Calculating sequence-dependent melting stability of duplex DNA oligomers and multiplex sequence analysis by graphs.

The analytical methods for characterizing DNA sequence-dependent thermodynamic stability have been reviewed. A set of n-n sequence stability parameters is presented. Examples in which these values are used to calculate the thermodynamic stability of short duplex DNA oligomers are presented. The problem of determining sets of isothermal sequences is addressed by representing DNA sequences as graphs. Representing DNA sequences by a graph descriptor with special mathematical properties minimizes the computational difficulty of determining the number of DNA sequences with identical predicted thermodynamic stability. This is achieved by replacement of a whole set of sequences by a single representative. Applications of this concept were demonstrated for sequences assembled from individual bases and sequences assembled from oligomeric blocks.

Thermal unfolding of ribonuclease A in phosphate at neutral pH: deviations from the two-state model.

The thermal denaturation of ribonuclease A (RNase A) in the presence of phosphate at neutral pH was studied by differential scanning calorimetry (DSC) and a combination of optical spectroscopic techniques to probe the existence of intermediate states. Fourier transform infrared (FTIR) spectra of the amide I' band and far-uv circular dichroism (CD) spectra were used to monitor changes in the secondary structure. Changes in the tertiary structure were monitored by near-uv CD. Spectral bandshape changes with change in temperature were analyzed using factor analysis. The global unfolding curves obtained from DSC confirmed that structural changes occur in the molecule before the main thermal denaturation transition. The analysis of the far-uv CD and FTIR spectra showed that these lower temperature-induced modifications occur in the secondary structure. No pretransition changes in the tertiary structure (near-uv CD) were observed. The initial changes observed in far-uv CD were attributed to the fraying of the helical segments, which would explain the loss of spectral intensity with almost no modification of spectral bandshape. Separate analyses of different regions of the FTIR amide I' band indicate that, in addition to alpha-helix, part of the pretransitional change also occurs in the beta-strands.

Enhanced prediction accuracy of protein secondary structure using hydrogen exchange Fourier transform infrared spectroscopy.

A novel equilibrium hydrogen exchange Fourier transform IR (HX-FTIR) spectroscopy method for predicting secondary structure content was employed using spectra obtained for a training set of 23 globular proteins. The IR bandshape and frequency changes resulting from controlled levels of H-D exchange were observed to be protein-dependent. Their analysis revealed these variations to be partly correlated to secondary structure. For each protein, a set of 6 spectra was measured with a systematic variation of the solvent H-D ratio and was subjected to factor analysis. The most significant component spectra for each protein, representing independent aspects of the spectral response to deuteration, were each subjected to a second factor analysis over the entire training set. Restricted multiple regression (RMR) analysis using the loadings of the principal components from 19 of these H-D analyses revealed an improvement in prediction accuracy compared with conventional bandshape-based analyses of FTIR data. Nearly a factor of 2 reduction in error for prediction of helix fractions was found using s1, the average spectral response for the H-D set. In some cases, significant error reduction for prediction of minor components was found using higher factors. Using the same analytical methods, prediction errors with this new deuteration-response-FTIR method were shown to be even better than those obtained by use of electronic circular dichroism (ECD) data for helix predictions and to be significantly lower for ECD-based sheet prediction, making these the best secondary structure predictions obtained with the RMR method. Tests of a limited variable selection scheme showed further improvements, consistent with previous results of this approach using ECD data.

Novel matrix descriptor for secondary structure segments in proteins: demonstration of predictability from circular dichroism spectra.

An extension to standard protein secondary structure predictions using optical spectra that encompasses the number and average lengths of segments of uniform secondary structure in the sequence is demonstrated. The connectivity and numbers of segments can be described by a matrix descriptor [sij] (i, j representing segment types such as helix and beta-sheet strands). Independent knowledge of the fractional concentration of each secondary structure type and of the total number of residues in the protein then with [sij] yields the average segment length of each type. The physical background for prediction of this extended structural descriptor from spectral data is summarized, rules for its generation from reference X-ray structures are defined, and formal variants of its form are discussed. Using a novel neural network approach to analyze a training set of electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectra for 23 proteins, matrix descriptors encompassing helix, sheet, and other forms are predicted. The results show that the matrix descriptor can be predicted to an accuracy comparable to that of conventionally predicted average fractional secondary structures. In this respect the ECD predictions of [sij] were significantly more accurate than the VCD ones, which may result from the longer range length dependence of the ECD bandshape and intensity. Summary results for a parallel analysis using Fourier transform infrared spectra indicate somewhat lower reliability than those for VCD.

