The structure of antimicrobial pexiganan peptide in solution probed by Fourier transform infrared absorption, vibrational circular dichroism, and electronic circular dichroism spectroscopy

Pexiganan (Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys), a 22 amino acid peptide, is an analogue of the magainin family of antimicrobial peptides present in the skin of the African clawed frog. Conformational analysis of pexiganan was carried out in different solvent environments for the first time. Organic solvents, trifluoroethanol (TFE) and methanol, were used to study the secondary structural preferences of this peptide in the membrane-mimicking environments. In addition, aqueous (D2O) and dimethyl sulfoxide (DMSO) solutions were also investigated to study the role of hydrogen bonding involved in the secondary structure formation. Fourier transform infrared absorption, vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) measurements were carried out under the same conditions to ascertain the conformational assignments in different solvents. All these spectroscopic measurements suggest that the pexiganan peptide has the tendency to adopt different structures in different environments. Pexiganan appears to adopt an alpha-helical conformation in TFE, a sheet-stabilized beta-turn structure in methanol, a random coil with beta-turn structure in D2O, and a solvated beta-turn structure in DMSO.     

Secondary structure of streptokinase in aqueous solution: a Fourier transform infrared spectroscopic study.

The secondary structure of streptokinase (Sk) in aqueous solution was quantitatively examined by using Fourier transform infrared (FT-IR) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy, were combined with band curve-fitting procedures to quantitate the spectral information from the amide I bands. Nine component bands were found under the broad, nearly featureless amide I bands which reflect the presence of various substructures. The relative areas of these component bands indicate an amount of beta-sheet between 30 and 37% and an alpha-helix content of only 12-13% in Sk. Further conformational substructures are assigned to turns (25-26%) and to "random" structures (15-16%). Additionally, the correlation of a pronounced component band near 1640 cm-1 (10-16% fractional area) with the possible presence of 3(10)-helices is discussed.     

Cluster analysis of protein fourier transform infrared spectra

Cluster analysis techniques were used to examine a set of Fourier transform infrared (FT-IR) spectra of bovine serum albumin (BSA) in the adsorbed and nonadsorbed states. The region from 1480 to 1600 cm^?1, comprising the amide II band, was used. Spectra were preprocessed to compensate for linear baseline variation, and the single linkage method of cluster analysis was applied. As expected, the spectra of adsorbed and nonadsorbed BSA fell into two distinct clusters. However, no further clustering was observed among the adsorbed BSA spectra on the basis of surface type, suggesting that surface specificity of the spectral changes induced in BSA by adsorption is not detectable above experimental variation. This work illustrates the value of using cluster analysis in the FT-IR study of proteins as a complement to other data analysis methods.     

Metal-dependent conformational changes in a recombinant vWF-A domain from human factor B: a solution study by circular dichroism, fourier transform infrared and (1)H NMR spectroscopy

Factor B is a key component of the alternative pathway of complement and is cleaved by factor D into the Ba and Bb fragments when complexed with the activated form of C3, namely C3b. The Bb fragment contains a von Willebrand factor type A (vWF-A) domain, which is composed of an open twisted almost-parallel beta-sheet flanked on both sides by seven alpha-helices A1 to A7, with a metal coordination site at its active-site cleft. Homology modelling of this vWF-A domain shows that the metal-binding site was present. Two recombinant vWF-A domains (Gly229-Ile444 and Gly229-Gln448) were examined by circular dichroism and Fourier transform infrared spectroscopy and indicated a significant conformational transition in the presence and absence of Mg(2+). Two upfield-shifted signals in the (1)H NMR spectrum were used as sensitive probes of the vWF-A protein structure, one of which was assigned to a methyl group and demonstrated metal- and pH-dependent properties between two distinct conformations. Temperature denaturation studies followed by spectroscopy showed that metal-binding caused the vWF-A structure to become significantly more stable. Ring current calculations based on a homology model for the vWF-A structure correlated one upfield-shifted signal with a methyl group on the alpha-helices in the vWF-A structure and the other one with individual single protons. An allosteric property of the vWF-A domain has thus been identified, and its implications for factor B activation were examined. Since the vWF-A domain after alpha-helix A7 is connected by a short link to the catalytic serine protease domain in the Bb fragment, the identification of a metal-free and a more stable metal-bound conformation for the vWF-A domain implies that the vWF-A interaction with C3b may alter its Mg(2+)-bound coordination in such a way as to induce conformational changes that may regulate the proteolytic activity of factor B.     

