repeated for E.coli in growth media. This simultaneous structure and force measurements were found to be feasible. To achieve our goal of correlated (not just simultaneous) studies we moved forward to vesicle systems.Our Lab has previously studied structural changes in BLG binding to lipid vesicles. We further that by studying structural transitions in single Giant Unilamellar Vesicles (GUV) as it binds to BLG. BLG was immobilized on a glass surface and was mechanically driven towards an optically trapped vesicle. The backscattered Raman signal from the vesicle was recorded as a function of proximity of the vesicle to BLG and we correlated conformational changes in the vesicle with its binding to BLG. We present these findings of our new experimental tool.

Inter-Residue Coupling of Model PPII Helices using 13c Isotopic Labeling

Characterization of poly-proline II (PPII) conformation on a site-specific basis has importance in developing a model for structure and stability in these systems. Coupling of selected residues for a series of related peptides having predominantly PPII conformations were measured using VCD and IR spectra of selected variants that were doubly labeled with 13C on the amide C=O. The characteristics of the 13C=O component of the amide I’IR band and their sensitivity to the local structure of the peptide are compared to predictions based on DFT level calculations for related structures and used to determine coupling between C=O groups along the backbone of this helical structure. Doubly labeled peptides have spectral shifts reflecting the mass change in addition to coupling between residues. In the PPII case the coupling is relatively weak, yet by combining IR and VCD along with DFT level calculations, we have been able to determine its coupling constants. Comparison of PPII structures with “random coils” can be done by comparing all Proline, mixed Ala-Pro and Lys-rich sequences. The shifts and couplings reflect the computations in all cases. 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 and VCD spectral patterns.