Predictions of protein secondary structures using factor analysis on Fourier transform infrared spectra: effect of Fourier self-deconvolution of the amide I and amide II bands.

Fourier self-deconvolution (FSD) was performed on protein amide I and II Fourier transform infrared (FTIR) spectra to test if the resultant increased band shape variation would lead to improvements in protein secondary structure prediction with our factor analysis based restricted multiple regression (RMR) methods. FTIR spectra of 23 proteins dissolved in H2O were measured and normalized to a constant amide I peak absorbance. The deconvolved spectra were renormalized by area so that the deconvolved spectra sets had the same area as before. Principal component analysis of the deconvolved spectra sets was carried out, which was followed by a selective multiple linear regression (RMR) analysis of the principal component loadings with regard to the fractional components (FC) of secondary structure. As compared to analyses based on the original spectra set, helix and sheet predictions were not noticeably improved by FSD; but, if a very large number of component spectra (16) were retained in the pool to select which loadings to be used in the RMR optimization, better predictions of turn and "other" resulted. The prediction quality varied depending on the deconvolution parameters used.

Vibrational circular dichroism spectra of proteins in the amide III region: measurement and correlation of bandshape to secondary structure.

Vibrational circular dichroism (VCD) spectra have been measured for 23 globular proteins dissolved in H2O/phosphate buffer over the 1400 to 1100 cm(-1) region which encompasses the amide III mode. Spectral responses characteristic of the dominant secondary structure type were found as broad features at approximately 1300 cm(-1), with the extreme forms having positive VCD for highly helical proteins and negative VCD for highly sheet-containing proteins. Quantitative correlation with secondary structure was carried out using previously developed factor analysis and restricted multiple regression (FA/RMR) techniques. Since the absorbance intensity of the amide III mode is difficult to determine due to overlap with other transitions, an alternative, absolute intensity-independent, simple structural analysis method was used. A linear regression was developed between the fractional components of secondary structure for the protein set and the overlap integrals of the normalized spectra from the set with that of a selected protein. The results of this simple method are quite comparable to those of the FA/ RMR approach for analysis with amide III VCD. On the other hand, test calculations with the new method when used with electronic CD spectra are not as good as FA/RMR due to its more intensity-dependent relationship with secondary structure.

Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals.

Aquaporins are integral membrane proteins found in diverse animal and plant tissues that mediate the permeability of plasma membranes to water molecules. Projection maps of two-dimensional crystals of aquaporin-1 (AQP1) reconstituted in lipid membranes suggested the presence of six to eight transmembrane helices in the protein. However, data from other sequence and spectroscopic analyses indicate that this protein may adopt a porin-like beta-barrel fold. In this paper, we use Fourier transform infrared spectroscopy to characterize the secondary structure of highly purified native and proteolyzed AQP1 reconstituted in membrane crystalline arrays and compare it to bacteriorhodopsin. For this analysis the fractional secondary structure contents have been determined by using several different algorithms. In addition, a neural network-based evaluation of the Fourier transform infrared spectra in terms of numbers of secondary structure segments and their interconnections [sij] has been performed. The following conclusions were reached: 1) AQP1 is a highly helical protein (42-48% alpha-helix) with little or no beta-sheet content. 2) The alpha-helices have a transmembrane orientation, but are more tilted (21 degrees or 27 degrees, depending on the considered refractive index) than the bacteriorhodopsin helices. 3) The helices in AQP1 undergo limited hydrogen/deuterium exchange and thus are not readily accessible to solvent. Our data support the AQP1 structural model derived from sequence prediction and epitope insertion experiments: AQP1 is a protein with at least six closely associated alpha-helices that span the lipid membrane.

Characterization of alanine-rich peptides, Ac-(AAKAA)n-GY-NH2 (n = 1-4), using vibrational circular dichroism and Fourier transform infrared. Conformational determination and thermal unfolding.

