Polarized Raman spectrum of single crystal uridylyl (3′–5′) adenosine: Local Raman tensors of some functional groups

Polarized Raman scattering measurements have been made of a single crystal of uridylyl(3′–5′)adenosine (UpA) by the use of a Raman microscope with 488.0 nm excitation. The UpA crystal belongs to space group P2₁ (monoclinic), and Raman intensities I_aa, I_bb, and I_c′c′, have been determined for each Raman band. These intensities correspond to the aa, bb, and c′c′ components of the crystal Raman tensor, where c′ is defined as an axis perpendicular to the crystallographic a axis in the ac plane. From these experimental data, and by taking the known crystal structure into account, anisotropic and isotropic molecular Raman tensors have been calculated for the following 11 normal modes: ring stretching modes of the adenine residue (protonated) at 1560, 1516, 1330, and 715 cm⁻¹; ring stretching modes of the uracil residue at 1696, 1657, 1615, 1228, and 790 cm⁻¹; PO⁻₂ symmetric stretching mode at 1080 cm⁻¹; P(—)O single bond stretching mode at 801 cm₋₁. These pieces of information of the Raman tensors are considered to be useful for estimating the orientations of the DNA and RNA strands in a biological complex from a polarized Raman spectroscopic measurement of such a complex. © 1998 John Wiley & Sons, Inc. Biopoly 45: 135–147, 1998     

Polarized Micro Raman Spectroscopic Studies of Nucleic Acid: Local Raman Tensors in Inosine-5′-monophosphoric Acid and the Orientation of Ekypoxanthine Residue in Poly(rI). Poly(rC) Fiber

Abstract

The polarized Raman spectra of a single crystal of the barium salt of inosine monophosphoric acid hexahydratc (Ba-IMP.6H₂O) have been observed with 488.0 nm excitation. For each Raman band, the relative intensities of aa, bb, cc, ab and bc tensor components have been determined. The tensor quotients from the crystal were augmented with measured depolarization ratios in solution. From these experimental data, the shape and orientation of the localized Raman scattering tensor were deduced for each of the normal modes of the hypoxanthine residue, phosphate moiety and ribose portion. The hypoxanthine residue gives a strong Raman band around 1553 cm^?1, which shows rather large depolarization ratio, p = 0.32, in aqueous solution, and shows a great scattering anisotropy in the single crystal of IMP. The shape and orientation of the Raman tensor associated to this 1553 cm^?1 vibration have been determined: one of its principal axes (y-axis) is directed along the long axis (N1-N7) of the hypoxanthine residue and the relative magnitudes of its components are given as r ₁ = α_xx/α_zz = ?1, r ₂ = α_yy/α_zz = 12. Next, a general relation has been shown between the orientation angles (θ and χ) of such a local Raman tensor in a uniaxial biological fiber and the anisotropy of Raman scattering intensities from the fiber. By the use of this relation, a discussion has been made of the orientation of the hypoxanthine residue in a poly(r1). poly(rC) duplex fiber.

     

Low-frequency dynamics and Raman scattering of crystals, of B-, A-, and Z-DNA, and fibers of C-DNA

Normal modes of vibration of DNA in the low-frequency region (10-300 cm-1 interval) have been identified from Raman spectra of crystals of B-DNA [d(CGCAAATTTGCG)], A-DNA [r(GCG)d(CGC) and d(CCCCGGGG)], and Z-DNA [d(CGCGCG) and d(CGCGTG)]. The lowest vibrational frequencies detected in the canonical DNA structures--at 18 +/- 2 cm-1 in the B-DNA crystal, near 24 +/- 2 cm-1 in A-DNA crystals, and near 30 +/- 2 cm-1 in Z-DNA crystals--are shown to correlate well with the degree of DNA hydration in the crystal structures, as well as with the level of hydration in calf thymus DNA fibers. These findings support the assignment [H. Urabe et al. (1985) J. Chem. Phys. 82, 531-535; C. Demarco et al. (1985) Biopolymers 24, 2035-2040] of the lowest frequency Raman band of each DNA to a helix mode, which is dependent primarily upon the degree of helix hydration, rather than upon the intrahelical conformation. The present results show also that B-, A-, C-, and Z-DNA structures can be distinguished from one another on the basis of their characteristic Raman intensity profiles in the region of 40-140 cm-1, even though all structures display two rather similar and complex bands centered within the intervals of 66-72 and 90-120 cm-1. The similarity of Raman frequencies for B-, A-, C-, and Z-DNA suggests that these modes originate from concerted motions of the bases (librations), which are not strongly dependent upon helix backbone geometry or handedness. Correlation of the Raman frequencies and intensities with the DNA base compositions suggests that the complex band near 90-120 cm-1 in all double-helix structures is due to in-plane librational motions of the bases, which involve stretching of the purine-pyrimidine hydrogen bonds. This would explain the centering of the band at higher frequencies in structures containing G.C pairs (greater than 100 cm-1) than in structures containing A.T pairs (less than 100 cm-1), consistent with the strengths of G.C and A.T hydrogen bonding.     

