Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy.

A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.     

Early stages of LDL oxidation: apolipoprotein B structural changes monitored by infrared spectroscopy

Changes in the conformation of apoliprotein B-100 in the early stages of copper-mediated low density lipoprotein oxidation have been monitored by infrared spectroscopy. During the lag phase no variation in structure is observed, indicating that copper binding to the protein does not significantly affect its structure. In the propagation phase, while hydroperoxides are formed but the protein is not modified, no changes in secondary structure are observed, but the thermal profile of the band corresponding to alpha-helix is displaced in frequency, indicating changes in tertiary structure associated with this conformation but not with beta-sheet components. When aldehyde formation starts, a decrease of approximately 3% in the area of bands corresponding to alpha-helix and beta-sheet is produced, concomitantly with an increase in beta-turns and unordered structure. The two bands corresponding to beta-turns vary as well under these conditions, indicating changes in these structures. Also at this stage the thermal profile shows variations in frequency for the bands corresponding to both alpha-helix and beta-sheet.The results are consistent with the hypothesis that as soon as the polyunsaturated fatty acids from the particle core are modified, this change is reflected at the surface, in the alpha-helical components contacting the monolayer.     

Secondary structures of rat lipolytic enzymes: circular dichroism studies and relation to hydrophobic moments

To explore the secondary structures of lingual and pancreatic lipases, circular dichroism measurements were performed. Maximum average ellipticities were used to calculate the percentage of alpha-helices, beta-sheets, and random coils. Lingual lipase had an ellipticity of -20235 +/- 140 deg cm2/dmol (mean +/- SE) at 220 nm suggesting 60% alpha-helix, 20% beta-sheet and 20% random coil structure, but the mean ellipticity for pancreatic lipase was -14093 +/- 82 deg cm2/dmol (mean +/- SE) at 210 nm suggesting a 34.8% alpha-helical, 25% beta-sheet and 40% random coil secondary structure. An alpha-helical stretch of residues with a large hydrophobic moment ("globular" alpha-helix by hydrophobic moment plot) from amino acids 382 through 389 at the COOH-terminal end of lingual lipase was noted. This sequence, absent in pancreatic lipase, may account for the avid binding of lingual lipase to fat emulsion particles.     

The conformation of T4 bacteriophage dihydrofolate reductase from circular dichroism

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

Transient non-native secondary structures during the refolding of alpha-lactalbumin detected by infrared spectroscopy

Stopped-flow Fourier-transform infrared spectroscopy (SF-FTIR) was used to identify native as well as non-native secondary structures during the refolding of the calcium-binding protein alpha-lactalbumin. Infrared absorbance spectra were recorded in real time after a pH jump induced refolding of the protein. In the presence of calcium, the refolding is fast with concerted appearance of secondary structures; in its absence, folding is much slower and intricate, with transient formation and disappearance of non-native beta-sheet. The possibility of detecting native as well as non-native structures at the same time is especially valuable in providing insight into the complexity of the refolding process of a protein.     

Secondary structure and dosage of soluble and membrane proteins by attenuated total reflection Fourier-transform infrared spectroscopy on hydrated films

Attenuated total reflection Fourier-transform infrared spectroscopy of thin hydrated films of soluble and membrane protein included in a phospholipid bilayer is shown to provide useful information as to the secondary structure of the protein. The analysis of the amide I band of deuterated samples by Fourier self-deconvolution followed by a curve fitting was performed by a new procedure in which all the input parameters are generated by the computer rather than by the investigator. The results of this analysis provide a correct estimation of the alpha-helix and beta-sheet structure content with a standard deviation of 8.6% when X-ray structures are taken as a reference. We also show that the orientation of the different secondary structures resolved by the Fourier self-deconvolution/curve-fitting procedure and of the phospholipid acyl chains can be simultaneously evaluated for membrane proteins reconstituted in a lipid bilayer. Of special interest for reconstitution of membrane proteins, the lipid/protein ratio can be accurately and quickly determined from the infrared spectrum.     

