Secondary structure perturbations in salt-induced protein precipitates

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na₂SO₄, were studied for twelve different proteins. α-helix and β-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of β-sheet structures with a concomitant loss of α-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of α-helix and β-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting β-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in α-helix content were influenced by the initial helical content and the dipole moment. The enhanced β-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.     

The Structure of Physarum polycephalum hemagglutinin I suggests a minimal carbohydrate recognition domain of legume lectin fold

Physarum polycephalum hemagglutinin I (HA1) is a 104-residue protein that is secreted to extracellular space. The crystal structure of HA1 has a β-sandwich fold found among lectin structures, such as legume lectins and galectins. Interestingly, the β-sandwich of HA1 lacks a jelly roll motif and is essentially composed of two simple up-and-down β-sheets. This up-and-down β-sheet motif is well conserved in other legume lectin-like proteins derived from animals, plants, bacteria, and viruses. It is more noteworthy that the up-and-down β-sheet motif includes many residues that make contact with the target carbohydrates. Our NMR data demonstrate that HA1 lacking a jelly roll motif also binds to its target glycopeptide. Taken together, these data show that the up-and-down β-sheet motif provides a fundamental scaffold for the binding of legume lectin-like proteins to the target carbohydrates, and the structure of HA1 suggests a minimal carbohydrate recognition domain.     

Vibrational analysis of peptides,polypeptides, and proteins. V. Normal vibrations of β-turns

The normal modes have been calculated for β-turns of types I, II, III, I′, II′, and III′. The complete set of frequencies is given for the first three structures; only the amide I, II, and III modes are given for the latter three structures. Calculations have been done for structures with standard dihedral angles, as well as for structures whose dihedral angles differ from these by amounts found in protein structures. The force field was that refined in our previous work on polypeptides. Transition dipole coupling was included, and is crucial to predicting frequency splittings in the amide I and amide II modes. The results show that in the amide I region, β-turn frequencies can overlap with those of the α-helix and β-sheet structures, and therefore caution must be exercised in the interpretation of protein bands in this region. The amide III modes of β-turns are predicted at significantly higher frequencies than those of α-helix and β-sheet structures, and this region therefore provides the best possibility of identifying β-turn structures. Amide V frequencies of β-turns may also be distinctive for such structures.     

The conformational analysis of peptides using fourier transform IR spectroscopy

Fourier transform infrared spectroscopy (FTIR) can be used for conformational analysis of peptides in a wide range of environments. Measurements can be performed in aqueous solution, organic solvents, detergent micelles as well as in phospholipid membranes. Information on the secondary structure of peptides can be derived from the analysis of the strong amide I band. Orientation of secondary structural elements within a lipid bilayer matrix can be determined by means of polarized attenuated total reflectance–FTIR spectroscopy. Hydrogen–deuterium exchange can be monitored by the analysis of the, amide II band. This review gives some example of peptide systems studied by FTIR spectroscopy. Studies on alamethicin and α-aminoisobutyric acid containing peptides have shown that FTIR spectroscopy is a sensitive tool for identifying 3₁₀-helical structures. Changes in the structure of the magainins upon interaction with charged lipids were detected using FTIR spectroscopy. Tachyplesin is an example of a β-sheet containing membrane active peptide. Polarized ir spectroscopy reveals that the antiparallel β-sheet structures of tachyplesin are oriented parallel to the membrane surface. Synthesis of peptides corresponding to functionally/structurally important regions of large proteins is becoming increasingly popular. FTIR spectroscopy has been used to analyze the structure of synthetic peptides corresponding to the ion-selective pore of the voltage-gated potassium channel. In biomembrane systems these peptides adopt a highly helical structure. Under conditions, where these peptides are aggregated the presence of some intermolecular β-sheet structure can also be detected. © 1994 John Wiley & Sons, Inc.     