Low-frequency Raman spectra of lysozyme.

New techniques in laser Raman spectroscopy are used to obtain spectra of aqueous solutions of lysozylme for frequency shifts as small as 5 cm^?1. In addition, Raman measurements are made on two crystalline forms of hen egg white lysozyme. The spectra obtained from the solution and from the crystal are found to be similar for frequencies above 100 cm^?1. However, a low-frequency band at 25 cm^?1 observed in crystalline lysozyme is not found in the solution, indicating that this band cannot be attributed to an internal molecular vibration.     

Structure-potential dependence of adsorbed enzymes and amino acids revealed by the surface enhanced Raman effect

Surface enhanced Raman scattering (SERS) of some enzymes (alkaline phosphatase, horseradish peroxidase and lactoperoxidase) and some amino acids (tryptophan, tyrosine and phenylalanine) on silver electrodes has been studied. The spectral band intensities of certain amino acids and amino acid residues were determined by their orientation on the surface and depended on the electrode potential (E).Abbreviations SERS surface enhanced Raman scattering - Trp tryptophan - Tyr tyrosine - Phe phenylalanine - E electrode potential - ORC oxidation-reduction cycle     

Polarized Raman spectroscopy of double-stranded RNA from bacteriophage phi6: local Raman tensors of base and backbone vibrations

Raman tensors for localized vibrations of base (A, U, G, and C), ribose and phosphate groups of double-stranded RNA have been determined from polarized Raman measurements on oriented fibers of the genomic RNA of bacteriophage phi6. Polarized Raman intensities for which electric vectors of both the incident and scattered light are polarized either perpendicular (I[bb]) or parallel (I[cc]) to the RNA fiber axis have been obtained by Raman microspectroscopy using 514.5-nm excitation. Similarly, the polarized Raman components, I(bc) and I(cb), for which incident and scattered vectors are mutually perpendicular, have been obtained. Spectra collected from fibers maintained at constant relative humidity in both H2O and D2O environments indicate the effects of hydrogen-isotopic shifts on the Raman polarizations and tensors. Novel findings are the following: 1) the intense Raman band at 813 cm(-1), which is assigned to phosphodiester (OPO) symmetrical stretching and represents the key marker of the A conformation of double-stranded RNA, is characterized by a moderately anisotropic Raman tensor; 2) the prominent RNA band at 1101 cm(-1), which is assigned to phosphodioxy (PO2-) symmetrical stretching, also exhibits a moderately anisotropic Raman tensor. Comparison with results obtained previously on A, B, and Z DNA suggests that tensors for localized vibrations of backbone phosphodiester and phosphodioxy groups are sensitive to helix secondary structure and local phosphate group environment; and 3) highly anisotropic Raman tensors have been found for prominent and well-resolved Raman markers of all four bases of the RNA duplex. These enable the use of polarized Raman spectroscopy for the determination of purine and pyrimidine base residue orientations in ribonucleoprotein assemblies. The present determination of Raman tensors for dsRNA is comprehensive and accurate. Unambiguous tensors have been deduced for virtually all local vibrational modes of the 300-1800 cm(-1) spectral interval. The results provide a reliable basis for future evaluations of the effects of base pairing, base stacking, and sequence context on the polarized Raman spectra of nucleic acids.     

