Individual tyrosine side-chain contributions to circular dichroism of ribonuclease

Experimental and theoretical studies using site-directed mutants of ribonuclease A (RNase A) offer more extensive information on the tyrosine side-chain contributions to the circular dichroism (CD) of the enzyme. Bovine pancreatic RNase A has three exposed tyrosine residues (Tyr73, Tyr76, and Tyr115) and three buried tyrosine residues (Tyr25, Tyr92 and Tyr97). The difference CD spectra between the wild type and the mutants at pH 7.0 (Deltaepsilon(277,wt) - Deltaepsilon(277,mut)) show bands with more negative DeltaDeltaepsilon(277) values for Y73F and Y115F than those for Y25F and Y92F and bands with positive DeltaDeltaepsilon(277) values for Y76F and Y97F. The theoretical calculations are in good semiquantitative agreement for all the mutants. The pH difference spectrum (pH 11.3-7.0) for the wild type shows a negative band at 295 nm and an enhanced positive band at 245 nm. The three mutants at buried tyrosine sites and one mutant at an exposed tyrosine site (Y76F) exhibit pH-difference spectra that are similar to that of the wild type. In contrast, two mutants at exposed tyrosine sites (Y73F and Y115F) exhibit diminished 295-nm negative bands and, instead of positive bands at 245 nm, negative bands are observed. Our results indicate that Tyr73 and Tyr115, two of the exposed tyrosine residues, are the largest contributors to the 277- and 245-nm CD bands of RNaseA, but the buried tyrosine residues and the one remaining exposed residue also contribute to these bands. Disulfide contributions to the 277- and 240-nm bands and the peptide contribution to the 240-nm band are confirmed theoretically.     

Recent contributions of electronic circular dichroism to the investigation of oligopeptide conformations

Recent applications in our laboratories of electronic circular dichroism to the study of peptide secondary structures and their changes under external stimuli are briefly reviewed. More specifically, this article deals with: 1). characterization of a novel peptide conformation; 2). origin of amino acid homo-chirality on Earth; 3). bend and helical peptides as spacers; and 4). transfer and propagation of chirality in peptides.     

A model for solvent contributions to polypeptide and protein circular dichroism

The possibility of bound solvent contributing directly to the circular dichroism of polypeptides and proteins is discussed. The model presented is based on the requirement of a breakdown in the planar symmetry of the amide environment. This symmetry breakdown is described in terms of the conformational states of the chain, and leads to necessary, but not sufficient, conditions for observable solvent contributions. Application of the model leads to the conclusion that, while some chain conformations are intrinsically incapable of roviding the required breakdown in the symmetry of solvent perturbations, other conformations force such a breakdown, notably poly-L -proline II and disordered chains.     

Aromatic side-chain contributions to the far ultraviolet circular dichroism of peptides and proteins

The rotational strength of the L_a transition in phenylalanine and tyrosine side chains has been calculated for dipeptides with various backbone and side-chain conformations. Similar calculations have also been performed for tripeptides in the β-turn conformation with aromatic residues at the corners of the turn. The interaction of the aromatic ring with neighboring peptides generates rotational strengths in the L_a transition of the order of 0.1 Debye-Bohr magneton. When the preferred backbone and side-chain conformations are considered, it is found that the most probable conformations have positive L_a bonds. This result accounts for the observation that the N-acyl amino acid amides of L -Tyr and L Phe have positive L_a bands. It also suggests that, although other interactions may affect the numerical value and even the sign, there will be a significant positive contribution to the rotational strength of aromatic residues in globular proteins from nearest-neighbor interactions. Calculations on proteins of known conformation at the nearest-neighbor level confirm the tendency toward positive L_a contributions for Phe and Tyr residues. This contribution can be of the order of 10% of the observed CD even in proteins with rather strong amide contributions. In some proteins, such as the gene 5 protein from bacteriophage fd and many snake-venom toxins, side-chain contributions from Tyr and Trp residues manifest themselves as positive CD bands in the 225–250-nm region. The magnitude of the nearest-neighbor contributions and the trend toward positive contributions are consistent with the observation of such CD bands in globular proteins. No special stacking interaction among aromatic side chains needs to be invoked.     

Tryptophan mutants of intestinal fatty acid-binding protein: ultraviolet absorption and circular dichroism studies.

An UV absorption and CD study of intestinal fatty acid-binding protein is presented. Since there are only two Trp residues in the molecule, two single-Trp mutants were prepared to deconvolute their signals. The individual contribution of the eight Phe and four Tyr residues was not established; however, Phe global contribution is relatively free of interferences from the other chromophores and was observed directly. CD spectra showed that Phe vibronic structure was unusually sharp and seems to monitor very specific details in the three-dimensional structure. The global signal from Tyr was assigned only approximately due to band broadening and overlapping. At the upper end of the CD spectrum, strong positive (1)L(b) Trp transitions from Trp 82 and strong negative (1)L(b) Trp transitions from Trp 6 were observed. (1)L(a) transitions were overall weak, positive for Trp 82 and negative for Trp 6, nearly cancelling each other out in the final spectrum. The above assignment is of practical and fundamental interest to monitor folding, binding, and molecular dynamics down to microdomain resolution. The assignment of Trp bands allowed comparison with previous data from CRABP1, another member of the IFABP family with 28% identical residues. It was found that structural homology extends beyond sequence and tertiary fold to include optical properties of equivalent Trp residues in the structure.     

