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.     

A circular dichroic study of helical structure in flagellar dynein

The circular dichroic spectra of outer arm dynein from sea urchin sperm flagella, of its separated alpha and beta heavy-chain complexes, and of the two major fragments produced by tryptic digestion of the beta heavy chain have been measured over the range 190-240 nm. Although the spectra show significant individuality, in all cases they qualitatively resemble those of typical globular proteins with mixed regions of alpha-helix and beta-sheet (alpha/beta-type structure) or with separate alpha-helix- and beta-sheet-rich regions (alpha+beta-type structure). Quantitative analyses of the spectra by both constrained and unconstrained least-squares curve-fitting procedures indicate that the intact dynein contains approximately 26% alpha-helix. The separated beta heavy-chain complex and its ATPase-containing amino-terminal domain (fragment A) both have spectra resembling that of intact dynein, and they appear to contain 32% and 23% alpha-helix, respectively. The carboxy-terminal domain of the beta heavy chain (fragment B) and the separated alpha heavy chain have significantly different spectra; however, they each appear to contain 26-36% alpha-helix. These data suggest that dynein does not contain an extensive alpha-helical domain, such as is found in the carboxy-terminal rod region of the other motor proteins myosin and kinesin.     

Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy

The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods. The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme. From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-sheet, 15% turns and 35% of non-repetitive structures, were estimated. A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23%. The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic. Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure.     

Intramolecular organization of Class I H-2 MHC antigens; localization of the alloantigenic determinants and the beta 2 m binding site to different regions of the H-2 Kb glycoprotein

The intramolecular organization of the membrane integrated Class I major histocompatibility complex (MHC) molecule H-2Kb (Kb) was analyzed. After the removal of the two carbohydrate moieties by glycosidase enzymes, proteolytic digestion of the Kb molecule yielded: 1) several fragments with the beta 2 microglobulin (beta 2 m) subunit still bound and 2) one fragment carrying alloantigenic activity but lacking the beta 2 m. Isolation of the beta 2 m binding fragments showed them to be derived from the C-2 domain by partial N-terminal sequence analysis. One fragment extended to the C-terminus and the other fragment had lost the transmembrane region. Such studies conclusively show that the beta 2 m subunit is bound in the third domain, i.e., C-2, of the Kb 44,000 m.w. heavy chain. The alloantigenic fragment also isolated from the proteolytic digest consists of the first 180 residues of the 44,000 m.w. heavy chain, i.e., domains N and C-1, and carried alloantigenic determinants detected by several monoclonal antibodies as well as alloantisera. The present studies indicate that the external region of the Class I molecules has two functional regions. The first 180 residues bear the recognition elements for the immune system, and the next 90 residues (180-270) are involved in binding to beta 2 m.     

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.     

Conformational analysis of thymidylate synthase from amino acid sequence and circular dichroism

Circular dichroism studies were carried out in the vacuum ultraviolet region for thymidylate synthase from Lactobacillus casei and its ligand complexes. The CD spectrum was analyzed for secondary structure by our method and the variable selection method, and both gave similar results. Our method predicts 33% alpha-helix, 25% (23% antiparallel and 2% parallel) beta-sheet, 20% turns, and 16% other structure. The secondary structure of this protein was also predicted from the amino acid sequence by four different methods. Though there is a variation in the prediction among these methods, the prediction of 32% alpha-helix and 23% beta-sheet by combining the four methods is in excellent agreement with our CD results. Further, the location of the predicted regions of alpha-helices and beta-strands along the sequence and the CD characteristics strongly suggest that this protein belongs to an alpha + beta structural class. Binding of the inhibitor FdUMP or the cofactor 5,10-methylenetetrahydrofolate did not change the CD spectrum. However, when both ligands were present, there was a significant change in the CD spectrum and the maximum changes occurred when the concentration of FdUMP was 1 mol/mol of enzyme. The addition of FdUMP and cofactor causes, respectively, a 5% and 6% decrease in beta-sheet and beta-turns and about an 8% increase in "other" structure.     