Circular dichroism and Fourier transform infrared spectroscopic studies on self-assembly of tetrapeptide derivative in solution and solvated film.

Aggregation of the hydrophobic peptide derivative Boc-Ala-Ile-Ile-Gly-OMe (1) was examined in methanol solution and in solvated film states. Formation of the peptide by self-assembly was evidenced using fluorescence [Mg salt of 8-anilino-naphthalenesulfonic acid (ANS) as an external probe] and circular dichroism (CD) spectroscopic techniques. In solution, peptide 1 formed as a stable aggregate at a concentration around 3 x 10(-4)m. The peptide gelled into a thin film for which we carried out CD and Fourier transform infrared (FTIR) measurements. Our spectroscopic study on peptide films at differing methanol concentrations indicates that the helical content of the peptide decreases with decreasing methanol concentration in solvated films. However, by reducing the methanol concentration we were able to observe a conformational transition from a predominantly helical turn to a beta-sheet structure via a random coil conformation. Our study focused on the aggregation of the alpha-helical turn-forming peptide derivative, which shows conformational transition on changing solvent concentration in the film form.     

Helical structure of sugar-carrying polystyrene in aqueous solution by circular dichroism

Radical polymerization of N-p-vinylbenzyl-D-lactonamide (VLA) gave an optically active helical polymer. The stereoregularity of poly(N-p-vinylbenzyl-D-lactonamide) (PVLA) measured by 13C NMR spectroscopy showed a well-resolved sharp-line width, which was assigned to the phenyl C-1 carbon of the isotactic polystyrene (PS). The helical structure of PVLA shown by circular dichroism (CD) indicated that the aromatic groups were chirally supramolecular-packed giving optically active disaccharide units in the side chain covalently linked via an amide linkage with PS, the original PS not being optically active. The intensity of CD for PVLA (a) decreased with increasing temperature due to the change in the conformation of the phenyl group or to the breakdown of intermolecular hydrogen bonding of amide groups and (b) increased in a mixture of water and alcohol due to the increased hydrophobicity. The CD intensity for maltose-carrying PS (PVMA) was slightly higher than that of PVLA CD due to the more hydrophobic property of PVMA than PVLA.     

Deglycosylation of alpha 1-proteinase inhibitor by endo-beta-N-acetylglucosaminidase F

The glycosidase endo-beta-N-acetylglucosaminidase F (endo F) from Flavobacterium meningosepticum was used for the deglycosylation of rat alpha 1-proteinase inhibitor (alpha 1 PI). alpha 1 PI containing three oligosaccharide side chains of the complex type was isolated from rat serum or from the medium of rat hepatocyte primary cultures. High-mannose-type alpha 1 PI or hybrid-type alpha 1 PI was isolated from the media of hepatocytes treated with 1-deoxymannojirimycin or swainsonine, respectively. The susceptibility of complex-type alpha 1 PI to endo F was studied in the presence of various detergents. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate and octyl glucopyranoside turned out to be most effective. In the absence of detergents, digestion of alpha 1 PI with high concentrations of endo F and/or long times of incubation led to the formation of alpha 1 PI with one and two oligosaccharide side chains. In the presence of 0.5% octyl glucopyranoside, the major cleavage products were unglycosylated alpha 1 PI and alpha 1 PI carrying one carbohydrate side chain. In contrast to the complex-type alpha 1 PI, the high-mannose type can be totally deglycosylated by endo F even in the absence of detergents. The susceptibility of the hybrid-type alpha 1 PI to endo F is between that of the complex and the high-mannose types.     

Qualitative studies of lung lavage alpha 1-proteinase inhibitor

A method is described which enables identification of the molecular size of alpha 1-proteinase inhibitor (alpha 1-PI) in biological fluids. This technique when applied to bronchoalveolar lavage fluids clearly demonstrates alpha 1-PI in three molecular forms; the native molecule (Mr approximately equal to ++54 000), a partially proteolysed form (Mr approximately equal to 49 000) and in a form suggestive of a complex with enzyme (Mr approximately equal to 82 000). Samples showing the presence of native alpha 1-PI inhibited more porcine pancreatic elastase than samples where no native alpha 1-PI was seen or where the predominant form was partially proteolysed alpha 1-PI (p less than 0.01). Although the predominant band of alpha 1-PI was more frequently the partially proteolysed form in current smokers (p less than 0.01), there was no clear difference in the inhibitory function of alpha 1-PI between current smokers and non-smokers and those with and without airflow obstruction.     