Vibrational circular dichroism (VCD) and Fourier transform IR (FTIR) were measured for a series of short alanine-based peptides having the general formula Ac-(AAKAA)n-GY-NH2 (n = 1-4) from 5 to 50 degrees C in D2O and at room temperature in both TFE and H2O. In both of these latter solvents, the dominant structural form at the lowest temperature for the longest oligomers is alpha-helical. The same is true for the n = 4 peptide in D2O, but under these more dilute aqueous conditions, the shorter (n = 3) peptides have mixed helix-coil structures and the n = 1 and 2 peptides are random coils. The VCD data do not support the 310-helix as a dominant contributor to the conformation of these oligomers in any of these solvents. These vibrational spectral data are consistent with lower-concentration electronic CD results and additionally indicate increased helical stability at higher concentrations. VCD amide I data for the 22mer (n = 4) in D2O indicate that the peptide undergoes a transition from a highly helical conformation at 5 degrees C to a dominant random coil structure at approximately 45 degrees C with a Tm of approximately 25 degrees C (effective midpoint). Factor analysis of the thermal data showed that three principal components were required to describe both the VCD and FTIR data for the n = 4 peptide in D2O. The transition is characterized by a gradual loss of contribution from a spectral component representing the alpha-helical fraction. The third component is evidence of an optically detected intermediate conformation best viewed as a mixed coil-helix structure resulting from end fraying of the helical peptide as the temperature is increased. The nature of the junction between the interior helix and frayed ends is not determined by these data and could involve local (phi and psi) angles mimicking a 310-helix that would provide consistency with ESR and NMR results from Millhauser and co-workers.

Protein structural segments and their interconnections derived from optical spectra. Thermal unfolding of ribonuclease T1 as an example.

A novel descriptor for protein structure is examined here that goes beyond predictions of the average fractional components (FC) of a few conformational types and represents the number and interconnection of segments of continuous, well-defined secondary structural elements such as alpha-helices and beta-sheets. This matrix descriptor can be predicted from optical spectra using neural network methods. The new matrix plus traditional FC descriptors can be quickly and generally obtained to provide a level of detail not previously derived from optical spectra and a discrimination between proteins that might otherwise be viewed as being very similar using just the FC descriptor. As an example of its potential utilization, this matrix descriptor approach was applied to an analysis of both the native state and the reversible thermal denaturation of ribonuclease T1 in H2O. Analyses of the FTIR spectral data indicate initial loss of the major helical segment at 50-55 degrees C but with little accompanying change in the number of sheet segments or the sheet FC values. Circular dichroism (CD) and vibrational CD data are also used to support this interpretation based on FC changes with temperature. Parallel analysis of the corresponding data for this protein in D2O demonstrates that the method is sensitive to the match between the degree of H-D exchange used to prepare samples for the unknown and the reference data set.

Predictions of secondary structure using statistical analyses of electronic and vibrational circular dichroism and Fourier transform infrared spectra of proteins in H2O.

Vibrational circular dichroism (VCD) and Fourier transform IR (FTIR) methods for prediction of protein secondary structure are systematically compared using selective regression analysis. VCD and FTIR spectra over the amide I and II bands of 23 proteins dissolved in H2O were analyzed using the principal component method of factor analysis (PC/FA) and regression fits to fractional components (FC) of secondary structure. Predictive capability was determined by computing structures for proteins sequentially left out of the regression. All possible combinations of PC/FA spectral parameters (coefficients) were used to form a full set of restricted multiple regressions (RMR) of PC/FA coefficients with FC values, both independently for each spectral data set as well as for the VCD and FTIR sets grouped together and with similarly obtained electronic CD (ECD) data. The distribution of predictive error for a set of the best RMR relationships that use a given number of spectral coefficients was used to select the optimal prediction algorithm. Minimum predictive error resulted for a small subset (three to six) of spectral coefficients, which is consistent with our earlier findings using VCD measured for proteins in 2H2O and ECD data. Subtracting the average absorption spectrum from all the training set FTIR spectra before analysis yields more variance in the FTIR band shape and improves the predictive ability of the best PC/FA RMR to near that for the VCD. Both methods (FTIR and VCD) using data for proteins in H2O are somewhat better predictors than amide I' (in 2H2O) VCD alone and, for helix, worse than ECD alone. Combining FTIR and VCD data did not dramatically change the prediction results. Predictions are improved by combining both with ECD data, indicating that the improvement is due to using their very different structural sensitivities. The coupled H2O-based spectral analyses and the mixed amide I' + II VCD plus ECD analysis are comparable for the helix and sheet components, indicating that partial deuteration is not a major source of prediction error.

