Thermal-induced changes in the secondary conformation of superoxide dismutase containing different metal ions

The thermal stability of three superoxide dismutases (SODs) with different metal ions (Mn, Cu/Zn, Fe) in the solid state was studied by a Fourier transform infrared (FT-IR) microspectroscopy combined with thermal analyzer. The IR spectra showed a maximum peak at 1652 cm(-1) for all the native SODs in the amide I band, suggesting a predominant random coil with less alpha-helix structures. By heating each sample, a shoulder at 1631 cm(-1) in the amide I band gradually appeared from 45 degrees C for Fe SOD and from 50 degrees C for Mn SOD but another shoulder at 1639 cm(-1) appeared from 50 degrees C for Cu/Zn SOD. The peak at 1631 cm(-1) is due to the intermolecular beta-sheet structure, but the peak at 1639 cm(-1) corresponds to the major intramolecular beta-sheet with less random coil structure. This reveals that in the first heating process the transformation from random coil/alpha-helix structure to beta-sheet structure initiated from around 45-50 degrees C. There was about 16-22% compositional change resulting from that transformation. However, both additional shoulders stood there and did not restore to their original spectra even with cooling to room temperature, suggesting the denaturation and irreversible properties of the solid SODs after heating. The thermal-dependent denaturation and irreversibility of Mn SOD, Cu/Zn SOD and Fe SOD were clearly evidenced by the increase in intramolecular and intermolecular beta-sheet structure.     

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.     

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.     

Synthesis and secondary structural studies of penta(acetyl-Hmb)A beta(1-40).

The Fmoc solid phase synthesis of A beta(1-40), a strongly aggregating peptide found in Alzheimer's disease brain, was performed using 2-hydroxy-4-methoxybenzyl (Hmb) backbone amide protection. Hmb-Gly residues were incorporated using N(alpha)-Fmoc-Hmb-Gly-OH rather than N,O-bisFmoc-Hmb-Gly-OPfp. Amino acid acylation of the sterically hindered Hmb-amino acids was monitored using 'semi-on-line' MALDI-TOF-MS in a novel application of this technique which significantly simplified the successful incorporation of these residues. Standard coupling conditions in N,N-dimethylformamide (DMF) were used throughout the synthesis. Comparative structural studies of acetyl-Hmb-protected and native A beta(1-40) were performed to investigate the structural basis of Hmb-mediated disaggregation. The incorporation of backbone amide protection was observed by circular dichroism spectroscopy and gel electrophoresis to strongly affect the solution structure of A beta(1-40). Despite the reported structure-breaking activity of Hmb groups, penta(acetyl-Hmb)A beta(1-40) was found to adopt both alpha-helix and intermolecular beta-sheet conformations. In 100% TFE a mixed alpha-helix/random coil structure was formed by the protected peptide indicating reduced alpha-helical propensity relative to A beta(1-40). The protected peptide formed beta-sheet structures in aqueous buffer. Gel electrophoresis indicated that, unlike native A beta(1-40), penta(acetyl-Hmb)A beta(1-40) did not form large aggregate species.     

pH- and thermal-dependent conformational transition of PGAIPG, a repeated hexapeptide sequence from tropoelastin