Computer-aided analysis of infrared, circular dichroism and absorption spectra

Marquardt and Powell optimization methods without constraintson the optimized spectral parameters were employed for decompositionof complex i.r., c.d. and absorption spectra into componentbands. The procedure resolved experimental spectra into eightcomponent bands and it can be easily adjusted for a larger setof component bands. The CPU time required for achievement ofsatisfactory convergence of parameters for eight component bandsis rather large even when using mainframe computers and thereforedivision of spectra into a few non-overlapped parts is advisable.The program also can be used for calculation of absorption,c.d. and difference spectra from formatted raw spectral data. Received on January 13, 1986; accepted on April 7, 1986     

Frequency analysis of infrared absorption and vibrational circular dichroism of proteins in D2O solution.

The IR absorption frequencies as derived from second derivatives of the Fourier transform IR spectra of the amide I' bands of globular proteins in D2O are compared to those obtained from band fitting of the vibrational circular dichroism (VCD) spectra. The two sets of frequencies are in very good agreement, yielding consistent ranges where amide I' VCD and IR features occur. Use of VCD to complement the IR allows one to add sign information to the frequency information so that features occurring in the overlapping frequency ranges that might arise from different secondary structures can be better discriminated. From this comparison, it is clear that correlation just of the frequency of a given IR transition to secondary structure can lead to a nonunique solution. Different sign patterns were identified for correlated groups of globular proteins in restricted frequency ranges that have been previously assigned to defined secondary structural elements. Hence, different secondary structural elements must contribute band components to a given frequency range.     

A novel mode of polymerization of alpha1-proteinase inhibitor

Patients homozygous for the Z mutant form of alpha1-proteinase inhibitor (alpha1-PI) have an increased risk for the development of liver disease because of the accumulation in hepatocytes of inclusion bodies containing linear polymers of mutant alpha1-PI. The most widely accepted model of polymerization proposes that a linear, head-to-tail polymer forms by sequential insertion of the reactive center loop (RCL) of one alpha1-PI monomer between the central strands of the A beta-sheet of an adjacent monomer. This model derives primarily from two observations: peptides that are homologous with the RCL insert into the A beta-sheet of alpha1-PI monomer and this insertion prevents alpha1-PI polymerization. Normal alpha1-PI monomer does not spontaneously polymerize; however, here we show that the disulfide-linked dimer of normal alpha1-PI spontaneously forms linear polymers in buffer. The monomers within this dimer are joined head-to-head. Thus, the arrangement of monomers in these polymers must be different from that predicted by the loop-A sheet model. Therefore, we propose a new model for alpha1-PI polymer. In addition, polymerization of disulfide-linked dimer is not inhibited by the presence of the peptide even though dimer appears to interact with the peptide. Thus, RCL insertion into A beta-sheets may not occur during polymerization of this dimer.     

Modeling the intact form of the alpha 1-proteinase inhibitor

The structure of the intact form of the serpin alpha 1-proteinase inhibitor has been modeled based on the assumption that the central strand s4A of the six-stranded beta-sheet A of the cleaved inhibitor is not incorporated into the sheet of intact alpha 1-proteinase inhibitor. This strand was removed from its position in the center of the sheet by suitable rotations about the backbone dihedrals of Lys343 using molecular graphics. The resulting structure was then annealed using molecular dynamics (MD) while applying progressive distance restraints to the reactive peptide bond (Met358-Ser359) for 50 ps. During this time, the disrupted beta-sheet reformed to create a five-stranded beta-sheet with strands 3 and 5 in a parallel arrangement. This change and accompanying structural rearrangements are largely confirmed by the X-ray structure of plakalbumin, whose structure reflects the overall structure of intact serpins. The successful modeling experiment demonstrates the utility of MD for making gross structural predictions based on related structures. The binding loop of the intact form is modeled to allow docking with serine proteinases, in particular thrombin, which most highly constrains the possible conformations of the binding loop.     

