Stacy A. Overman

Polarized Raman spectra of oriented fibers of A DNA and B DNA: anisotropic and isotropic local Raman tensors of base and backbone vibrations

Polarized Raman spectra of oriented fibers of calf thymus DNA in the A and B conformations have been obtained by use of a Raman microscope operating in the 180 degrees back-scattering geometry. The following polarized Raman intensities in the spectral interval 200-1800 cm-1 were measured with both 514.5 and 488.0 nm laser excitations: (1) Icc, in which the incident and scattered light are polarized parallel to the DNA helical axis (c axis); (2) Ibb, in which the incident and scattered light are polarized perpendicular to c; and (3) Ibc and Icb, in which the incident and scattered light are polarized in mutually perpendicular directions. High degrees of structural homogeneity and unidirectional orientation were confirmed for both the A and B form fibers, as judged by comparison of the observed Raman markers and intensity anisotropies with measurements reported previously for oligonucleotide single crystals of known three-dimensional structures. The fiber Raman anisotropies have been combined with solution Raman depolarization ratios to evaluate the local tensors corresponding to key conformation-sensitive Raman bands of the DNA bases and sugar-phosphate backbone. The present study yields novel vibrational assignments for both A DNA and BDNA conformers and also confirms many previously proposed Raman vibrational assignments. Among the significant new findings are the demonstration of complex patterns of A form and B form indicator bands in the spectral intervals 750-900 and 1050-1100 cm-1, the identification of highly anisotropic tensors corresponding to vibrations of base, deoxyribose, and phosphate moieties, and the determination of relatively isotropic Raman tensors for the symmetrical stretching mode of phosphodioxy groups in A and B DNA. The present fiber results provide a basis for exploitation of polarized Raman spectroscopy to determine DNA helix orientation as well as to probe specific nucleotide residue orientations in nucleoproteins, viruses, and other complex biological assemblies.

Raman Spectroscopy of Proteins

A protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide backbone and its side chains. The spectral positions, intensities, and polarizations of the Raman bands are sensitive to protein secondary, tertiary, and quaternary structures and to side ‐chain orientations and local environments. In favorable cases, the Raman spectrum serves as an empirical signature of protein three‐dimensional structure, intramolecular dynamics, and intermolecular interactions. Here, the strengths of Raman spectroscopy are illustrated by considering recent applications that address (1) subunit folding and recognition in assembly of the icosahedral capsid of bacteriophage P22, (2) orientations of subunit main chains and side chains in native filamentous viruses, (3) roles of cysteine hydrogen bonding in the folding, assembly, and function of virus structural proteins, and (4) structural determinants of protein/DNA recognition in gene regulatory complexes. Conventional Raman, UV‐resonance Raman, and polarized Raman techniques are surveyed.

Structural studies of viruses by Raman spectroscopy. Novel vibrational assignments for proteins from Raman spectra of viruses

Raman spectroscopy and ultraviolet resonance Raman spectroscopy are versatile methods for probing details of protein structure and dynamics in complex biological assemblies, including viruses. The information contained in the Raman spectrum of a virus is ordinarily interpreted on the basis of an understanding that has been developed from detailed spectroscopic investigations of simpler and better characterized molecular structures, such as small globular proteins, peptides and related model compounds and their isotopic derivatives. The model systems approach serves generally as the foundation for reliable band assignments and has been the key to successful application of Raman methods to supramolecular assemblies. However, the converse approach is also possible. The supramolecular assembly may serve as a ‘model compound’ and its Raman signature may provide novel spectra–structure correlations applicable to isolated protein subunits, simpler proteins or related small molecules. Thus, the Raman spectrum of a virus particle can yield new insights into protein vibrational assignments. Here, a number of new vibrational assignments that have emerged from Raman studies of filamentous viruses are identified. The Raman bands in question have not been identified previously in proteins and are demonstrated to originate either from vibrations of the protein main-chain (CαH marker, 1340–1350 cm-1) or from aromatic amino acid side chains (phenylalanine marker, 827 cm-1; tyrosine singlet, 850–855 cm-1; tryptophan marker, 1560 cm-1). The present results, which are considered in the light of existing correlations between data on Raman spectra and protein structure, suggest that much remains to be learned about the structural significance of protein Raman bands.