Nanosecond temperature jump and time-resolved Raman study of thermal unfolding of ribonuclease A

A nanosecond temperature jump (T-jump) apparatus was constructed and combined with time-resolved Raman measurements to investigate thermal unfolding of a protein for the first time. The 1.56-microm heat pulse with 9 ns width at 10 Hz was obtained through the two-step stimulated Raman scattering in D(2) gas involving seeding and amplification. To achieve uniform temperature rise, the counter-propagation geometry was adopted for the heat pulse. The temperature rise was determined by anti-Stokes to Stokes intensity ratios of the 317 and 897 cm(-1) bands of MoO(4)(2-) ions in an aqueous solution. The T-jump as large as 9 degrees C in 10 ns was attained. The unfolding of bovine pancreatic ribonuclease A was monitored with time-resolved Raman spectra excited at 532 nm. The C-S stretching band of Met residues exhibited 10% change of that expected from the stationary state temperature-difference spectra in the initial 200 ns following T-jump and another 10% in 5 ms. The Raman intensity of SO(4)(2-) ions around 980 cm(-1) increased at 100 micros, presumably due to some conformational changes of the protein around the active site. The S-S stretches and tyrosine doublet displayed little changes within 5 ms. Thus, the conformational changes in the initial step of unfolding are not always concerted.     

Raman spectroscopy of chlorophyll d from Acaryochloris marina

The Raman spectroscopy of chlorophyll (Chl) d isolated from Acaryochloris marina has been measured in the range of 250-3200 cm(-1) at 77 K following excitation of its B(x) band at 488 nm. A peak at 1659 cm(-1) of medium intensity arising from Cz=O stretching vibration in the formyl group 3(1) specific to Chl d was observed clearly. Peaks due to other Cz=O stretching vibrations of the 13(1) keto-, 13(3) ester- and 17(3) groups have also been observed with much weaker intensities. Intense Raman peaks in the range of 1000-1800 cm(-1) are reported and homologous comparison with corresponding Raman shifts of Chl a, Chl b and BChl a are presented.     

Measurement of hemoglobin oxygen saturation using Raman microspectroscopy and 532-nm excitation.

The resonant Raman enhancement of hemoglobin (Hb) in the Q band region allows simultaneous identification of oxy- and deoxy-Hb. The heme vibrational bands are well known at 532 nm, but the technique has never been used to determine microvascular Hb oxygen saturation (So(2)) in vivo. We implemented a system for in vivo noninvasive measurements of So(2). A laser light was focused onto areas of 15-30 microm in diameter. Using a microscope coupled to a spectrometer and a cooled detector, Raman spectra were obtained in backscattering geometry. Calibration was performed in vitro using blood at several Hb concentrations, equilibrated at various oxygen tensions. So(2) was estimated by measuring the intensity of Raman signals (peaks) in the 1,355- to 1,380-cm(-1) range (oxidation state marker band nu(4)), as well as from the nu(19) and nu(10) bands (1,500- to 1,650-cm(-1) range). In vivo observations were made in microvessels of anesthetized rats. Glass capillary path length and Hb concentration did not affect So(2) estimations from Raman spectra. The Hb Raman peaks observed in blood were consistent with earlier Raman studies using Hb solutions and isolated cells. The correlation between Raman-based So(2) estimations and So(2) measured by CO-oximetry was highly significant for nu(4), nu(10), and nu(19) bands. The method allowed So(2) determinations in all microvessel types, while diameter and erythrocyte velocity could be measured in the same vessels. Raman microspectroscopy has advantages over other techniques by providing noninvasive and reliable in vivo So(2) determinations in thin tissues, as well as in solid organs and tissues in which transillumination is not possible.     

Estimation of protein secondary structure from the laser Raman amide I spectrum

A new method for estimating protein secondary structure from the laser Raman spectrum has been developed whereby the amide I Raman band of a protein is analyzed directly as a linear combination of amide I bands of proteins whose secondary structures are known. For 14 proteins, analyzed by removing each one from the reference set and analyzing its structure in terms of the remaining proteins, the average correlation coefficients between the Raman and X-ray diffraction estimates of helix, beta-strand, turn, and undefined were 0.98, 0.98, 0.82 and 0.35, respectively. Significant correlations were also observed for distinctions between alpha-helix (0.98) and disordered helix (0.82), and between parallel (0.82) and antiparallel (0.97) beta-sheets. The average standard deviation of these Raman estimates from the X-ray values is less than 4%. In addition, a singular value analysis of 20 Raman amide I spectra indicates that there may be as many as nine significant independent pieces of information present in the amide I region.     