Stoichiometry and Preferential Interaction: Two Components of the Principle for Protein Structure Organization

Abstract

The effect of pressure on the conformational structure of amyloid β (1–40) peptide (Aβ(1–40)), exacerbated with or without temperature, was determined by Fourier transform infrared (FT-IR) microspectroscopy. The result indicates the shift of the maximum peak of amide I band of intact solid Aβ(1–40) from 1655 cm^?1 (α-helix) to 1647–1643 cm^?1 (random coil) with the increase of the mechanical pressure. A new peak at 1634 cm^?1 assigned to β-antipar- allel sheet structure was also evident. Furthermore, the peak at 1540 cm^?1 also shifted to 1527 (1529) cm^?1 in amide II band. The former was assigned to the combination of α-helix and random coil structures, and the latter was due to β-sheet structure. Changes in the composition of each component in the deconvoluted and curve-fitted amide I band of the compressed Aβ(1–40) samples were obtained from 33% to 22% for α-helix/random coil structures and from 47% to 57% for β-sheet structure with the increase of pressure, respectively. This demonstrates that pressure might induce the conformational transition from α-helix to random coil and to β-sheet structure. The structural transformation of the compressed Aβ(1–40) samples was synergistically influenced by the combined effects of pressure and temperature. The thermal-induced formation of β-sheet structure was significantly dependent on the pressures applied. The smaller the pressure applied the faster the β-sheet structure transformed. The thermal-dependent transition temperatures of solid Aβ(1–40) prepared by different pressures were near 55–60 °C.     

Vibrational circular dichroism spectra of three conformationally distinct states and an unordered state of poly(L-lysine) in deuterated aqueous solution

Fourier transform ir vibrational circular dichroism (VCD) spectra in the amide I′ region of poly(L-lysine) in D₂O solutions have confirmed the existence of three distinct conformational states and an unordered conformational state in this homopolypeptide. Characteristic VCD spectra are presented for the right-handed α-helix, the antiparallel β-sheet, an extended helix conformation previously referred to as the so-called “random coil,” and a completely unordered conformation characterized by the absence of any amide I′ VCD. VCD for the antiparallel β-sheet in solution and the unordered chain conformation are presented for the first time. Each of the four different VCD spectra is unique in appearance and lends weight to the view that VCD has the potential to become a sensitive new probe of the secondary structure of proteins in solution.     

Determination of secondary structure of normal fibrin from human peripheral blood

The secondary structure of human fibrin from normal donors and from bovine and suilline plasma was studied by Fourier transform ir spectroscopy and a quantitative analysis of its secondary structure was suggested. For this purpose, a previously experimented spectrum deconvolution procedure based on the use of the Conjugate Gradient Minimisation Algorithm with the addition of suitable constraints was applied to the analysis of conformation-sensitive amide bands. This procedure was applied to amide I and III analysis of bovine and suilline fibrin, obtained industrially, and to amide III analysis of human fibrin clots. The analysis of both amide I and III in the first case was useful in order to test the reliability of the method. We found bovine, suilline, and human fibrin to contain about 30% α-helix (amide I and III components at 1653 cm⁻¹, and 1312 and 1284 cm⁻¹, respectively), 40% β-sheets (amide I and III components at 1625 and 1231 cm⁻¹, respectively) and 30% turns (amide I and III components at 1696, 1680, 1675 cm⁻¹, and 1249 cm⁻¹, respectively). The precision of the quantitative determination depends on the amount of these structures in the protein. Particularly, the coefficient of variation is < 10% for percentage values of amide I and III components > 15 and 5%, respectively. The good agreement of our quantitative data, obtained separately by amide I and amide III analysis, and consistent with a previous fibrinogen (from commercial sources) study that reports only information about fibrin β-sheet content obtained by factor analysis, leads us to believe that the amounts of secondary structures found (α-helix, β-sheets, and turns) are accurate. © 1997 John Wiley & Sons, Inc. Biopoly 41: 545–553, 1997.     