The secondary structure of PGAIPG (Pro-Gly-Ala-IIe-Pro-Gly), a repeated hexapeptide of tropoelastin, in buffer solution of different pH was determined by using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The thermal-dependent structural change of PGAIPG in aqueous solution or in solid state was also examined by thermal FTIR microspectroscopy. The conformation of PGAIPG in aqueous solution exhibited a pH-dependent structural characterization. A predominant peak at 1614 cm(-1) (aggregated beta-sheet) with a shoulder near 1560 cm(-1) (beta-sheet) appeared in pH 5.5-8.5 buffer solutions. A new broad shoulder at 1651 cm(-1) (random coil and/or alpha-helix) with 1614 cm(-1) was observed in the pH 4.5 buffer solution. However, the broad shoulder at 1651 cm(-1) was converted to a maximum peak at 1679 cm(-1) (beta-turn/antiparallel beta-sheet) when the pH shifted from 4.5 to 3.5, but the original pronounced peak at 1614 cm(-1) became a shoulder. Once the pH was lowered to 2.5, the IR spectrum of PGAIPG was dominated by major absorption at 1679 cm(-1) with a minor peak at 1552 cm(-1) (alpha-helix/random coil). The result indicates that the pH was a predominant factor to transform PGAIPG structure from aggregated beta-sheet (pH 8.5) to beta-turn/intermolecular antiparallel beta-sheet (pH 2.5). Moreover, a partial conformation of PGAIPG with minor alpha-helix/random coil structures was also explored in the lower pH buffer solution. There was no thermal-dependent structural change for solid-state PGAIPG. The thermal-induced formation of aggregated beta-sheet for PGAIPG in aqueous solution was found from 28 to 30 degrees C, however, which might be correlated with the formation of an opaque gel that turned from clear solution. The formation of aggregated beta-sheet structure for PGAIPG beyond 30 degrees C might be due to the intermolecular hydrogen bonded interaction between the hydrophobic PGAIPG fragments induced by coacervation.     

Pressure dependence of human fibrinogen correlated to the conformational alpha-helix to beta-sheet transition: an Fourier transform infrared study microspectroscopic study

We used Fourier transform infrared (FTIR) microspectroscopy to investigate pressure-induced conformational changes in secondary structure of fibrinogen (FBG). Solid state FBG was compressed on a KBr pellet (1KBr method) or between two KBr pellets (2KBr method). The peak positions of the original and second-derivative ir spectra of compressed FBG samples prepared by the 1KBr method were similar to FBG sample without pressure. When FBG was prepared by the 2KBr method and pressure was increased up to 400 kg/cm(2), peaks at 1625 (intermolecular beta-sheet) and 1611 (beta-sheet aggregates structure and/or the side-chain absorption of the tyrosine residues) cm(-1) were enhanced. The peaks near 1661 (beta-sheet) and 1652 (alpha-helix) cm(-1) also exhibited a marked change with pressure. A linear correlation was found between the peak intensity ratio of 1611/1652 cm(-1) (r = 0.9879) or 1625/1652 cm(-1) (r = 0.9752) and applied pressure. The curve-fitted compositional changes in secondary structure of FBG also indicate that the composition of the alpha-helix structure (1657-1659 cm(-1)) was gradually reduced with the increase in compression pressure, but the composition of the beta-sheet structure (1681, 1629, and 1609 cm(-1)) gradually increased. This indicates that pressure-induced conformational changes in FBG include not only transformations from alpha-helix to beta-sheet structure, but also unfolding and denaturation of FBG and the formation of aggregates.     

Protein structural changes in keratin fibers induced by chemical modification using 2-iminothiolane hydrochloride: a Raman spectroscopic investigation

For the purpose of investigating in detail the influence of chemical modification using 2-iminothiolane hydrochloride (2-IT) on keratin fibers, the structure of cross-sections at various depths of white human hair, treated with 2-IT and then oxidized, was directly analyzed without isolating the cuticle and cortex, using Raman spectroscopy. In particular, the beta-sheet and/or random coil content (beta/R) and the alpha-helix (alpha) content in human hair fibers were estimated by amide I band analysis. The S-S band intensity, amide III (unordered) band intensity, and beta/R content existing from the cuticle region to the center of cortex region of virgin white human hair remarkably increased by performing the chemical modification using 2-IT. On the other hand, not only the S-S band intensity, but also S-O band intensity existing throughout the cortex region of the bleached (damaged) white human hair increased by performing chemical modification using 2-IT. In particular, beta/R content existing throughout the cortex region of the bleached white human hair decreased, while the skeletal C-C stretch (alpha) band intensity at 935 cm(-1) and the alpha content remarkably increased. This indicates a secondary structural change from the random coil form to the alpha-helix form in the proteins existing throughout the cortex region. From these experiments, we concluded that the formation of new disulfide (-SS-) groups resulting from chemical modification using 2-IT induced the secondary structural changes of proteins existing throughout the cortex region.     