The Infrared and Raman Spectra of the Duplex of d(GGTATACC) in the Crystal Show Bands Due to Both the A-form and the B-form of DNA

Abstract

The deoxyoligonucleotide, d(GGTATACC), forms a duplex structure that crystallizes in the DNA A form. This has been shown by both X-ray diffraction studies and Raman spectroscopy (1,2). The presence of the DNA B form has been reported using diffuse X-ray scattering from a crystal of the closely related sequence d(GGBrUABrUACC)(3). In this paper the infrared spectrum of the d(GGTATACC) crystal is presented and curve resolution of both the Raman and IR spectra have been carried out. The percentage of A and B forms have been estimated. The %B form in the crystal has been estimated from the IR spectra to be about 15% and from Raman to be about 20%. Moreover the IR spectrum of the A conformation in the crystal is slightly different from the IR spectrum of the A conformation in polynucleotide fibers in particular in the region of the phosphate stretching vibrations and of the in-plane double bond vibrations of the bases. We show that it is feasible to obtain IR as well as Raman spectra of small crystals of oligonucleotides and that this is a good method of identifying all of the different conformations that may be in the crystal.     

The infrared and Raman spectra of the duplex of d(GGTATACC) in the crystal show bands due to both the A-form and the B-form of DNA

The deoxyoligonucleotide, d(GGTATACC), forms a duplex structure that crystallizes in the DNA A form. This has been shown by both X-ray diffraction studies and Raman spectroscopy (1,2). The presence of the DNA B form has been reported using diffuse X-ray scattering from a crystal of the closely related sequence d(GGBrUABrUACC)(3). In this paper the infrared spectrum of the d(GGTATACC) crystal is presented and curve resolution of both the Raman and IR spectra have been carried out. The percentage of A and B forms have been estimated. The %B form in the crystal has been estimated from the IR spectra to be about 15% and from Raman to be about 20%. Moreover the IR spectrum of the A conformation in the crystal is slightly different from the IR spectrum of the A conformation in polynucleotide fibers in particular in the region of the phosphate stretching vibrations and of the in-plane double bond vibrations of the bases. We show that it is feasible to obtain IR as well as Raman spectra of small crystals of oligonucleotides and that this is a good method of identifying all of the different conformations that may be in the crystal.     

Conformational dependence of the Raman scattering intensities from polynucleotides

The intensity of Raman scattering from the various Raman active vibrations of poly-(riboadenylic acid), poly(ribocytidylic acid), poly(ribouridylic acid), and poly(riboinosinic acid) in moderately dilute solutions were examined as the temperature was changed to alter their conformation. It was found that certain highly intense, highly polarized Raman bands from the totally symmetric, i.e., in-plane, ring vibrations of the nucleic acid bases become less intense as the chains become more ordered in solution. Since these vibrations occur at frequencies which are markedly different for each type of base, Raman spectroscopy appears to provide a new method for the characterizing of the average conformation of each of the bases in solution. A theory for the resonant Raman effect is given in which it is shown that, a decrease in resonant Raman intensity is to be expected if one obtains a decrease in the intensity of the corresponding ultraviolet absorption band with which the incident light is resonant. If it is assumed that certain Raman bands derive their intensity predominantly from the first few ultraviolet absorption intensities, then a qualitative explanation of our observed conformational dependence of the ordinary Raman intensities can be obtained.     

Laser Raman investigations of intact single muscle fibers. Protein conformations

Raman spectra, in the frequency region of the protein vibrations, of intact single muscle fibers of the giant barnacle are presented. Strong bands at 1521 and 1156 cm-1 in the spectra are attributed to resonance-enhanced Raman bands of membrane-bound beta-carotene. Many bands of the myofibrillar proteins are also observed, and at least three spectral features confirm that these proteins adopt a predominantly alpha-helical structure: (1) the amide I band at 1648 cm-1, (2) the weak scattering in the amide III region, and (3) a strong skeletal C-C stretching band at 939 cm-1. Deuterated fibers have also been examined in order to find the exact shape of the amide III band. The presence in the fibers of paramyosin, which is only found in catch muscles, is also apparent from the spectra.     