Determination of the secondary structure of proteins from the amide I band of the laser Raman spectrum

The amide I band in the laser Raman spectrum of proteins has been resolved into six components, each representing residues in a different type of secondary structure. These structure types are ordered or bihydrogen-bonded helix (believed to be located in the center of helical segments), disordered or monohydrogen-bonded helix (believed to be located at the ends of helical segments), antiparallel beta sheet, parallel beta sheet, reverse turn, and undefined. The Raman spectrum representing 100% of each type of residue conformation has been computed from the solvent-subtracted Raman spectra of ten proteins with known secondary structure, plus poly-l-lysine using a least-squares solution of the overdetermined system of equations. Linear combinations of these reference spectra were then fitted to the experimental amide I spectra of these and other proteins to estimate the fractions of residues in these conformations. Statistical tests suggest that the discrimination between bihydrogen-bonded helix and monohydrogen-bonded helix is significant as is the discrimination between parallel and antiparallel β-sheet. However, the discrimination between random structure and turns has not yet been accomplished by these studies. The absolute difference between X-ray and Raman estimates of structure for 17 protein samples is generally less than 6%. We conclude that detailed and reasonably accurate estimates of secondary structure can be derived from the amide I spectra of proteins.     

Determination of the secondary structure of isomeric forms of human serum albumin by a particular frequency deconvolution procedure applied to fourier transform IR analysis

A new deconvolution procedure was applied to the analysis of Fourier transform in spectra of human serum albumin secondary structure in the native state and in states denatured by heat and acid treatment. The deconvolution method is based on the use of the Conjugate Gradient Minimization Algorithm, with the addition of suitable constraints directly obtained by the application to the measured spectrum of the second derivative operator. This method computes central band frequency, bandwidth, and amplitude of the different spectral components of conformation-sensitive amide bands. In the specific case, it was applied to analysis of the amide I band, and the quantitative determination of the different secondary structures (α-helix, β-sheet, β-turns, and random) was attempted for all the samples examined. The precision of the quantitative determination depends on the amounts of these structures present in the protein. The coefficient of variation is <10% for values of amide I component >15%. The accuracy was tested by comparing, by means of linear regression, the results obtained for human serum albumin, hemoglobin, α-chymotrypsin, and cytochrome c, using our method, with those obtained by x-ray crystallography and CD; the results obtained by other vibrational spectroscopic approaches were also compared. The fit standard error between x-ray and ir secondary structure values estimated by our method is 2.5% for α-helix, 7.16% for β structures, and 5.1% for other structures (turns and random coils). Quantitative results are given for the secondary structures (α-helix, turns, and β-strands) present in the native state (turns and β-strands up to now unknown in aqueous solution), together with the percentages of these structures and additional ones (random coils and β-sheets) formed during denaturization. © 1996 John Wiley & Sons, Inc.     

Characterization of β-turns in cyclic hexapeptides in solution by fourier transform IR spectroscopy

The β-turn represents a structural element frequently encountered in globular proteins. However, in spite of various theoretical and experimental studies the ir signature bands of pure β-turns are still not established beyond doubt. Although considerable information exists now on the ir spectra of β-helical and β-sheet structures, the lack of knowledge concerning turn structures in general, and that of β-turns in particular, presents a major uncertainty in the estimation of global protein secondary structures from ir spectroscopic data. To obtain more specific information about the characteristic amide bands in β-turns, we report herein an ir spectroscopic analysis of a series of five cyclic pseudo-hexapeptides known to form β-turns from previous CD and nmr studies [A. Perczel, M. Hollósi, B. M. Foxman, and G. D. Fasman (1991) Journal of the American Chemical Society, Volume 113, pp. 9772-9784 ]. We show here that in these cyclic peptides the amide groups involved in β-turns that comprise a ten-membered hydrogen-bonded ring (and represent the first H-bond pair in a β-sheet), give rise to characteristic amide I bands in the range 1638–1646 cm^?1, with the exact position depending on the solvent and the nature of the side-chain substituents. © 1993 John Wiley & Sons, Inc.     