Structural study of the catalytic domain of PKCzeta using infrared spectroscopy and two-dimensional infrared correlation spectroscopy

The secondary structure of the catalytic domain from protein kinase C zeta was studied using IR spectroscopy. In the presence of the substrate MgATP, there was a significant change in the secondary structure. After heating to 80 degrees C, a 14% decrease in the alpha-helix component was observed, accompanied by a 6% decrease in the beta-pleated sheet; no change was observed in the large loops or in 3(10)-helix plus associated loops. The maximum increase with heating was observed in the aggregated beta-sheet component, with an increase of 14%. In the presence of MgATP, and compared with the sample heated in its absence, there was a substantial decrease in the 3(10)-helix plus associated loops and an increase in alpha-helix. Synchronous 2D-IR correlation showed that the main changes occurred at 1617 cm(-1), which was assigned to changes in the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in alpha-helix. In the presence of MgATP, the main correlation was between aggregated beta-sheet and the large loops component. The asynchronous 2D-correlation spectrum indicated that a number of components are transformed in intermolecularly aggregated beta-sheet, especially the alpha-helix and beta-sheet components. It is interesting that changes in 3(10)-helix plus associated loops and in alpha-helix preceded changes in large loops, which suggests that the open loops structure exists as an intermediate state during denaturation. In summary, IR spectroscopy revealed an important effect of MgATP on the secondary structure and on the thermal unfolding process when this was induced, whereas 2D-IR correlation spectroscopy allowed us to show the establishment of the denaturation pathway of this protein.     

Dependence of elevated human leukocyte antigen class I molecule expression on increased heavy chain, light chain (beta 2-microglobulin), transporter associated with antigen processing, tapasin, and peptide

Human leukocyte antigen (HLA) class I molecule expression was investigated by DNA-mediated gene transfer. Cell surface expression was increased up to 75% by transfection of HLA-A2 or HLA-B8 heavy chain genes but not genes encoding light chains (beta(2)-microglobulin (beta(2)m)), transporter associated with antigen processing (TAP), or tapasin. Interferon (IFN) treatment further increased expression of transfected heavy chains, suggesting that IFN inducible molecules support heavy chain expression. IFN induces beta(2)m, TAP, and tapasin mRNAs. Transfected heavy chain expression increased upon cotransfection with genes encoding TAP1 and TAP2 but not individual TAP subunits, beta(2)m, or tapasin. Tetracycline inducible heavy chain gene expression was also increased by IFN treatment or TAP cotransfection, suggesting that IFN-induced TAP supports heavy chain maturation. Expression of a mutant that does not interact strongly with TAP, HLA-A2-T134K, was also increased by IFN. Inhibition of TAP-dependent peptide transport by ICP47 reduced heavy chain expression. Expression of HLA-A2, but not HLA-B8, was restored in ICP47 cells by HLA-A2-binding (IP-30) signal peptides. However, these peptides did not further increase transfected HLA-A2 expression, suggesting that peptide availability does not limit heavy chain expression in the absence of ICP47. These results suggest that cytokine-induced TAP supports maturation of HLA class I molecules through combined chaperone and peptide supply functions.     

An N-linked glycan modulates the interaction between the CD1d heavy chain and beta 2-microglobulin

Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with beta(2)-microglobulin (beta(2)m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to beta(2)m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d-beta(2)m heterodimers. None of the four were indispensable for assembly or the ability to bind alpha-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present alpha-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(alpha1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-beta(2)m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with beta(2)m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from beta(2)m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from beta(2)m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and beta(2)m, which may explain these unusual properties.     

Influence of secondary structure of polyglycine on some conformational properties

Values of four conformational properties, namely unperturbed dimension [r2]0, dipole moment [mu 2], mean squared optical anisotropy [gamma 2], and molar Kerr constant [mK], have been calculated for polyglycine chains allowing several combinations of the secondary structure with the aim of studying the dependence of these magnitudes on the secondary structure of the chain. Two different approaches to the secondary structure have been used. In the first, chains with all their units in a given conformation (random coil, alpha-helix or beta-sheet) are interrupted at several positions by one unit in a different conformation. In the second, chains with varying composition of two conformations alpha-helix/beta-sheet and beta-sheet/random coil were allowed and the results obtained compared with previous work for alpha-helix/random coil chains.     