Comparison of and limits of accuracy for statistical analyses of vibrational and electronic circular dichroism spectra in terms of correlations to and predictions of protein secondary structure.

This work provides a systematic comparison of vibrational CD (VCD) and electronic CD (ECD) methods for spectral prediction of secondary structure. The VCD and ECD data are simplified to a small set of spectral parameters using the principal component method of factor analysis (PC/FA). Regression fits of these parameters are made to the X-ray-determined fractional components (FC) of secondary structure. Predictive capability is determined by computing structures for proteins sequentially left out of the regression. All possible combinations of PC/FA spectral parameters (coefficients) were used to form a full set of restricted multiple regressions with the FC values, both independently for each spectral data set as well as for the two VCD sets and all the data grouped together. The complete search over all possible combinations of spectral parameters for different types of spectral data is a new feature of this study, and the focus on prediction is the strength of this approach. The PC/FA method was found to be stable in detail to expansion of the training set. Coupling amide II to amide I' parameters reduced the standard deviations of the VCD regression relationships, and combining VCD and ECD data led to the best fits. Prediction results had a minimum error when dependent on relatively few spectral coefficients. Such a limited dependence on spectral variation is the key finding of this work, which has ramifications for previous studies as well as suggests future directions for spectral analysis of structure. The best ECD prediction for helix and sheet uses only one parameter, the coefficient of the first subspectrum. With VCD, the best predictions sample coefficients of both the amide I' and II bands, but error is optimized using only a few coefficients. In this respect, ECD is more accurate than VCD for alpha-helix, and the combined VCD (amide I' + II) predicts the beta-sheet component better than does ECD. Combining VCD and ECD data sets yields exceptionally good predictions by utilizing the strengths of each. However, the residual error, its distribution, and, most importantly, the lack of dependence of the method on many of the significant components derived from the spectra leads to the conclusion that the heterogeneity of protein structure is a fundamental limitation to the use of such spectral analysis methods. The underutilization of these data for prediction of secondary structure suggests spectral data could predict a more detailed descriptor.

Detection and characterization of triple-helical pyrimidine-purine-pyrimidine nucleic acids with vibrational circular dichroism.

Vibrational circular dichroism (VCD) spectra were measured in the C = O stretching region for poly(U)*poly(A).poly(U), poly(dT)*poly(dA).poly(dT), and poly(U)*poly(dA). poly(dT). These VCD spectra of the triple-helical structure were dramatically different from those of the corresponding duplexes. The VCD indicates that a very similar base-pair structure is present in these triplexes. The same sign pattern was found for poly(C+)*poly(I).poly(C), which implies a generality of structure than can result from the steric constraint of the triple helix conformation. By contrast, the corresponding duplexes are quite different in terms of their VCD. The transitions between triplex, duplex, and single-stranded forms were studied as a function of temperature and interpreted using factor analysis. The relative stabilities of the triplexes lie in the order RNA > DNA > hybrid. Nondegenerate dipole-coupling calculations for a U*A.U oligomer were carried out for the C = O stretching modes to model the spectral changes observed. The experimental absorbance spectra indicate that the bases have nonequivalent H-bonds which can be achieved if a reverse Hoogsteen base-pairing scheme is assumed. The computational VCD results with such a scheme were in better qualitative agreement with experiment than those using the expected Hoogsteen base-pairing scheme.

The dynamic behavior of annexin V as a function of calcium ion binding: a circular dichroism, UV absorption, and steady-state and time-resolved fluorescence study.

