Conformational study of sequential Lys and Leu based polymers and oligomers using vibrational and electronic CD spectra

Vibrational CD (VCD) and electronic CD (ECD) spectra of some sequential Lys and Leu based oligo- and polypeptides were studied as a function of added salt and (for ECD) as a function of concentration in aqueous solution. For these samples, the VCD spectra can only be measured at relatively high concentrations under which the well-known salt-induced transition to a β-sheet form can be observed for the KL based species, but only the end-state α-helical conformation is obvious for the LKKL based samples. ECD concentration dependence demonstrates that, at high concentration with no added or with added salt, LKKL based oligomers and polymers give α-helical spectra. These data provide evidence of aggregation induced secondary structure formation in an exceptionally simple peptide system. Similarly, the KL based oligomers and polymers give β-sheet like spectra at high concentration or at high salt. These systems further provide model systems under “normal” aqueous conditions that yield VCD band shapes that correlate to the major secondary structural types of polypeptides. They are in substantial agreement with those spectra obtained on homopolypeptides and on proteins, confirming the relative independence of the VCD technique from side-chain and solvent effects. © 1994 John Wiley & Sons, Inc.     

Vibrational circular dichroism of polypeptides. II. Solution amide II and deuteration results

Vibrational CD (VCD) of amide I and II vibrations of several α-helical polypeptides have been measured in solution. For the amide II as well as the amide I [previously published: Lal, B.B. & Nafie, L.A. (1982) Biopolymers 21 , 2161] we find the VCD to be characteristic of the polypeptide secondary structure. Amide II bands of right-handed α helices were all found to have negative VCD and to have their maximum rotational strength for the parallel (low-energy) component. However, left-handed α helices formed from L -amino acids gave positive amide II bands at higher frequencies than found for the right-handed helices, indicating that the VCD was sensitive to the stereochemical difference. The amide-I VCD spectra of some deuterated right-handed α-helical polypeptides have a new negative feature to low frequency that does not reflect theoretical predictions but also appears to be stereochemically sensitive. Amide-II and amide-A VCD of a few deuterated polypeptides imply retention of the secondary-structure-dependent characteristics seen in the hydrogenated VCD.     

Interaction of amphipathic polypeptides with phospholipids: characterization of conformations and the CD of oriented beta-sheets

The effects of interaction with phospholipids on the conformation of the alternating copolymer, poly(Leu-Lys), and the random copolymer poly(Leu⁵⁰, Lys⁵⁰) have been investigated by CD and ir spectroscopy. Poly(Leu-Lys) undergoes a partial unordered → β-sheet transition in solution in the presence of lysolecithin. On addition of lysolecithin plus cholate, an unordered → α -helix transition is observed. In films deposited from these solutions, poly(Leu-Lys) adopts the anti-parallel β-sheet conformation, as in aqueous solutions at moderate ionic strength. Polarized ir spectra showed that the plane of the β-sheet in such films deviates from the plane of the film by no more than 14°. The random copolymer, poly(Leu⁵⁰, Lys⁵⁰), is α-helical in the presence of lysolecithin and lysolecithin plus cholate, regardless of whether the sample is a solution or a film. CD measurements on the poly(Leu-Lys) films provide information about the component of the CD tensor for light propagating normal to the plane of the β-sheet. These measurements show (1) a negative n → π* CD band (214 nm maximum) with higher intensity than the average CD for isotropic solution; and (2) a positive band in the π → π* region (195 nm maximum), which is weaker than that in the isotropic spectrum.     

The influence of nearest-neighboring amino acid residues on aspects of secondary structure of proteins. Attempts to locate -helices and -sheets

The influence of nearest-neighbor pairs of amino acids (n ? 1) and (n + 1) on the conformation of amino acid (n) in proteins has been studied. From experimental data on eleven proteins of known three dimensional structures, our definition of an α-helical domain in the Φ,Ψ plot has been reexamined and found to be satisfactory. On the same principle, a regular β-sheet domain has been delineated. We then revised our 20 × 20 table of frequencies of occurrences of various conformations tabulating three values: α-helical, β-sheet, and neither. These frequencies were then used to locate the helixbreaking positions in cytochrome b₅, papain, thermolysin, and calcium-binding protein. In conjuction with the helical wheel method, they were useful for predicting the locations of most α-helical segments. Similarly the β-sheet breaking positions in papain were located and most of the β-sheets found by X-ray diffraction were close to or between these positions. Data on β-sheets are extremely sparse so that extensive tests were not possible. The application of this method to abnormal hemoglobins suggested possible distortions of helices and in several instances correlated with abnormal properties of the hemoglobins and association with disease. The variable region of human immunoglobin heavy chains was found to have a very low α-helical content though β-sheet structures might exist.     