Raman study of crystalline peptides containing beta turns

The Raman spectra of crystalline H-ProLeuGlyNH₂ which has a type II β turn, crystalline S-benzylCysProLeuGlyNH₂ which has a type I β-turn, and crystalline gramicidin S which has two β turns and β-sheet structure in its conformation, were investigated. The amide I and amide III bands of the peptides with β turns were generally different from those which are diagnostic for α-helix and β-sheet conformations. The patterns of the amide I and amide III bands, when examined together, indicate that Raman spectra can provide diagnostic evidence for β-turn structure in peptides.     

Dissociation of β-Sheet Stacking of Amyloid β Fibrils by Irradiation of Intense,Short-Pulsed Mid-infrared Laser

Structure of amyloid β (Aβ) fibrils is rigidly stacked by β-sheet conformation, and the fibril state of Aβ is profoundly related to pathogenesis of Alzheimer’s disease (AD). Although mid-infrared light has been used for various biological researches, it has not yet been known whether the infrared light changes the fibril structure of Aβ. In this study, we tested the effect of irradiation of intense mid-infrared light from a free-electron laser (FEL) targeting the amide bond on the reduction of β-sheet content in Aβ fibrils. The FEL reduced entire contents of proteins exhibiting β-sheet structure in brain sections from AD model mice, as shown by synchrotron-radiation infrared microscopy analysis. Since Aβ_1-42 fibril absorbed a considerable FEL energy at amide I band (6.17 μm), we irradiated the FEL at 6.17 μm and found that β-sheet content of naked Aβ_1-42 fibril was decreased using infrared microscopic analysis. Consistent with the decrease in the β-sheet content, Congo-red signal is decreased after the irradiation to Aβ_1-42 fibril. Furthermore, electron microscopy analysis revealed that morphologies of the fibril and proto-fibril were largely changed after the irradiation. Thus, mid-infrared light dissociates β-sheet structure of Aβ fibrils, which justifies exploration of possible laser-based therapy for AD.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Raman scattering of chymotrypsinogen A, ribonuclease, and ovalbumin in the aqueous solution and solid state

The Raman spectra of three globular proteins, beef pancreas chymotrypsinogen A, beef pancreas ribonuclease, and hen egg white ovalbumin have been obtained in the solid state and aqueous solution. X-ray diffraction and circular dichroism evidence have indicated that these proteins have a low α-helical content and a large fraction of the residues in the unordered and β-sheet conformation. The frequencies and intensities of the amide I and amide III lines are consistent with assignments based on the Raman spectra of polypeptides. The intense amide III lines observed in all the spectra would be expected for proteins with a low fraction of the residues in the α-helical conformation. Several spectra changes occur upon dissolution of the proteins in water and may be associated with further hydration of the proteins. The spectrum of thermally denatured chymotrypsinogen is presented. A 3 cm^–1 decrease in the frequency of the amide I line of the protein dissolved in D₂O upon heating was observed. This observation is consistent with a denaturation mechanism allowing only slight changes in the secondary structure but an increase in solvent penetration upon going from the native to the reversibly denatured state.     

Flow-induced conformational changes and phase behavior of aqueous poly-L-lysine solutions

Poly-L -lysine exists as an α-helix at high pH and a random coil at neutral pH. When the α-helix is heated above 27°C, the macromolecule undergoes a conformational transition to a β-sheet. In this study, the stability of the secondary structure of poly-L -lysine in solutions subjected to shear flow, at temperatures below the α-helix to β-sheet transition temperature, were examined using Raman spectroscopy and CD. Solutions initially in the α-helical state showed time-dependent increases in viscosity with shearing, rising as much as an order of magnitude. Visual observation and turbidity measurements showed the formation of a gel-like phase under flow. Laser Raman measurements demonstrated the presence of small amounts of β-sheet structure evidenced by the amide I band at 1666 cm⁻¹. CD measurements indicated that solutions of predominantly α-helical conformation at 20°C transformed into 85% α-helix and 15% β-sheet after being sheared for 20 min. However, on continued shearing the content of β-sheet conformation decreased. The observed phenomena were explained in terms of a “zipping-up” molecular model based on flow enhanced hydrophobic interactions similar to that observed in gel-forming flexible polymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 239–246, 1998     