Structure study of recombinant RGD-hirudin by vibrational and circular dichroism spectroscopy

The secondary structure of a new type of recombinant RGD-hirudin, which has the activities of anti-thrombin and anti-platelet aggregation, has been studied by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy and circular dichroism (CD) methods. The distribution of various secondary structure elements was determined using only a very small amount of sample protein. It was found that the recombinant RGD-hirudin contains about 26% extended chain, 21% beta-turn and 53% unordered structure, leaving no alpha-helix. The results showed that the regular secondary structure of recombinant RGD-hirudin is increased compared with wild-type hirudin. The RGD segment that is located at the end of a long arm of a beta-sheet is thought to play an important role in the additional function of anti-platelet aggregation. Throughout the experiments, FT-IR, Raman spectroscopy and CD generated mutually reinforcing results.     

Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.

Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.     

Effect of glycine substitutions on alpha5(IV) chain structure and structure-phenotype correlations in Alport syndrome

The phenotype variety caused by glycine substitutions in alpha5(IV) chain in X-linked Alport syndrome (XLAS) prompted the complexity of structure changes of alpha5(IV) chain that was little to know now. In this study, we expressed a domain of alpha5(IV) chain containing different glycine substitutions (G1015V and G1030S, respectively) which were revealed in two XLAS pedigrees with different phenotype severities and the corresponding domain of a control in Escherichia coli. The recombinant proteins were characterized by immunoblot and mass spectrometry and analyzed the secondary structure by using circular dichroism (CD) spectroscopy. CD analysis showed that the recombinant protein containing G1015V mutation identified in the pedigree of juvenile-onset XLAS exhibited 12.9% alpha-helix that was not found in the control recombinant protein. The spectrum of the recombinant protein containing G1030S mutation identified in the pedigree of adult-onset XLAS was slightly different from that of the control, that is, mostly with the random coil and the beta-sheet, while without alpha-helix. These results demonstrated that two kinds of glycine substitutions, although in the same domain of alpha5(IV) chain, displayed the distinctly different secondary structures. The changes of the secondary structure could explain the phenotypic diversities of XLAS, which would be hardly understood solely by analyzing genomic DNA or mRNA of alpha5(IV) chain.     

Effect of hydrophobic surfactant proteins SP-B and SP-C on phospholipid monolayers. Protein structure studied using 2D IR and beta correlation analysis

We have applied two-dimensional infrared (2D IR) and betanu correlation spectroscopy to in-situ IR spectroscopy of pulmonary surfactant proteins SP-B and SP-C in lipid-protein monolayers at the air-water interface. For both SP-B and SP-C, a statistical windowed autocorrelation method identified two separate surface pressure regions that contained maximum amide I intensity changes: 4-25 mN/m and 25-40 mN/m. For SP-C, 2D IR and betanu correlation analyses of these regions indicated that SP-C adopts a variety of secondary structure conformations, including alpha-helix, beta-sheet, and an intermolecular aggregation of extended beta-sheet structure. The main alpha-helix band split into two peaks at high surface pressures, indicative of two different helix conformations. At low surface pressures, all conformations of the SP-C molecule reacted identically to increasing surface pressure and reoriented in phase with each other. Above 25 mN/m, however, the increasing surface pressure selectively affected the coexisting protein conformations, leading to an independent reorientation of the protein conformations. The asynchronous 2D IR spectrum of SP-B showed the presence of two alpha-helix components, consistent with two separate populations of alpha-helix in SP-B-a hydrophobic fraction associated with the lipid chains and a hydrophilic fraction parallel to the membrane surface. The distribution of correlation intensity between the two alpha-helix cross peaks indicated that the more hydrophobic helix fraction predominates at low surface pressures whereas the more hydrophilic helix fraction predominates at high surface pressures. The different SP-B secondary structures reacted identically to increasing surface pressure, leading to a reorientation of all SP-B subunits in phase with one another.     

Calnexin,calreticulin, and ERp57 cooperate in disulfide bond formation in human CD1d heavy chain

Members of the CD1 family of membrane glycoproteins can present antigenic lipids to T lymphocytes. Like major histocompatibility complex class I molecules, they form a heterodimeric complex of a heavy chain and beta(2)-microglobulin (beta(2)m) in the endoplasmic reticulum (ER). Binding of lipid antigens, however, takes place in endosomal compartments, similar to class II molecules, and on the plasma membrane. Unlike major histocompatibility complex class I or CD1b molecules, which need beta(2)m to exit the ER, CD1d can be expressed on the cell surface as either a free heavy chain or associated with beta(2)m. These differences led us to investigate early events of CD1d biosynthesis and maturation and the role of ER chaperones in its assembly. Here we show that CD1d associates in the ER with both calnexin and calreticulin and with the thiol oxidoreductase ERp57 in a manner dependent on glucose trimming of its N-linked glycans. Complete disulfide bond formation in the CD1d heavy chain was substantially impaired if the chaperone interactions were blocked by the glucosidase inhibitors castanospermine or N-butyldeoxynojirimycin. The formation of at least one of the disulfide bonds in the CD1d heavy chain is coupled to its glucose trimming-dependent association with ERp57, calnexin, and calreticulin.     