Structural changes and aggregation process of Cu/containing amine oxidase in the presence of 2,2,2'-trifluoroethanol

Conformational and structural changes of lentil seedlings amine oxidase (LSAO) were studied in the presence of trifluoroethanol (TFE) by spectroscopic and analytical techniques. At TFE concentrations up to 5%, the induction of a structural transition from beta-sheet to alpha-helix and up to 10% TFE a structural transition from alpha-helix to beta-sheet as well as inactivation of the enzyme are observed. At TFE concentrations between 10-35%, LSAO proves to be prone to aggregation and beyond 35% TFE leads to a non-native protein structure with a high alpha-helix content. The obtained results revealed that the aggregation of LSAO is strongly linked to the nature of secondary structures.     

Protein structural perturbation and aggregation on homogeneous surfaces

We have demonstrated that globular proteins, such as hen egg lysozyme in phosphate buffered saline at room temperature, lose native structural stability and activity when adsorbed onto well-defined homogeneous solid surfaces. This structural loss is evident by alpha-helix to turns/random during the first 30 min and followed by a slow alpha-helix to beta-sheet transition. Increase in intramolecular and intermolecular beta-sheet content suggests conformational rearrangement and aggregation between different protein molecules, respectively. Amide I band attenuated total reflection/Fourier transformed infrared (ATR/FTIR) spectroscopy was used to quantify the secondary structure content of lysozyme adsorbed on six different self-assembled alkanethiol monolayer surfaces with -CH3, -OPh, -CF3, -CN, -OCH3, and -OH exposed functional end groups. Activity measurements of adsorbed lysozyme were in good agreement with the structural perturbations. Both surface chemistry (type of functional groups, wettability) and adsorbate concentration (i.e., lateral interactions) are responsible for the observed structural changes during adsorption. A kinetic model is proposed to describe secondary structural changes that occur in two dynamic phases. The results presented in this article demonstrate the utility of the ATR/FTIR spectroscopic technique for in situ characterization of protein secondary structures during adsorption on flat surfaces.     

Secondary structural analysis of the Na(+),K(+)-ATPase and its Na(+) (E(1)) and K(+) (E(2)) complexes by FTIR spectroscopy

The Na(+),K(+)-ATPase is an integral membrane protein which transports sodium and potassium cations against an electrochemical gradient. The transport of Na(+) and K(+) ions is presumably connected to an oscillation of the enzyme between the two conformational states, the E(1) (Na(+)) and the E(2) (K(+)) conformations. The E(1) and E(2) states have different affinities for ligand interaction. However, the determination of the secondary structure of this enzyme in its sodium and potassium forms has been the subject of much controversy. This study was designed to provide a quantitative analysis of the secondary structure of the Na(+),K(+)-ATPase in its sodium (E(1)) and potassium (E(2)) states in both H(2)O and D(2)O solutions at physiological pH, using Fourier transform infrared (FTIR) with its self-deconvolution and second derivative resolution enhancement methods, as well as curve-fitting procedures. Spectroscopic analysis showed that the secondary structure of the sodium salt of the Na(+),K(+)-ATPase in H(2)O solution contains alpha-helix 19.8+/-1%, beta-sheet 25.6+/-1%, turn 9.1+/-1%, and beta-anti 7.5+/-1%, whereas in D(2)O solution, the enzyme shows alpha-helix 16.8+/-1%, beta-sheet 24.5+/-1.5%, turn 10.9+/-1%, beta-anti 9.8+/-1%, and random coil 38.0+/-2%. Similarly, the potassium salt in H(2)O solution contains alpha-helix 16.6+/-1%, beta-sheet 26.4+/-1.5%, turn 8.9+/-1%, and beta-anti 8.1+/-1%, while in D(2)O solution it shows alpha-helix 16.2+/-1%, beta-sheet 24.5+/-1.5%, turn 10.3+/-1%, beta-anti 9.0+/-1%, and random coil 40+/-2%. Thus the main differences for the sodium and potassium forms of the Na(+),K(+)-ATPase are alpha-helix 3.2% in H(2)O and 0.6% in D(2)O, beta-sheet (pleated and anti) 1.5% in H(2)O and random structure 2% (D(2)O), while for other minor components (turn structure), the differences are less than 1%.     