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.     

Special Interaction of Anionic Phosphatidic Acid Promotes High Secondary Structure in Tetrameric Potassium Channel

Anionic phosphatidic acid (PA) has been shown to stabilize and bind stronger than phosphatidylglycerol via electrostatic and hydrogen bond interaction with the positively charged residues of potassium channel KcsA. However, the effects of these lipids on KcsA folding or secondary structure are not clear. In this study, the secondary structure analyses of KcsA potassium channel was carried out using circular dichroism spectroscopy. It was found that PA interaction leads to increases in α-helical and β-sheet content of KcsA protein. In PA, KcsA α-helical structure was further stabilized by classical membrane-active cosolvent trifluoroethanol followed by reduction in the β-sheet content indicating cooperative transformation from the β-sheet to an α-helical structure. The data further uncover the role of anionic PA in KcsA folding and provide mechanism by which strong hydrogen bonds/electrostatic interaction among PA headgroup and basic residues on lipid binding domains may induce high helical structure thereby altering the protein folding and increasing the stability of tetrameric assembly.     

A circumventing role for the non-native intermediate in the folding of β-lactoglobulin

Folding experiments have suggested that some proteins have kinetic intermediates with a non-native structure. A simple G ?o model does not explain such non-native intermediates. Therefore, the folding energy landscape of proteins with non-native intermediates should have characteristic properties. To identify such properties, we investigated the folding of bovine β-lactoglobulin (βLG). This protein has an intermediate with a non-native α-helical structure, although its native form is predominantly composed of β-structure. In this study, we prepared mutants whose α-helical and β-sheet propensities are modified and observed their folding using a stopped-flow circular dichroism apparatus. One interesting finding was that E44L, whose β-sheet propensity was increased, showed a folding intermediate with an amount of β-structure similar to that of the wild type, though its folding took longer. Thus, the intermediate seems to be a trapped intermediate. The high α-helical propensity of the wild-type sequence likely causes the folding pathway to circumvent such time-consuming intermediates. We propose that the role of the non-native intermediate is to control the pathway at the beginning of the folding reaction.     

Secondary structural changes in the intact and the disulfide Bridges cleaved β-lactoglobulin A and B in solutions of urea,guanidine hydrochloride,and sodium dodecyl sulfate

The relative proportions of α-helix, β-sheet, and unordered form in β-lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of β-lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% α-helix and 41% β-sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased β-sheet up to 48% but did not affect the α-helical proportion. The α-helical proportions of nonreduced β-lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the α-helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The β-sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

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.     

Analysis of Interactions of Buried Polar Side Chains in β-Proteins

Qualitative and quantitative analysis of polar side chains inaccessible to water molecules, as well as their interactions in 100 globular β-sheet proteins, was performed. It was shown that completely buried polar side chains are widespread in β-proteins, with their vast majority being involved in side chain-side chain or side chain-main chain interactions. An analysis of frequency of occurrence of different side chain-partner pairs demonstrated that these interactions are selective. The results were compared with similar data obtained earlier for α-helical proteins.     

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     

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     

Identification and removal of proteins that co-purify with infectious prion protein improves the analysis of its secondary structure

Prion diseases are neurodegenerative disorders associated with the accumulation of an abnormal isoform of the mammalian prion protein (PrP). Fourier transform infrared spectroscopy (FTIR) has previously been used to show that the conformation of aggregated, infectious PrP (PrP(Sc) ) varies between prion strains and these unique conformations may determine strain-specific disease phenotypes. However, the relative amounts of α-helix, β-sheet and other secondary structures have not always been consistent between studies, suggesting that other proteins might be confounding the analysis of PrP(Sc) secondary structure. We have used FTIR and LC-MS/MS to analyze enriched PrP(Sc) from mouse and hamster prion strains both before and after the removal of protein contaminants that commonly co-purify with PrP(Sc) . Our data show that non-PrP proteins do contribute to absorbances that have been associated with α-helical, loop, turn and β-sheet structures attributed to PrP(Sc) . The major contaminant, the α-helical protein ferritin, absorbs strongly at 1652 cm(-1) in the FTIR spectrum associated with PrP(Sc) . However, even the removal of more than 99% of the ferritin from PrP(Sc) did not completely abolish absorbance at 1652 cm(-1) . Our results show that contaminating proteins alter the FTIR spectrum attributed to PrP(Sc) and suggest that the α-helical, loop/turn and β-sheet secondary structure that remains following their removal are derived from PrP(Sc) itself.     