Trifluoroethanol-induced conformational change of tetrameric and monomeric soybean agglutinin: Role of structural organization and implication for protein folding and stability

2,2,2-Trifuoroethanol (TFE)-induced conformational structure change of a β-sheet legume lectin, soybean agglutinin (SBA) has been investigated employing its exclusive structural forms in quaternary (tetramer) and tertiary (monomer) states, by far- and near-UV CD, FTIR, fluorescence, low temperature phosphorescence and chemical modification. Far-UV CD results show that, for SBA tetramer, native atypical β-conformation transforms to a highly α-helical structure, with the helical content reaching 57% in 95% TFE. For SBA monomer, atypical β-sheet first converts to typical β-sheet at low TFE concentration (10%), which then leads to a nonnative α-helix at higher TFE concentration. From temperature-dependent studies (5–60 °C) of TFE perturbation, typical β-sheet structure appears to be less stable than atypical β-sheet and the induced helix entails reduced thermal stability. The heat induced transitions are reversible except for atypical to typical β-sheet conversion. FTIR results reveal a partial α-helix conversion at high protein concentration but with quantitative yield. However, aggregation is detected with FTIR at lower TFE concentration, which disappears in more TFE. Near-UV CD, fluorescence and phosphorescence studies imply the existence of an intermediate with native-like secondary and tertiary structure, which could be related to the dissociation of tetramer to monomer. This has been further supported by concentration dependent far-UV CD studies. Chemical modification with N-bromosuccinimide (NBS) shows that all six tryptophans per monomer are solvent-exposed in the induced α-helical conformation. These results may provide novel and important insights into the perturbed folding problem of SBA in particular, and β-sheet oligomeric proteins in general.     

Raman spectroscopic study of poly (β-benzyl-L-aspartate) and sequential polypeptides

Poly-β-benzyl-L -aspartate (poly[Asp(OBzl)]) forms either a lefthanded α-helix, β-sheet, ω-helix, or random coil under appropriate conditions. In this paper the Raman spectra of the above poly[Asp(OBzl)] conformations are compared. The Raman active amide I line shifts from 1663 cm^?1 to 1679 cm^?1 upon thermal conversion of poly[Asp(OBzl)] from the α-helical to β-sheet conformation while an intense line appearing at 890 cm^?1 in the spectrum of the α-helix decreases in intensity. The 890 cm^?1 line also displays weak intensity when the polymer is dissolved in chloroform–dichloroacetic acid solution and therefore is converted to the random coil. This line probably arises from a skeletal vibration and is expected to be conformationally sensitive. Similar behavior in the intensity of skeletal vibrations is discussed for other polypeptides undergoing conformational transitions. The Raman spectra of two cross-β-sheet copolypeptides, poly(Ala-Gly) and poly(Ser-Gly), are examined. These sequential polypeptides are model compounds for the crystalline regions of Bombyx mori silk fibroin which forms an extensive β-sheet structure. The amide I, III, and skeletal vibrations appeared in the Raman spectra of these polypeptides at the frequencies and intensities associated with β-sheet homopolypeptides. Since the sequential copolypeptides are intermediate in complexity between the homopolypeptides and the proteins, these results indicate that Raman structure–frequency correlations obtained from homopolypeptide studies can now be applied to protein spectra with greater confidence. The perturbation scheme developed by Krimm and Abe for explaining the frequency splitting of the amide I vibrations in β-sheet polyglycine is applied to poly(L -valine), poly-(Ala-Gly), poly(Ser-Gly), and poly[Asp(OBzl)]. The value of the “unperturbed” frequency, V₀, for poly[Asp(OBzl)] was significantly greater than the corresponding values for the other polypeptides. A structural origin for this difference may be displacement of adjacent hydrogen-bonded chains relative to the standard β-sheet conformation.     