Purification and structural characterization of herpes simplex virus glycoprotein C

Purification of herpes simplex virus glycoprotein C (gC) in microgram amounts yielded sufficient material for an analysis of its secondary structure. Purification was facilitated by using the mutant virus gC-3, which bears a point mutation that interrupts the putative hydrophobic membrane anchor sequence, causing the secretion of gC-3 protein into the cell culture medium. gC-3 protein was purified by size fractionation of concentrated culture medium from infected cells on a gel filtration column of Sephacryl S-200, followed by immunoaffinity chromatography on a column constructed of gC-specific monoclonal antibodies cross-linked to a protein A-Sepharose CL-4B matrix. Purified gC-3 had a molecular weight of 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size expected for gC, was reactive with gC-specific monoclonal antibodies in protein immunoblots, and contained amino acid sequences characteristic of gC as determined by radiochemical amino acid microsequence analyses. Polyclonal antisera obtained from a rabbit immunized with gC-3 reacted with wild-type gC in immunoprecipitation, enzyme immunoassay, and immunoelectroblot (western blot) assays. Deglycosylation by treatment with trifluoromethanesulfonic acid reduced the molecular weight of gC-3 by approximately 35%. Analyses of both native and deglycosylated gC-3 by Raman spectroscopy showed that the native molecule consists of about 17% alpha-helix, 24% beta-sheet, and 60% disordered secondary structures, whereas deglycosylated gC-3 consists of about 8% alpha-helix, 10% beta-sheet, and 81% disordered structures. These data were in good agreement with the 11% alpha-helix, 18% beta-sheet, 61% beta-turn, and 9% disordered structures calculated from Chou-Fasman analysis of the primary sequence of gC-3.     

Secondary structure and 3D homology modeling of grass carp (Ctenopharyngodon idellus) major histocompatibility complex class I molecules

No information to date is available on the structure of fish major histocompatibility complex (MHC) class I and beta2-microglobulin (beta2m) proteins. In the present study, grass carp (Ctenopharyngodon idellus) MHC class I (Ctid-MHC I) and beta(2)-microglobulin (Ctid-beta2m) genes were expressed as soluble maltose binding protein (MBP)-proteins and purified in a pMAL-p2X/Escherichia coli TB1 system. The expressed proteins were purified on amylase affinity columns followed by DEAE-Sepharose. The purified products were identified by Western blotting with anti-MBP polyclonal antibodies. The MBP-Ctid-MHC I and MBP-Ctid-beta2m were cleaved separately with Factor Xa, mixed together and purified on DEAE-Sepharose. The secondary structures were analyzed by circular dichroism (CD) spectrophotometry. The three-dimensional (3D) structure of their peptide-binding domain (PBD) was modeled based sequence homology. The sequence lengths of the alpha-helix, beta-sheet, turn, and random coil in the Ctid-MHC I protein were 79aa, 75aa, 20aa, and 99aa, respectively. In the 97aa of Ctid-beta2m, the contents of the alpha-helix, beta-sheet, turn, and random coil were 0aa, 41aa, 12aa, and 44aa, respectively. The Ctid-beta2m protein displayed a typical beta-sheet. Homology modeling of the Ctid-MHC I and Ctid-beta2m proteins demonstrated similarities with the structure of human MHC class I proteins.     

Minimal model for studying prion-like folding pathways

The Monte Carlo technique is used to simulate the energy landscape and the folding kinetics of a minimal prion-like protein model. We show that the competition between hydrogen-bonding and hydrophobic interactions yields two energetically favored secondary structures, an alpha-helix and a beta-hairpin. Folding simulations indicate that the probability of reaching the alpha-helix form from a denatured random conformation is much higher than the probability of reaching the beta-sheet form, even though the beta-sheet has a lower energy. The existence of a lower energy beta-sheet state gives the possibility for the normal alpha-helix structure to take a structural transformation into the beta-sheet structure under external influences.     

Fourier transform infrared spectroscopic studies on the secondary structure of the Ca2+-ATPase of sarcoplasmic reticulum

Fourier transform infrared spectroscopy has been applied to the study of the secondary structure of the Ca2+-ATPase of sarcoplasmic reticulum. An attempt is made to quantitatively assess the various secondary structures present. Values of 45% alpha-helix, 32% beta-sheet and 23% turns were obtained. A comparison is made of these results and those obtained using other techniques such as CD and Raman spectroscopy. The various assumptions inherent in the present procedure are discussed. The effect of various ligands, e.g. Ca2+, vanadate, ATP and phosphate, upon the structure were investigated. Upon binding these ligands no marked spectral changes were observed.     

