Amide Modes of the α-Helix:  Raman Spectroscopy of Filamentous Virus fd Containing Peptide ¹³C and ²H Labels in Coat Protein Subunits

Abstract

The filamentous virus fd consists of a single-stranded DNA genome sheathed by 2700 copies of a 50-residue alpha-helical subunit (protein pVIII) and serves as a model assembly of alpha-helices. To advance vibrational assignments for the alpha-helix, we have investigated Raman spectra of fd virions containing 13C and 2H (deuterium) labels at various main-chain sites of the pVIII subunits. 13C was introduced at specific peptide carbonyls, while deuterium was introduced at selected alpha-carbon (Calpha) and amide nitrogen positions. Interpretation of the Raman spectra reveals a previously unrecognized alpha-helix band in the spectral interval 730-745 cm-1, tentatively assigned to a carbonyl in-plane bending mode (amide IV). Experimental evidence has also been obtained for a distinctive alpha-helix marker near 1345 cm-1, assigned to a coupled Calpha-H bending and Calpha-C stretching mode. The fd virions containing 13C-labeled carbonyls exhibit unexpectedly complex amide I profiles, consisting of multiple band components. Amide I splitting resulting from 13C substitution of carbonyls is attributed to decoupling of transition-dipole interactions normally occurring in the extended pVIII helix. The present study identifies novel conformation-dependent Raman bands in a native alpha-helix assembly, confirms amide I and amide III assignments proposed previously for filamentous viruses, and facilitates new Raman assignments for the packaged ssDNA. The alpha-helix markers identified here should also be useful in conformation analyses of other proteins by Raman spectroscopy.

Raman Microscopy and X-ray Diffraction, a Combined Study of Fibrillin-rich Microfibrillar Elasticity

“…2. The major peaks have been labeled with the appropriate wave number, and the structural features to which they correspond are detailed in Tables I and II (12,(15)(16)(17)(18). The spectra of the native and relaxed tissue showed a high degree of homology to each other.…”

“…It is therefore possible that the environment of at least some of these residues changes following tissue extension. Furthermore, a number of other structural features can also contribute to the peak at 1343 cm Ϫ1 , including main chain deformations, meaning that definitive changes in tryptophan hydrophilicity may not be readily apparent from this particular part of the spectra (17).…”

Raman Spectroscopy of Proteins

“…32 A distinctive a-helix marker has also been observed for this protein at 1345 cm 1 , which has been assigned to the C a -H bend coupled with the C a -C stretch. respectively, and Figure 4(d), (e), and (g) shows the difference spectra, a b, a d, and a f, respectively, which denote the T-minus-R difference spectra of quaternary structures of HbA.…”

Dependence of the Raman signature of genomicB-DNA on nucleotide base sequence

“…Additional details of this instrumentation have been described previously. 22 The effective spectral resolution was 3 cm Ϫ1 . All samples were maintained at room temperature (21°C).…”

“…The advantages of this experimental design have been noted recently in applications to proteins and viruses. 21,22 This Raman instrumentation, which generates essentially noise-free data and high spectral resolution, permits the detection of previously unrecognized Raman bands of B DNA. The difference spectra computed from these data are of unprecedented accuracy, revealing novel Raman band shifts and significant Raman intensity changes as a function of even modest differences in DNA base content.…”