FTIR spectra of solid poly-l-lysine in the stretching NH mode range

Three bands at 3270 cm(-1), 3200 cm(-1) and 3030 cm(-1) are found in the IR stretching proton (nu(1)) mode spectral range in spectra of solid poly-l-lysine (PLL). Strong quantitative changes of these bands are observed in samples dried from water solutions with different pH. The narrow band at 3270 cm(-1), which is strong in the spectrum of PLL precipitated from pH=12 alkaline medium, is assigned to the nu(1) peptide proton mode of NH-CO (amide A) of the beta-sheet structure type. The band at 3200 cm(-1), which is intensified in PLL precipitated from pH=1 acidic medium, relates to the nu(1) peptide mode in the random coil structure. The band at 3030 cm(-1), whose peak intensity increases two-fold in going from alkaline to acidic medium, is assigned to the nu(1) modes of protonated NH(3)(+) side chain groups. The frequencies of all bands were used for estimating H-bond energy relying on an empirical correlation between this property and the red shift of the nu(1) band. The enthalpy of the secondary structure transition from beta-sheet to the random coil, which is observed in PLL at the change of pH from 11 to 1 amounts to 4.7 kJ mol(-1).     

4,4(')-Dianilino-1,1(')-binaphthyl-5,5(')-disulfonate: report on non-beta-sheet conformers of Alzheimer's peptide beta(1-40)

The venerable fluorescent probe of protein hydrophobic regions, 4,4(')-dianilino-1,1(')-binaphthyl-5,5(')-disulfonate (bis-ANS), unexpectedly increases in fluorescence with soluble beta(1-40) in acidic buffer solutions but reacts weakly with amyloid fibrils while other hydrophobic probes react with the fibrils. CD analysis correlates reaction with the probe with random coil/mixed conformations and alpha-helical forms of beta(1-40) in buffer solutions but less so with soluble beta-sheet forms or amyloid fibrils. The kinetics of the fluoroalcohol-induced interconversion of conformers can be followed by changes in bis-ANS fluorescence. Formation of the beta-sheet form in aqueous buffer is limited by a slow component (minutes) while fluoroalcohol-promoted changes between beta-sheet and alpha-helix occur over seconds. Variants of beta(1-40) such as beta(1-42) or the Dutch E22Q mutation of beta(1-40) and fragments beta(1-28), beta(12-28), beta(10-20 amide), and beta(10-35 amide) react with bis-ANS under conditions that do not support fibril formation. Primary amino acid sequence is important as beta(1-11) does not cause bis-ANS fluorescence while beta(1-16) does, but hydrophobicity is not as beta(25-35) and beta(15-20 amide) are unreactive. bis-ANS is a useful biophysical tool for characterizing particular, but not all, soluble Abeta conformations distinct from the fibrillar form of amyloid peptides detected by Thioflavin T.     

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.     

Temperature effect on the structural stability, similarity, and reversibility of human serum albumin in different states

In order to investigate the thermal stability of human serum albumin (HAS) in three different states (aqueous solution, cast film, and solid powder), Fourier transform infrared (FTIR) spectroscopy was applied to determine the protein secondary structural changes of these HSA samples under non-isothermal or isothermal condition. The structural similarity of HSA before and after thermal treatment was also studied to estimate the thermo-reversible property of the HSA in these different states. The results indicate that with the increase of temperature, the maximum peaks at 1652 and 1547 cm(-1) (alpha-helix) shifted to 1647 and 1542 cm(-1) (random coil), respectively. An additional peak at 1620 cm(-1) assigned to intermolecular beta-sheet structure clearly appeared with temperature. The alpha-helix content was found to be reduced in favor of the formation of intermolecular hydrogen-bonded antiparallel beta-sheet structure beyond 60 degrees C in the heating process. From the data of structural similarity, HSA sample whether in solid powder or cast film form exhibited a better thermo-reversible property than HSA in aqueous solution even heating to 200 degrees C.     

Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.     