Secondary conformations and temperature effect on structural transformation of amyloid beta (1-28), (1-40) and (1-42) peptides

Secondary structure of three amyloid b-peptides [A beta(1-28), A beta(1-40) and A beta(1-42)] in the solid state was respectively determined by Fourier transform infrared (FT-IR) microspectroscopy. Their thermal-dependent structural transformation were also investigated by FT-IR microspectroscopy equipped with a thermal analyzer. The present result demonstrates that the solid-state A beta(1-28), A beta(1-40) and A beta(1-42) peptides showed a significant IR spectral difference in the amide I and II bands. The secondary conformation of A beta(1-28) peptide was the combination of major beta-sheet and minor alpha-helix with little random coil structures, but A beta(1-40) peptide showed the co-existence of major beta-sheet and minor random coil with little alpha-helix structures. A beta(1-42) peptide mainly consisted of the predominant b-sheet structure. Although the intact A beta(1-28), A beta(1-40) or A beta(1-42) peptide exhibits a different secondary structure, a similar beta-conformation may form after thermal treatment. A thermal-dependent transition was found for solid A beta(1-28) and A beta(1-40) peptides near 40 degrees C and 45 degrees C, respectively. There was no transition temperature for solid A beta(1-42) peptide, however, due to only a very little level of alpha-helix and random coil structure containing in the solid A beta(1-42) peptide. The thermal denaturation plays an important role in the structural transformation from alpha-helix/random coil to beta-sheet.     

Pressure-induced transformation of alpha-helix to beta-sheet in the secondary structures of amyloid beta (1-40) peptide exacerbated by temperature

The effect of pressure on the conformational structure of amyloid beta (1-40) peptide (A beta(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 A beta(1-40) from 1655 cm(-1) (alpha-helix) to 1647-1643 cm(-1) (random coil) with the increase of the mechanical pressure. A new peak at 1634 cm(-1) assigned to beta-antiparallel 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 alpha-helix and random coil structures, and the latter was due to beta-sheet structure. Changes in the composition of each component in the deconvoluted and curve-fitted amide I band of the compressed A beta(1-40) samples were obtained from 33% to 22% for alpha-helix/random coil structures and from 47% to 57% for beta-sheet structure with the increase of pressure, respectively. This demonstrates that pressure might induce the conformational transition from alpha-helix to random coil and to beta- sheet structure. The structural transformation of the compressed A beta(1-40) samples was synergistically influenced by the combined effects of pressure and temperature. The thermal-induced formation of beta-sheet structure was significantly dependent on the pressures applied. The smaller the pressure applied the faster the beta-sheet structure transformed. The thermal-dependent transition temperatures of solid A beta(1-40) prepared by different pressures were near 55-60 degrees C.     

Effect of protease digestion on the secondary structure of sarcoplasmic reticulum Ca2(+)-ATPase as seen by FT-i.r. spectroscopy

1. Upon controlled protein cleavage the catalytic activity of the Ca2(+)-ATPase from sarcoplasmic reticulum is drastically reduced concomitantly with small but significant changes in secondary structure as seen by Fourier transformed infrared (FT-i.r.) spectroscopy, although no loss of protein bound to the membrane is found. 2. FT-i.r. band fitting procedures show a reduction in the beta-sheet and turns content of the protein which is accompanied by an increase in alpha-helix and/or random structure. 3. These changes in the secondary structure of the protein appear to be well correlated to the tryptic digestion pattern and also to changes in the ATP hydrolysis rate of the Ca2(+)-ATPase. 4. It is concluded that these small changes reflect the disruption of key domains of the protein, which lie outside of the membrane matrix, leading to loss of enzymatic activity. 5. FT-i.r. spectroscopy appears to be a very useful technique to study changes in secondary structure of proteins.     