The binding of calcium ions to annexin V in the absence of phospholipids has been studied by UV-difference spectroscopy, circular dichroism, and steady-state and time-resolved fluorescence. In the absence of calcium, the unique tryptophan 187, located in domain III of annexin V, is surrounded by a strongly hydrophobic environment, as indicated by its "blue" fluorescence emission maximum (325 nm). This corresponds well with the description of the structure determined by X-ray crystallography of several crystal forms. The Trp187 time-resolved fluorescence decay shows the existence of a fast (picosecond) excited-state reaction which can involve the formation of an H-bond between the indole NH group and the proximate epsilon-OH and/or alpha-carbonyl groups of Thr224. Titration with calcium tends to stabilize the overall structure, as shown by circular dichroism, while leading to large modifications of the local structure around Trp187 making it accessible to the solvent as shown by UV-difference spectra, circular dichroism spectra, and the displacement of its fluorescence emission maximum at saturating concentrations of calcium (350 nm). A rapid (picosecond) formation of an excited-state complex, probably involving one or a few water molecules of the solvation shell, is observed. These observations correlate well with the conformational change observed in crystal structures obtained in high calcium concentrations, involving the removal of Trp187 from the buried position to the surface of the molecule [Sopkova, J., Renouard, M., & Lewit-Bentley, A. (1993) J. Mol. Biol. 234, 816-825; Concha, N. O., Head, J. F., Kaetzel, M. A., Dedman, J. R., & Seaton, B. A. (1993) Science 261, 1321-1324]. In the solvent-exposed conformation, the indole ring becomes mobile in the subnanosecond and nanosecond time range. This conformational change and the increase in local flexibility can be important for the accommodation of the protein on the surface of phospholipid membranes.

Empirical studies of protein secondary structure by vibrational circular dichroism and related techniques. Alpha-lactalbumin and lysozyme as examples.

Vibrational circular dichroism (VCD) has been shown to be sensitive to secondary structure in proteins and peptides and has been used as the basis for quantitative secondary-structure-prediction algorithms. However, the accuracy of these algorithms is not matched by the apparent qualitative sensitivity of the VCD spectra. This report provides examples of the use of VCD to follow structural change spectrally and to clarify the qualitative nature of the structural changes underlying the spectral variation. The VCD spectra and the complementary UV electronic CD (ECD) and FTIR spectra of alpha-lactalbumin (LA) have been studied as a function of pH, denaturation, Ca2+ ion and solvent conditions for several species. Spectral data for lysozyme were compared with those of LA because of their very similar crystal structures. In fact, these proteins in D2O-based pH 7 solution have quite different spectra using these optical techniques. Even for the LA proteins, the human differs from the bovine and goat species. Furthermore, under low pH conditions, where the LAs are in a reversibly denatured, molten globule form, the spectra are more similar, species variation is minimal and the spectral differences from lysozyme are in fact smaller. Our data are consistent with native, pH 7, alpha-lactalbumin having a less well organized structure than lysozyme, possibly in a dynamic sense. Conversely, in the low-pH, molten globule form of LA, tertiary structure is lost which could relax constraints that might distort the helical segments in the native form. The differences between the interpretation of our results and those from X-ray and NMR data may be due to motional sampling of various geometries in LA which all contribute to the spectral signatures seen in optical spectra but whose contributions are washed out in NMR or frozen out in the crystal structure. Part of this flexibility may relate to the rather large 3(10)-helical content in the LA protein structure. Fluctionality may have specific functional effects, perhaps allowing LA to bind better to beta-galactosyl transferase and form the biologically active lactose synthetase complex.

Quantitative analysis of vibrational circular dichroism spectra of proteins. Problems and perspectives.

Experimental and computational aspects of the quantitative analysis of vibrational circular dichroism (VCD) of proteins are discussed. Experimentally, the effect of spectral resolution, sample concentration, cell selection and spectral normalization effects are considered. The influence of random intensity variations on the results of quantitative analysis of amide I' VCD are shown to be minor up to a 15% variation in spectral intensity. A computational algorithm, based on factor analysis of the spectra and multiple linear regression calculation of fractions of secondary structures (FC), was designed to analyse quantitatively the details of the VCD spectra-structure relationship. It also enabled the results of VCD measured independently for the amide I' and amide II regions to be combined. Our study is based primarily on the optimization of the calculation to predict FC values for proteins not included in the reference data set used for regression. The best prediction is obtained with the function using only part of the observable independent VCD spectral components. Inclusion of all components actually reduces the prediction accuracy of the analysis. Spectroscopic reasons for such behaviour and the consequences of the interdependence of the crystallographic FC values on the spectra-structure analysis are discussed. Finally, the possibility of utilizing VCD spectra to obtain quantitative structural information about the protein beyond the conventional secondary structure composition is explored. A matrix descriptor of super-secondary structure features for proteins is designed, and preliminary results for prediction of this descriptor from amide I' VCD spectra are presented. These latter calculations use a novel design of the back-propagation neural network.