Effects of ELF magnetic field on membrane protein structure of living HeLa cells studied by Fourier transform infrared spectroscopy

The effects of exposure to a 50 Hz magnetic field (maximum of 41.7 to 43.6 mT) on the membrane protein structures of living HeLa cells were studied using attenuated total reflection infrared spectroscopy. One min of such exposure shifted peak absorbance of the amide I band to a smaller wave number, reduced peak absorbance of the amide II band, and increased absorbance at around 1600 cm(-1). These results suggest that exposure to the ELF magnetic field has reversible effects on the N-H inplane bending and C-N stretching vibrations of peptide linkages, and changes the secondary structures of alpha-helix and beta-sheet in cell membrane 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.     

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

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

NMR studies of amyloid beta-peptides: proton assignments, secondary structure, and mechanism of an alpha-helix----beta-sheet conversion for a homologous, 28-residue, N-terminal fragment.

Beta-peptide is a major component of amyloid deposits in Alzheimer's disease. We report here a proton nuclear magnetic resonance (NMR) spectroscopic investigation of a synthetic peptide that is homologous to residues 1-28 of beta-peptide [abbreviated as beta-(1-28)]. The beta-(1-28) peptide produces insoluble beta-pleated sheet structures in vitro, similar to the beta-pleated sheet structures of beta-peptide in amyloid deposits in vivo. For peptide solutions in the millimolar range, in aqueous solution at pH 1-4 the beta-(1-28) peptide adopts a monomeric random coil structure, and at pH 4-7 the peptide rapidly precipitates from solution as an oligomeric beta-sheet structure, analogous to amyloid deposition in vivo. The NMR work shown here demonstrates that the beta-(1-28) peptide can adopt a monomeric alpha-helical conformation in aqueous trifluoroethanol solution at pH 1-4. Assignment of the complete proton NMR spectrum and the determination of the secondary structure were arrived at from interpretation of two-dimensional (2D) NMR data, primarily (1) nuclear Overhauser enhancement (NOE), (2) vicinal coupling constants between the amide (NH) and alpha H protons, and (3) temperature coefficients of the NH chemical shifts. The results show that at pH 1.0 and 10 degrees C the beta-(1-28) peptide adopts an alpha-helical structure that spans the entire primary sequence. With increasing temperature and pH, the alpha-helix unfolds to produce two alpha-helical segments from Ala2 to Asp7 and Tyr10 to Asn27. Further increases in temperature to 35 degrees C cause the Ala2-Asp7 section to become random coil, while the His13-Phe20 section stays alpha-helical. A mechanism involving unfavorable interactions between charged groups and the alpha-helix macrodipole is proposed for the alpha-helix----beta-sheet conversion observed at midrange pH.     

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%.     

Characterization of a partially denatured state of a protein by two-dimensional NMR: reduction of the hydrophobic interactions in ubiquitin

A stable, partially structured state of ubiquitin, the A-state, is formed at pH 2.0 in 60% methanol/40% water at 298 K. Detailed characterization of the structure of this state has been carried out by 2D NMR spectroscopy. Assignment of slowly exchanging amide resonances protected from the solvent in the native and A-state shows that gross structural reorganization of the protein has not occurred and that the A-state contains a subset of the interactions present in the native state (N-state). Vicinal coupling constants and NOESY data show the presence of the first two strands of the five-strand beta-sheet that is present in the native protein and part of the third beta-strand. The hydrophobic face of the beta-sheet in the A-state is covered by a partially structured alpha-helix, tentatively assigned to residues 24-34, that is considerably more flexible than the alpha-helix in the N-state. There is evidence for some fixed side-chain--side-chain interactions between these two units of structure. The turn-rich area of the protein, which contains seven reverse turns and a short piece of 3(10) helix, does not appear to be structured in the A-state and is approaching random coil.     

Structure of the pore-forming transmembrane domain of a ligand-gated ion channel

The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedo has been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3-6 kDa in size that are resistant to peptide (1)H/(2)H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm(-)1, respectively, in both (1)H(2)O and (2)H(2)O. The amide I band is very similar to the amide I bands observed in the spectra of alpha-helical proteins, such as myoglobin and bacteriorhodopsin, that lack beta structure and exhibit much less beta-sheet character than is observed in proteins with as little as 20% beta sheet. Curve-fitting estimates 75-80% alpha-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane alpha-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane alpha-helices.     

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.