Raman tensors for the tryptophan side chain in proteins determined by polarized Raman microspectroscopy of oriented N-acetyl-l-tryptophan crystals

Abstract Polarized Raman spectra have been obtained from oriented single crystals of N -acetyl-l-tryptophan by use of a Raman microscope and 488.0 nm argon excitation. The crystal, of orthorhombic space group P2 1 2 1 2 1 , provides the relative Raman intensities I aa , I bb and I cc corresponding to the aa, bb and cc components of the crystal Raman tensor. The polarized Raman spectra of the crystal have been combined with depolarization ratios from solution spectra of randomly oriented N -acetyl-l-tryptophan and l-tryptophan to yield Raman tensors for each of the following vibrational normal modes of the indole moiety: N 1 H stretch (≈3416 cm −1 ), WI (≈1617 cm −1 ), W 2 (≈1576 cm −1 ), W 3 (≈1557 cm −1 ), W 4 (≈1487 cm −1 ), W 5 (≈1458 cm −1 ), W 6 (≈1424 cm −1 ), W 7 (≈1357 cm −1 ), W 7′ (≈1332 cm −1 ), W 16 (≈1010 cm −1 ) and W 18 (≈757 cm −1 ). These Raman tensors determined for the tryptophan residue in N -acetyl-l-tryptophan are proposed as being transferable to tryptophan side chains in proteins. A knowledge of Raman tensors for the tryptophan side chain should facilitate the determination of indole ring orientation in biological complexes amenable to investigation by the method of polarized Raman microspectroscopy.

Orientations of Tyrosines 21 and 24 in Coat Subunits of Ff Filamentous Virus: Determination by Raman Linear Intensity Difference Spectroscopy and Implications for Subunit Packing

Virions of the Ff group of bacteriophages (fd, f1, M13) are morphologically identical filaments (approximately 6-nm diameter x approximately 880-nm length) in which a covalently closed, single-stranded DNA genome is sheathed by approximately 2700 copies of a 50-residue alpha-helical subunit (pVIII). Orientations of pVIII tyrosines (Tyr21 and Tyr24) with respect to the filament axis have been determined by Raman linear intensity difference (RLID) spectroscopy of flow-oriented mutant virions in which the tyrosines were independently mutated to methionine. The results show that the twofold axis of the phenolic ring (C1-C4 line) of Tyr21 is inclined at 39.5 +/- 1.4 degrees from the virion axis, and that of Tyr24 is inclined at 43.7 +/- 0.6 degrees. The orientation determined for the Tyr21 phenol ring is close to that of a structural model previously proposed on the basis of fiber x-ray diffraction results (Protein Data Bank, identification code 1IFJ). On the other hand, the orientation determined for the Tyr24 phenol ring differs from the diffraction-based model by a 40 degrees rotation about the Calpha-Cbeta bond. The RLID results also indicate that each tyrosine mutation does not greatly affect the orientation of either the remaining tyrosine or single tryptophan (Trp26) of pVIII. On the basis of these results, a refined model is proposed for the coat protein structure in Ff.

A Structural Model for the Single-Stranded DNA Genome of Filamentous Bacteriophage Pf1

The filamentous bacteriophage Pf1, which infects strain PAK of Pseudomonas aeruginosa, is a flexible filament ( approximately 2000 x 6.5 nm) consisting of a covalently closed DNA loop of 7349 nucleotides sheathed by 7350 copies of a 46-residue alpha-helical subunit. The subunit alpha-helices, which are inclined at a small average angle ( approximately 16 degrees ) from the virion axis, are arranged compactly around the DNA core. Orientations of the Pf1 DNA nucleotides with respect to the filament axis are not known. In this work we report and interpret the polarized Raman spectra of oriented Pf1 filaments. We demonstrate that the polarizations of DNA Raman band intensities establish that the nucleotide bases of packaged Pf1 DNA are well ordered within the virion and that the base planes are positioned close to parallel to the filament axis. The present results are combined with a previously proposed projection of the intraviral path of Pf1 DNA [Liu, D. J., and Day, L. A. (1994) Science 265, 671-674] to develop a novel molecular model for the Pf1 assembly.