Spectroscopic study of the temperature-dependent conformation of glucoamylase.

Vibrational circular dichroism, electronic circular dichroism and infrared absorption with Fourier self-deconvolution have been used for a conformational study of the small form, G2, of glucoamylase, 1,4-alpha-D-glucan glucohydrolase from Aspergillus niger (EC 3.2.1.3) in aqueous solution. From the temperature dependence of spectra measured from 25 degrees C to 60 degrees C it was seen that the helical content is relatively constant to 50 degrees C and then sharply decreases by a factor of more than three by 60 degrees C. This decrease in helix is primarily compensated by a rise in the fraction of beta-sheet; but bend, turn and 'other' components also increase. By comparison of the three techniques, it was determined that the electronic CD analysis was quantitatively in error due to interference by glycosidic residues. The inherent resolution of the vibrational techniques, FTIR and VCD, avoids such interference.

Frequency analysis of infrared absorption and vibrational circular dichroism of proteins in D2O solution.

The IR absorption frequencies as derived from second derivatives of the Fourier transform IR spectra of the amide I' bands of globular proteins in D2O are compared to those obtained from band fitting of the vibrational circular dichroism (VCD) spectra. The two sets of frequencies are in very good agreement, yielding consistent ranges where amide I' VCD and IR features occur. Use of VCD to complement the IR allows one to add sign information to the frequency information so that features occurring in the overlapping frequency ranges that might arise from different secondary structures can be better discriminated. From this comparison, it is clear that correlation just of the frequency of a given IR transition to secondary structure can lead to a nonunique solution. Different sign patterns were identified for correlated groups of globular proteins in restricted frequency ranges that have been previously assigned to defined secondary structural elements. Hence, different secondary structural elements must contribute band components to a given frequency range.

Vibrational circular dichroism studies of epidermal growth factor and basic fibroblast growth factor.

Vibrational circular dichroism (VCD) studies are reported for two unrelated recombinant growth factor proteins: epidermal growth factor and basic fibroblast growth factor (bFGF). NMR, electronic CD, and bFGF X-ray studies indicate that these two proteins are primarily composed of beta-sheet and loop secondary structure elements with no detectable alpha-helices. Two reports on solution conformation of these proteins using FTIR absorption spectroscopy with subsequent resolution enhancement confirmed the presence of a large fraction of a beta-sheet conformation but in addition indicated the presence of large absorption bands in the 1650-1656 cm-1 region, which are typically assigned to alpha-helices. The VCD spectra of both proteins have band shapes that strongly resemble those of other high beta-sheet fraction proteins, such as the trypsin family of proteins. Quantitative analysis of the VCD spectra also indicates that these proteins are predominantly in beta-sheet and extended ("other") conformations with very little alpha-helix fraction. These results agree with the CD interpretation and affirm that the FTIR peaks in the region 1650-1656 cm-1 can be assigned to loops. This study provides an example of the limitations of using FTIR frequencies alone for examination of protein secondary structure.

Relationships between secondary structure fractions for globular proteins. Neural network analyses of crystallographic data sets.

The relationship between the fractions of protein secondary structural components as determined from X-ray crystallographic data by the procedures of Kabsch and Sander (KS) and of Levitt and Greer (LG) is analyzed by neural network analysis of these two tabulations of literature data. A linear relationship between the KS and LG reductions of X-ray data to secondary structure descriptors is demonstrated by a regression analysis of the relationships between these sets of structural parameters. Back-propagation neural network analysis was then used to derive equations for determination of the most probable fractions of beta-sheet, bend, turn, and "other" conformations given the fraction of alpha-helix in a globular protein. The deviation of the X-ray values for beta-sheet from that determined with these equations was shown to have a variance that exponentially decreased with increasing fraction of alpha-helix. A second neural network analysis showed that knowledge of both the alpha-helical and beta-sheet fractions in a protein significantly reduces the uncertainty in prediction of the other components of the secondary structure. These analyses provide insight into the nature of the data sets derived from crystal structures. Since these complications of crystal structure data are commonly used as reference information for quantitative evaluation of spectra (for example, FTIR, Raman, and electronic or vibrational circular dichroism) in terms of secondary structure, such internal correlations in the reference sets may have significant effects on the stability of spectroscopic analyses derived from them.