Differential activity and structure of highly similar peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2

Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and used as a model for the 95% identical commercially available horseradish peroxidase HRP A2. The crystal structure of ATP A2 at 1.45 A resolution at 100 K showed a water molecule only 2.1 A from heme iron [Ostergaard, L., et al. (2000) Plant Mol. Biol. 44, 231-243], whereas spectroscopic studies of HRP A2 in solution at room temperature [Feis, A., et al. (1998) J. Raman Spectrosc. 29, 933-938] showed five-coordinated heme iron, which is common in peroxidases. Presented here, the X-ray crystallographic, single-crystal, and solution resonance Raman studies at room temperature confirmed that the sixth coordination position of heme iron of ATP A2 is essentially vacant. Furthermore, electronic absorption and resonance Raman spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plant HRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highly flexible compared with other plant peroxidases. Ostergaard et al. (2000) also demonstrated that ATP A2 expression and lignin formation coincide in Arabidopsis tissues, and docking of lignin precursors into the substrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates. In contrast, the additional methoxy group of the sinapyl moiety gave rise to steric hindrance, not only in A2 type peroxidases but also in all peroxidases. We confirm these predictions for ATP A2, HRP A2, and HRP C. The specific activity of ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady-state study at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1 = 1.0 microM(-1) x s(-1). The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2, and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 +/- 0.3, 4.0 +/- 0.2, 0.70 +/- 0.03, and 0.04 +/- 0.2 microM(-1) x s(-1) for HRP A2, respectively, and 4.6 +/- 0.2, 2.3 +/- 0.1, 0.25 +/- 0.01, and 0.01 +/- 0.004 microM(-1) x s(-1) for ATP A2, respectively. The structural origin of the differential reactivity is discussed in relation to glycosylation and amino acid substitutions. The results are of general importance to the use of homologous models and structure determination at low temperatures.     

Effects of organic solutes on the Raman spectra of barnacle muscle fibers

The Raman spectra observed from barnacle muscle fibers are quite complex because the cytoplasm of these cells contains several proteins and solutes. An extraction procedure was used to separate organic solutes from the contractile proteins. Glycine, trimethylamine oxide, taurine, and alanine were found to contribute to the Raman spectra of barnacle muscle fibers, while spectra of lobster fibers reveal the presence of betaine in addition. We have observed that the increase in osmolarity of the intracellular fluid caused by the augmentation of the salinity of sea water (density, 1.023-1.030) in which the barnacles were kept, induces a reduction of intensity of the amide I band. To distinguish among the different parameters which are modified by the sea water salinity, observations were made on glycerinated barnacle muscle fibers. The reduction of intensity of the amide I band in the Raman spectra of glycerinated muscle fibers was also observed with the addition of taurine (0.08 M) in the external relaxing solution. Therefore, under these experimental conditions, the Raman scattering intensity in the amide I region assigned to the alpha-helix conformation (1645-1650 cm-1) is increased when the concentration of organic electrolytes is reduced. However, as no significant decrease of the scattering intensity in the 1660-1670 cm-1 region where the amide I bands of either beta-sheet or disordered conformations normally appear was observed, the increase of intensity of the amide I band centered at 1645 cm-1 is assigned to a change of orientation of alpha-helical segments of the myosin molecules. Our results suggest that organic solutes influence the position of the S-2 segments relative to the thick filaments.     