Orientation and Interactions of an Essential Tryptophan (Trp-38) in the Capsid Subunit of Pf3 Filamentous Virus

The filamentous bacteriophage Pf3 consists of a covalently closed DNA single strand of 5833 nucleotides sheathed by approximately 2500 copies of a 44-residue capsid subunit. The capsid subunit contains a single tryptophan residue (Trp-38), which is located within the basic C-terminal sequence (-RWIKAQFF) and is essential for virion assembly in vivo. Polarized Raman microspectroscopy has been employed to determine the orientation of the Trp-38 side chain in the native virus structure. The polarized Raman measurements show that the plane of the indolyl ring is tilted by 17 degrees from the virion axis and that the indolyl pseudo-twofold axis is inclined at 46 degrees to the virion axis. Using the presently determined orientation of the indolyl ring and side-chain torsion angles, chi(1) (N-C(alpha)-C(beta)-C(gamma)) and chi(2,1) (C(alpha)-C(beta)-C(gamma)-C(delta1)), we propose a detailed molecular model for the local structure of Trp-38 in the Pf3 virion. The present Pf3 model is consistent with previously reported Raman, ultraviolet-resonance Raman and fluorescence results suggesting an unusual environment for Trp-38 in the virion assembly, probably involving an intrasubunit cation-pi interaction between the guanidinium moiety of Arg-37 and the indolyl moiety of Trp-38. Such a C-terminal Trp-38/Arg-37 interaction may be important for the stabilization of a subunit conformation that is required for binding to the single-stranded DNA genome during virion assembly.

Unfolding Thermodynamics of the Δ-Domain in the Prohead I Subunit of Phage HK97: Determination by Factor Analysis of Raman Spectra

An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Delta-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Delta-domain is thought to resemble a phage scaffolding protein, by virtue of its highly alpha-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Delta-domain secondary structure and to quantify the thermodynamic parameters of Delta-domain unfolding. The results are compared for the Delta-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the alpha-helicity (approximately 70%), median melting temperature (T(m)=58 degrees C), enthalpy (DeltaH(m)=50+/-5 kcal mol(-1)), entropy (DeltaS(m)=150+/-10 cal mol(-1) K(-1)), and average cooperative melting unit (n(c) approximately 3.5) of the in situ Delta-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Delta-domain, despite an enhanced helical secondary structure ( approximately 90% alpha-helix), exhibits diminished thermostability (T(m)=40 degrees C; DeltaH(m)=27+/-2 kcal mol(-1); DeltaS(m)=86+/-6 cal mol(-1) K(-1)) and noncooperative unfolding ( approximately 1) vis-à-vis the in situ Delta-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Delta-domains. The present results clarify the key role of the gp5 Delta-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.

Orientations of Tyrosine Residues (Y21 and Y24) in Coat Protein Subunits of the Filamentous Virus fd

The filamentous virus fd is an important model for membrane protein assembly and is used extensively as a cloning vector and vehicle for peptide display. The threadlike virion (∼6 × 880 nm) comprises a single-stranded DNA genome sheathed by ∼2700 copies of a 50-residue α-helical subunit (pVIII). pVIII contains two tyrosines, Y21 and Y24. Tyrosine side-chain orientations in the virion have been examined by polarized Raman microspectroscopy of oriented fibers of wild type fd and of mutants in which either Y21 or Y24 is replaced by methionine. Polarized Raman spectra were collected for two fiber orientations in the laboratory frame of reference (abc, where c is parallel to the virion or fiber axis). In one orientation, designated cc, electric vectors of the exciting (514.5 nm) and scattered radiation are parallel to c; in the other (bb), these vectors are parallel to b. The intensity ratio, I cc /I bb , was determined for each Raman band.