Secondary structure of the mammalian 70-kilodalton heat shock cognate protein analyzed by circular dichroism spectroscopy and secondary structure prediction

Heat shock proteins are rapidly synthesized when cells are exposed to stressful agents that cause protein damage. The 70-kDa heat shock induced proteins and their closely related constitutively expressed cognate proteins bind to unfolded and aberrant polypeptides and to hydrophilic peptides. The structural features of the 70-kDa heat shock proteins that confer the ability to associate with diverse polypeptides are unknown. In this study, we have used circular dichroism (CD) spectroscopy and secondary structure prediction to analyze the secondary structure of the mammalian 70-kDa heat shock cognate protein (hsc 70). The far-ultraviolet CD spectrum of hsc 70 indicates a large fraction of alpha-helix in the protein and resembles the spectra one obtains from proteins of the alpha/beta structural class. Analysis of the CD spectra with deconvolution methods yielded estimates of secondary structure content. The results indicate about 40% alpha-helix and 20% aperiodic structure within hsc 70 and between 16-41% beta-sheet and 21-0% beta-turn. The Garnier-Osguthorpe-Robson method of secondary structure prediction was applied to the rat hsc 70 amino acid sequence. The predicted estimates of alpha-helix and aperiodic structure closely matched the values derived from the CD analysis, whereas the predicted estimates of beta-sheet and beta-turn were midway between the CD-derived values. Present evidence suggests that the polypeptide ligand binding domain of the 70-kDa heat shock protein resides within the C-terminal 160 amino acids [Milarski, K. L., & Morimoto, R. I. (1989) J. Cell Biol. 109, 1947-1962].(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational change in a single molecular species, beta3, of beta-conglycinin in acidic ethanol solution

Several physicochemical experiments were done to obtain further information on the conformational changes occurring in beta-conglycinin in acidic-ethanol solution, using a single molecular species of this protein, beta3. By far-UV circular dichroism (CD), a transition from beta-sheet to alpha-helical structure was observed upon addition of acidic-ethanol, and the alpha-helix content was found to reach 76% in 70% ethanol (pH 2). From analyses of near-UV CD and difference absorption spectra, it was found that the tertiary structure of the beta3 species was significantly altered at ethanol concentrations between 10 and 20%. The profiles of binding of 1-anilinonaphthalene-8-sulfonic acid to the beta3 species during acidic-ethanol denaturation were indicative of the existence of intermediate conformers in the molten globule-like denaturation state. By measuring Fourier transform infrared spectra and estimating the Stokes radius by dynamic light scattering, the beta3 molecules were found to aggregate with an increase in ethanol concentration.     

Primary sequence and structural analysis of sterol carrier protein 2 from rat liver: homology with immunoglobulins

Sterol carrier protein 2 (SCP2) is involved in the later steps of cholesterol biosynthesis and in the intracellular transport of cholesterol. In the present investigation, the amino acid sequence of SCP2 from rat liver has been determined. It is a single polypeptide chain with 122 amino acid residues. Secondary structure prediction indicates an amphipathic alpha-helix region for residues 21-34 and antiparallel beta-sheet structure for residues 35-95. A major finding is the significant homology which exists over approximately 80 residues between SCP2 and the variable domains of the heavy chain of immunoglobulin G.     

Determination of alpha-helix and beta-sheet stability in the solid state: a solid-state NMR investigation of poly(L-alanine)

The relative stability of alpha-helix and beta-sheet secondary structure in the solid state was investigated using poly(L-alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. (13)C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the alpha-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% beta-sheet and 45% alpha-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% beta-sheet and 40% alpha-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% alpha-helix/50% beta-sheet at 100 degrees C when starting from a mostly alpha-helical state. No change was observed upon heating a beta-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with beta-sheet approximately 260 J/mol more stable than alpha-helix in solid-state PLA.     

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.