Nucleotide sequence of bacteriophage fd DNA.

The sequence of the 6,408 nucleotides of bacteriophage fd DNA has been determined. This allows to deduce the exact organisation of the filamentous phage genome and provides easy access to DNA segments of known structure and function.     

The conformation of T4 bacteriophage dihydrofolate reductase from circular dichroism

The secondary and tertiary structure of T4 bacteriophage dihydrofolate reductase is investigated by vacuum ultraviolet circular dichroism (CD) spectroscopy and probability analysis of the primary amino acid sequence. The far ultraviolet CD spectrum of the enzyme in the range of 260-178 nm is analyzed by the generalized inverse and variable selection methods developed by our laboratory. Variable selection yields an average content of 26% alpha-helix, 21% antiparallel beta-sheet, 10% parallel beta-sheet, 20% beta-turns, and 32% "other" structures within the T4 protein. The characteristic peaks of the CD spectrum indicate that the enzyme has a lot of antiparallel beta-sheet, which is typical of the alpha + beta tertiary class of globular proteins. The secondary structure of the protein is also analyzed by using four statistical methods on the amino acid sequence. Although the secondary structures predicted by each individual statistical method vary to a considerable extent, the fractions of each structure jointly predicted by a majority of the methods are in excellent agreement with our CD analysis. The alternating arrangement for some segments of alpha-helix and beta-sheet predicted from primary structure to be within the enzyme is characteristic of proteins containing parallel beta-sheet. This supports our conclusion that the protein contains both parallel and antiparallel beta-sheet structures, but finding both types of beta-sheet also means that the protein may have the variation on alpha/beta tertiary structure recently found in EcoRI endonuclease and thymidylate synthase. These observations, in conjunction with other physical properties of the T4 reductase, suggest that the enzyme perhaps shares an evolution in common with the dihydrofolate reductases derived from type I R-plasmids rather than with the host-cell protein.     

Nuclear magnetic resonance of the filamentous bacteriophage fd.

The filamentous bacteriophage fd and its major coat protein are being studied by nuclear magnetic resonance (NMR) spectroscopy. 31P NMR shows that the chemical shielding tensor of the DNA phosphates of fd in solution is only slightly reduced in magnitude by motional averaging, indicating that DNA-protein interactions substantially immobilize the DNA packaged in the virus. There is no evidence of chemical interactions between the DNA backbone and the coat protein, since experiments on solid virus show the 31P resonances to have the same principle elements of its chemical shielding tensor as DNA. 1H and 13C NMR spectra of fd virus in solution indicate that the coat proteins are held rigidly in the structure except for some aliphatic side chains that undergo relatively rapid rotations. The presence of limited mobility in the viral coat proteins is substantiated by finding large quadrupole splittings in 2H NMR of deuterium labeled virions. The structure of the coat protein in a lipid environment differs significantly from that found for the assembled virus. Data from 1H and 13C NMR chemical shifts, amide proton exchange rates, and 13C relaxation measurements show that the coat protein in sodium dodecyl sulfate micelles has a native folded structure that varies from that of a typical globular protein or the coat protein in the virus by having a partially flexible backbone and some rapidly rotating aromatic rings.     

Study of the circular dichroism of bacteriophage phi 6 and phi 6 nucleocapsid

We have studied the CD of both bacteriophage ?6 and ?6 nucleocapsid in order to assess the in situ state of the viral, double-stranded RNA (dsRNA). The results showed that packaged ?6 RNA is slightly hyperdichroic (4–5%) at 264 nm relative to dsRNA in solution. Also, the CD of dsRNA within both the virus and the nucleocapsid was unlike CD spectra of any of the three types of dsRNA condensed in vitro, as described in the accompanying paper. Using a curve-fitting program described elsewhere (Edmondson, S.P. & Gray, D.M. (1983) Nucleic Acids Res. 11 , 175–192), we have fit reference CD spectra of ?6 RNA and protein to measured CD spectra of ?6 intact phage and nucleocapsid. Results of the computer analysis indicated that ?6 RNA in situ is spectrally similar to dsRNA that is slightly dehydrated.     

