Ahmed Lakhani

Comparison of vibrational circular dichroism instruments: development of a new dispersive VCD.

A dispersive vibrational circular dichroism (VCD) instrument has been designed and optimized for the measurement of mid-infrared (MIR) bands such as the amide I and amide II vibrational modes of peptides and proteins. The major design considerations were to construct a compact VCD instrument for biological molecules, to increase signal-to-noise (S/N) ratio, to simultaneously collect and digitize the sample transmission and polarization modulation signals, and to digitally ratio them to yield a VCD spectrum. These were realized by assembling new components using design factors adapted from previous VCD instruments. A collection of spectra for peptides and proteins having different dominant secondary structures (alpha-helix, beta-sheet, and random coil) measured for identical samples under the same conditions showed that the new instrument had substantially improved S/N as compared with our previous dispersive VCD instrument These instruments both provide protein VCD for the amide I that are comparable to or somewhat better than those measurable with commercial Fourier transform (FT) VCD instruments if just the amide I band in the spectra is obtained at modest resolution (8 cm−1) with the same total data collection time on each type of instrument.

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.

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.