Polarized Raman scattering from oriented single microcrystals of d(A5T5)2 and d(pTpT)

Polarized Raman spectra have been obtained from single microcrystals of the duplex of the decamer d(A₅T₅)₂ using a Raman microscope. This is the first report of Raman spectra from a crystal of a deoxyoligomer that contains only long, nonalternating sequences of adenine and thymine. Sequences containing d(A)_n and d(T)_n are of interest in view of recent suggestions that they induce bends in DNA and that they might exist in a nonstandard B-conformation. Polarized Raman spectra of a crystal of d(pTpT) have also been obtained. Both crystals display Raman bands whose intensities are very sensitive to the orientation of the crystal with respect to the direction of polarization of the incident laser beam. These spectra indicate that the helical axes of the oligonucleotides are parallel to the long axes of the crystals and that the d(A₅T₅)₂ is not appreciably bent in the crystal. The Raman spectrum from the d(pTpT) crystal indicates that all of the furanose ring puckers are in a C2′-endo configuration since only the C2′-endo marker band at 835 ± 5 cm^?1 is present. Crystals of d(A₅T₅)₂ show measurable Raman intensities in both the 838- and 816-cm^?1 bands. This indicates the presence of both the C2′-endo and C3′-endo, or possibly other non-C2′-endo, furanose conformations. The 816-cm^?1 band is weak so that only a small fraction of the residues are estimated to be in the non-C2′-endo conformation. In both the d(pTpT) and d(A₅T₅)₂ crystals the intensity of the bands due to vibrations of the backbone show only a small dependence on orientation of the crystals. This result is explained by the low symmetry of the puckered sugar rings. It is concluded that Raman spectra obtained from oligonucleotide crystals in which the orientation of the crystal axes to the laser polarization is not carefully controlled may contain intensity artifacts that are due to polarization effects.     

In-situ confocal Raman observation of structural changes of insulin crystals in sequential dehydration process

In-situ confocal Raman spectroscopy combined with relative humidity (RH) control technique was used to study the sequential dehydration process of insulin crystals. By gradually decreasing the ambient RH of the insulin crystal, the content of the hydration water in the crystal was quantitatively controlled. Tyrosine (Tyr) residues were very sensitive to the micro-environmental changes, and four Raman features 828cm(-1), 852cm(-1), 1174cm(-1) and 1206cm(-1) of Tyr were employed to monitor the dehydration process. Taking advantage of the ratios I(852)/I(828) at different RH values, the mole fractions of the 'exposed' and 'buried' Tyr residues were estimated. Moreover, using the ratio I(1174)/I(1206) as an indicator of the dehydration process, three RH regions were discriminated. This is believed to imply that different types of the hydration water were lost step by step, i.e. firstly the 'second-layer' and 'first-layer' classes, then the 'contact' class, and finally, the 'inside' class. In addition, the profile of the amide I band was observed to gradually change with RH. By band fitting of the amide I region, changes in secondary structure were quantitatively determined. And the results showed that nearly 17% of α-helix converted into β-sheet with RH decreasing from 92% to 2%.     

Quantitative surface-enhanced resonance Raman scattering of phthalocyanine-labelled oligonucleotides

The evaluation of phthalocyanine labels for the surface-enhanced resonance Raman scattering (SERRS) detection of oligonucleotides is reported. Three phthalocyanine-labelled oligonucleotides were assessed, each containing a different metal centre. Detection limits for each labelled oligonucleotide were determined using two excitation frequencies where possible. Limits of detection as low as 2.8 × 10⁻¹¹mol.dm⁻³ were obtained which are comparable to standard fluorescently labelled probes used in previous SERRS studies. The identification of two phthalocyanine-labelled oligonucleotides without separation was also demonstrated indicating their suitability for multiplexing. This study extends the range of labels suitable for quantitative surface-enhanced resonance Raman scattering with silver nanoparticles and offers more flexibility and choice when considering SERRS for quantitative DNA detection.     

Polarization sensitive coherent anti-Stokes Raman scattering spectroscopy of the amide I band of proteins in solutions.

Polarization sensitive coherent anti-Stokes Raman scattering (PCARS) spectroscopy is a fruitful technique to study Raman vibrations of diluted molecules under off-electron resonant conditions. We apply PCARS as a direct spectroscopic method to investigate the broad amide I band of proteins in heavy water. In spontaneous Raman spectroscopy, this band is not well resolved. We fit a number of spectra taken of each protein under different polarization conditions, with a single set of parameters. It then appears that some substructure is observed in the amide I band. From this substructure, we determine the percentage of alpha-helix, beta-sheet, and random coil for the proteins lysozyme, albumin, ribonuclease A, and alpha-chymotrypsin.