Secondary structure of the murine histocompatibility alloantigen H-2Kb: relationship between heavy chain, beta 2-microglobulin, and antigenic reactivity

The far-ultraviolet circular dichroism (CD) spectra of the extracellular portion (papain-cleaved fragment) of the histocompatibility antigen H-2Kb and its noncovalently associated components, heavy chain and beta 2-microglobulin (beta 2m), indicate that the antigen is highly structured, containing about 30% alpha-helix, 41% beta-sheet, and 29% random coil. Separation of beta 2m from the heavy chain produced a decrease in heavy chain alpha-helix and beta-sheet structure which correlated with a loss of alloantigenic reactivity. Reconstitution of the heavy chain-beta 2m complex resulted in an increase in secondary structure which was greater than the sum of the free chains and the recovery of considerable alloantigenic reactivity. This suggests that some of the secondary structure and much of the alloantigenic reactivity may depend on conformation associated with the binding of beta 2m to heavy chain. A prediction of heavy chain secondary structure based on Chou-Fasman analysis of the primary amino acid sequence agreed with results from CD measurements and suggested that the segments of alpha-helix and beta-sheet structure are distributed throughout the molecule.     

Protein unfolding at interfaces: slow dynamics of alpha-helix to beta-sheet transition

A two-phase sequential dynamic change in the secondary structure of hen egg lysozyme (Lys) adsorbed on solid substrates was observed. The first phase involved fast conversion of alpha-helix to random/turns (within the first minute or at very low coverage or high substrate wettability) with no perceptible change in beta-sheet content. The second phase (1-1200 min), however, involved a relatively slow conversion from alpha-helix to beta-sheet without a noticeable change in random/turns. An important finding of this work is that the concentration of lysozyme in the adsorbed state has a substantial effect on the fractional content of secondary structures. Attenuated total reflection Fourier transform infrared (ATR/FTIR) spectroscopy, along with a newly-developed optimization algorithm for predicting the content of secondary structure motifs, was used to correlate the secondary structure and the amount of adsorbed lysozyme with the surface wettability of six different flat nanoporous substrates. Although three independent variables, surface wettability, solution concentration and time for adsorption, were used to follow the fractional structural changes of lysozyme, the results were all normalized onto a single plot with the amount adsorbed as the universal independent variable. Consequently, lateral interactions among proteins likely drive the transition process. Direct intermolecular force adhesion measurements between lysozyme and different functionalized self-assembled alkanethiol monolayers confirm that hydrophobic surfaces interact strongly with proteins. The lysozyme-unfolding pathway during early adsorption appears to be similar to that predicted by published molecular modeling results.     

Conformational study of silklike peptides modified by the addition of the calcium-binding sequence from the shell nacreous matrix protein MSI60 using 13C CP/MAS NMR spectroscopy

The calcium-binding site of the pearl oyster (Pinctada fucata) nacreous layer matrix protein MSI60 was introduced between different Ala-Gly repeating regions derived from the primary sequences of several silk fibroins. Several different organic solvents whose effect on the repetitive domains of silk peptides is well-understood were used to modify the secondary structure of the flanking Ala-Gly repeating regions. The local conformations of the flanking Ala-Gly repeating regions as well as the calcium-binding motif, MSI60, were determined by 13C CP/MAS NMR spectroscopy. The secondary structures of the polyalanine, poly(Ala), domains were modified by the solvent treatments in a predictable fashion, suggesting that only the solvent treatment and not the conformation of the MSI60 domain affected the conformation of poly(Ala) regions. Ala-Gly domains behaved differently, taking random coil conformation regardless of the choice of solvent, indicating that their secondary structure is affected by the central MSI60 domain. The conformation of the MSI60 domain is not altered by the solvent treatments, suggesting that it may retain its ability to bind calcium ions. This was confirmed using a calcium-binding assay. The assay further showed that the calcium-binding capability of MSI60 in the synthetic peptides was most effective when the flanking domain was in the beta-sheet structure.     

Prediction and Fourier transform infrared spectroscopy estimation of the secondary structure of a Bacillus licheniformis endo-beta-1,3-1,4-D-glucanase.

The secondary structure of a recombinant Bacillus licheniformis endo-beta-1,3-1,4-D-glucanase (EC.3.2.1.73) has been estimated by Fourier Transform Infrared Spectroscopy and also predicted by the algorithm of Chou and Fasman. From the curve fitting of the deconvolved IR spectrum, the most probable distribution of the secondary structural classes appears to be about 40% beta-sheet, 25% reverse turn, 24% non-ordered and 11% alpha-helix. From theoretical prediction of secondary structure the protein would present 37% beta-sheet, 31% reverse turn, 22% non-ordered and 10% alpha-helix.