Concentration-dependent transitions govern the subcellular localization of islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is a peptide hormone cosecreted with insulin by pancreatic β-cells. In type II diabetes, IAPP aggregates in a process that is associated with β-cell dysfunction and loss of β-cell mass. The relationship between IAPP's conformational landscape and its capacity to mediate cell death remains poorly understood. We have addressed these unknowns by comparing the cytotoxic effects of sequence variants with differing α-helical and amyloid propensities. IAPP was previously shown to oligomerize cooperatively on binding to lipid bilayers. Here, comparable transitions are evident in cell culture and are associated with a change in subcellular localization to the mitochondria under toxic conditions. Notably, we find that this toxic gain of function maps to IAPP's capacity to adopt aggregated membrane-bound α-helical, and not β-sheet, states. Our findings suggest that upon α-helical mediated oligomerization, IAPP acquires cell-penetrating peptide (CPP) properties, facilitating access to the mitochondrial compartment, resulting in its dysfunction.     

Contents to volume 178

UV CD and IR spectra of the water-soluble bacteriochlorophyll-protein antenna isolated from Prosthecochloris aestuarii indicate that about 50% of the protein is in a β-sheet conformation while for the dominant antenna complexes isolated from bacteria (B800-850) and from green plants (LHC), the α-helix (45%) is more abundant than the β-sheet (~ 10%) conformation. Furthermore, IR dichroism studies show that the α-helical segments of a large variety of intrinsic membrane Chl-protein complexes (antenna and reaction centers) are tilted on the average at 30–35° away from the membrane normal. The observation that in these complexes the Chl planes are also tilted at about the same angle suggests that the transmembrane orientation of the α-helices determines the positioning of the Chl molecules in photosynthetic membranes.     

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.     

The surface of β-sheet proteins contains amphiphilic regions which may provide clues about protein folding

A major bottleneck in the field of biochemistry is our limited understanding of the processes by which a protein folds into its native conformation. Much of the work on this issue has focused on the conserved core of the folded protein. However, one might imagine that a ubiquitous motif for unaided folding or for the recognition of chaperones may involve regions on the surface of the native structure. We explore this possibility by an analysis of the spatial distribution of regions with amphiphilic α-helical potential on the surface of β-sheet proteins. All proteins, Including β-sheet proteins, contain regions with amphiphilic α-helical potential. That is, any α-helix formed by that region would be amphiphilic, having both hydrophobic and hydrophilic surfaces. In the three-dimensional structure of all β-sheet proteins analyzed, we have found a distinct pattern in the spatial distribution of sequences with amphiphilic α-helical potential. The amphiphilic regions occur in ring shaped clusters approximately 20 to 30 Å in diameter on the surface of the protein. In addition, these regions have a strong preference for positively charged amino acids and a lower preference for residues not favorable to α-helix formation. Although the purpose of these amphiphilic regions which are not associated with naturally occurring α-helix is unknown, they may play a critical role in highly conserved processes such as protein folding. © 1996 Wiley-Liss, Inc.     

Secondary structure of proteins associated in thin films

The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L -lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy¹ and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all α-helical myoglobin, decreased the molar ellipticity of the α/β RNase A, and increased the molar ellipticity of all-β Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less α-helix and more β-sheet structure. The results suggest that the protein α-helices are less stable in films and that the secondary structure may rearrange into β-sheets at high temperature. Two heterooligomeric peptides and poly (L -lysine), all in solution at neutral pH with “random coil” conformation, formed films with variable degrees of their secondary structure in β-sheets or β-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a “random” structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable α-helix or β-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent. © 1993 John Wiley & Sons, Inc.     

Vibrational circular dichroism of polypeptides. IV. Film studies of L-alanine homo-oligopeptides

Vibrational CD (VCD) and ir absorption data are reported for a series of films of Boc-(L -Ala)_n-OMe homo-oligopeptides (n = 3–7) in the amide I and A regions. The data evidenced a sharp change between n = 3 and n = 4, which parallels the onset of β-structure formation, and another between n = 5 and n = 6, which parallels the full development of β-structure. This represents the first report of the application of VCD to oligopeptide conformation. The data resembled earlier reported film VCD studies of higher-molecular-weight polypeptides of known β-structure.     

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.