Spectroscopic origin of conformation-sensitive contributions to polysaccharide optical activity: Vacuum-ultraviolet circular dichroism of agarose

The vacuum-uv CD of agarose solid films has been measured to 145 nm and shows a positive band near 180 nm and a larger negative band at around 152 nm. The positive band remains accessible in aqueous solution and has been used to characterize changes in molecular conformation and interaction during the sol–gel transition. The temperature profile of vacuum-uv CD shows sharp, discontinuous changes around the melting and setting points of the gel, which are interpreted in terms of cooperative intermolecular association through double helices, and pronounced hysteresis, which is discussed in terms of helix–helix aggregation.     

Studies of the structure of bacteriophage lambda cro protein in solution. Analysis of the circular dichroism data

The secondary and tertiary structures of bacteriophage cro protein were studied by circular dichroism. The pH dependence of this structure was investigated: cro protein is stable over pH 4.5-10.5. At these pH-values cro protein contains approximately 35% alpha-helix, approximately 20% antiparallel beta-structure and approximately 15% beta-turn, while the remaining part of the protein molecule is in the irregular state. The secondary and tertiary structures of the protein are modified abruptly at more acid and more alkaline pH-values. The curves characterizing the secondary and tertiary structures of the protein are symbatic. The effect of Gu-HCl on the secondary and tertiary structures of cro protein at 22 degrees C and pH 7.2 was studied also. The conformational transition occurs within 0.6-1.9 M Gu-HCl. The changes in the secondary and tertiary structures of the protein have a symbatic character. Thermal denaturation of cro protein was examined. A possible mechanism of the protein denaturation is discussed.     

Method of oriented circular dichroism.

We present a new method for determining the orientation of alpha-helical sections of proteins or peptides in membrane. To apply this method, membranes containing proteins must be prepared in a multilayer array. Circular dichroism (CD) spectra of the multilayer sample are then measured at the normal as well as oblique incident angles with respect to the bilayer planes; we call such spectra oriented circular dichroism (OCD). The procedure of OCD measurement, particularly the ways to avoid the spectral artifacts due to the effects of dielectric interfaces, linear dichroism and birefringence, and the method of data analysis are described in detail. To illustrate the method, we analyze the OCD of alamethicin in diphytanoylphosphatidylcholine multilayers. We conclude unambiguously that the helical section of alamethicin is parallel to the membrane normal when the sample is in the full-hydration state, but the helical section rotates to the plane of membrane when the sample is in a low-hydration state. We also obtained the parallel and perpendicular CD spectra of alpha-helix, and found them to be in agreement with previous theoretical calculations based on the exciton theory. These spectra are useful for analyzing protein orientations in future experiments.     

The circular dichroism of lysozyme.

The circular dichroism spectra of hen egg white lysozyme, and of lysozyme derivatives in which tryptophan residues 62 or 108, or both, are selectively oxidized, have been measured as a function of pH over the range of 200 to 310 nm. Neither Trp-62 nor Trp-108 is principally responsible for the positive rotational strength in the 280 to 300 nm region. The spectrum in the 200 to 230 nm region is nearly the same in the native protein and in the derivatives, and is little affected by binding of saccharide. These results are used to reinterpret the circular dichroism spectra of the lysozymes and alpha-lactalbumins.     

Magnetic circular dichroism study on synthetic polynucleotides, bacteriophage structure, and DNA's

The MCD (magnetic circular dichroism) spectra of Ap, ApA, ApApA, poly A, Up, UpU, poly U and double-stranded poly A:U alternating copoly A–U and alternating deoxyribopoly A–T were measured with a Cary 61 spectropolarimeter fitted with a Varian superconducting magnet at a field strength of 50 Kgauss. The MCD spectra of T2 and T5 DNA at various stages of heal denaturation were measured as a function of hyperchromicity of the sample. MCD spectra of the intact and degraded T2 and T5 phages were used to study the degree of alteration of the DNA inside the phages versus the DNA in vitro. The results for the adenine polymers show that the main MCD bands, B_2u(271 nm), B_1u(252 nm), and E_1u(212 nm), show a decrease in specific magnitude as the length of the polymer is increased, reflecting the degree of stacking of the polymer. In contrast, the uridine series of polymers shows little change of the MCD bands, indicating that there is little interaction between the bases regardless of the length of the polymers. The MCD spectra of poly A:U, alternating poly r(A–U): (A–U), and alternating poly d(A–T):(A–T) show significant differences among themselves in the magnitude of the B_2u band and when compared with the sum of the spectrum for the poly A plus poly U. This may indicate the selective effect of hydrogen bonding on the B_2u band. Alternatively, the difference may be due to the absence of an n → π* transition in the double-stranded polymer. Measurements of denatured T2 and To DNA's show increases in all MCD bands. The T2 DNA internally packed in phage shows an increase of the B_2u and E_1ubands, the B_2u remaining unchanged. The internal T5 DNA shows an increase of the B_1u band only. Thus, the internal DNA structure is altered in a manner quite different from a simple denaturation caused by hydrogen bond breaking. Furthermore, different MCD bands indicate that different modes of DNA packing exist for T2 and T5 phages.