Conformational analysis of a snake neurotoxin by prediction from sequence, circular dichroism, and raman spectroscopy.

The secondary structure of the major neurotoxin from the sea snake Lapemis hardwickii was investigated by several methods of conformational analysis: structure prediction, circular dichroism, and laser Raman spectroscopy. From the primary structure, secondary structure prediction yielded two regions of β-sheet structure at residues 1–7 and 41–45. β-Turns were predicted at residues 14–17, 20–23, 30–33, 37–40, and 46–49. From the predictions, the toxin appears to be composed of approximately 20% β-sheet and 33% β-turn. The CD spectrum of the native toxin appears to be a hybrid of model spectra for β-sheet and β-turn proteins. The pH perturbation studies on the toxin observed by CD demonstrated that the toxin is a very stable molecule except at extremely high or low pH values. The Raman data indicated that the toxin contains both antiparallel β-sheet and β-turn structure. Using two methods of secondary structure quantitation from Raman spectra the molecule was calculated to contain 35% β-sheet from one method and 27% from the other. Overall, the various methods demonstrate that the toxin is composed of β-sheet and β-turn structure with little or no α-helix present. From the comparison of these different techniques appreciation can be gained for the necessity of several methods when identifying and quantitating secondary structure.     

A distinct utility of the amide III infrared band for secondary structure estimation of aqueous protein solutions using partial least squares methods

Fourier transform infrared spectroscopy is becoming an increasingly important method to study protein secondary structure. The amide I region of the protein infrared spectrum is the widely used region, whereas the amide III region has been comparatively neglected due to its low signal. Since there is no water interference in the amide III region and, more importantly, the different secondary structures of proteins have more resolved differences in their amide III spectra, it is quite promising to use the amide III region to determine protein secondary structure. In our current study, a partial least squares (PLS) method was used to predict protein secondary structures from the protein IR spectra. The IR spectra of aqueous solutions of 16 different proteins of known crystal structure have been recorded, and the amide I, the amide III, and the amide I combined with the amide III region of these proteins were used to set up the calibration set for the PLS algorithm. Our results correlate quite well with the data from X-ray studies, and the prediction from the amide III region is better than that from amide I or combined amide I and amide III regions.     

A new approach to secondary structure evaluation: Secondary structure prediction of porcine adenylate kinase and yeast guanylate kinase by CD spectroscopy of overlapping synthetic peptide segments

A new approach for evaluating the secondary structure of proteins by CD spectroscopy of overlapping peptide segments is applied to porcine adenylate kinase (AK1) and yeast guanylate kinase (GK3). One hundred seventy-six peptide segments of a length of 15 residues, overlapping by 13 residues and covering the complete sequences of AK1 and GK3, were synthesized in order to evaluate their secondary structure composition by CD spectroscopy. The peptides were prepared by solid phase multiple peptide synthesis method using the 9-fluorenylmethoxycarbonyl/tert-butyl strategy. The individual peptide secondary structures were studied with CD spectroscopy in a mixture of 30% trifluoroethanol in phosphate buffer (pH 7) and subsequently compared with x-ray data of AK1 and GK3. Peptide segments that cover α-helical regions of the AK1 or GK3 sequence mainly showed CD spectra with increasing and decreasing Cotton effects that were typical for appearing and disappearing α-helical structures. For segments with dominating β-sheet conformation, however, the application of this method is limited due to the stability and clustering of β-sheet segments in solution and due to the difficult interpretation of random-coiled superimposed β-sheet CD signals. Nevertheless, the results of this method especially for α-helical segments are very impressive. All α-helical and 71% of the β-sheet containing regions of the AK1 and GK3 could be identified. Moreover, it was shown that CD spectra of consecutive peptide content reveal the appearance and disappearance of α-helical secondary structure elements and help localizing them on the sequence string. © 1997 John Wiley & Sons, Inc. Biopoly 41: 213–231, 1997