Conformational analysis of hemopexin by fourier-transform infrared and circular dichroism spectroscopy

Hemopexin is a serum glyco-protein that binds heme with the highest known affinity of any characterized heme-binding protein and plays an important role in receptormediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far-UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier-transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% β-structure, 15% α-helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme-binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis-histidyl heme–hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme-binding function and the native structure of the hemeprotein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor-mediated endocytosis. © 1994 Wiley-Liss, Inc.     

Differential scanning calorimetric,circular dichroism,and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans

The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64 degrees C and an exothermic event centered near 75 degrees C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications.     

vpu transmembrane peptide structure obtained by site-specific fourier transform infrared dichroism and global molecular dynamics searching.

The recently developed method of site-directed Fourier transform infrared dichroism for obtaining orientational constraints of oriented polymers is applied here to the transmembrane domain of the vpu protein from the human immunodeficiency virus type 1 (HIV-1). The infrared spectra of the 31-residue-long vpu peptide reconstituted in lipid vesicles reveal a predominantly alpha-helical structure. The infrared dichroism data of the (13)C-labeled peptide yielded a helix tilt beta = (6.5 +/- 1.7) degrees from the membrane normal. The rotational pitch angle omega, defined as zero for a residue located in the direction of the helix tilt, is omega = (283 +/- 11) degrees for the (13)C labels Val(13)/Val(20) and omega = (23 +/- 11) degrees for the (13)C labels Ala(14)/Val(21). A global molecular dynamics search protocol restraining the helix tilt to the experimental value was performed for oligomers of four, five, and six subunits. From 288 structures for each oligomer, a left-handed pentameric coiled coil was obtained, which best fits the experimental data. The structure reveals a pore occluded by Trp residues at the intracellular end of the transmembrane domain.     

Involvement of hyposialylated immunoglobulins in the inactivation of alpha 1-proteinase inhibitor

The elastase inhibitory capacity of alpha 1-proteinase inhibitor (alpha 1-PI) was measured, using a direct and reproducible method, with phagocytic cells maintained in the tissue culture plate through the assay. The oxidative inactivation of alpha 1-PI is known to be mediated by the action of myeloperoxidase (MPO). The fact that hyposialylated IgG (hs IgG) induce the release of MPO prompted us to investigate the effects of such hs IgG on the inhibitory capacity of alpha 1-PI. The results show that 1-PI inactivation was observed only when phagocytic cells were activated by aggregated hs IgG, and not by unaggregated hs IgG. These observations indicate that hyposialylation should be completed by aggregation to perpetuate the oxidative reactions characteristic of inflammatory diseases.     

Fourier transform infrared spectroscopic analysis of protein secondary structures

Infrared spectroscopy is one of the oldest and well established experimental techniques for the analysis of secondary structure of polypeptides and proteins. It is convenient, non-destructive, requires less sample preparation, and can be used under a wide variety of conditions. This review introduces the recent developments in Fourier transform infrared （FTIR） spectroscopy technique and its applications to protein structural studies. The experimental skills, data analysis, and correlations between the FTIR spectroscopic bands and protein secondary structure components are discussed. The applications of FTIR to the second- ary structure analysis, conformational changes, structural dynamics and stability studies of proteins are also discussed.     

High-level production and isolation of human recombinant alpha 1-proteinase inhibitor in yeast

The cDNA coding for mature human alpha 1-proteinase inhibitor (alpha 1-PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated alpha 1-PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2mu and the leu2D selection marker. Biologically active recombinant alpha 1-PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high-Mr yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE-agarose, Zn-chelate agarose, kappa-chain agarose, heparin-agarose and aminohexyl-agarose.     

Comparative properties of human alpha-1-proteinase inhibitor glycosylation variants

Variant forms of human alpha-1-proteinase inhibitor (alpha-1-PI), obtained by the treatment of human Hep G2 cells with specific inhibitors of glycosylation were tested for both inhibitory activity and heat stability. All were found to have the same second-order association rate with human neutrophil elastase, indicating a lack of importance of the carbohydrate moiety. In contrast, incompletely glycosylated forms of alpha-1-PI were found to be heat sensitive relative to the mature protein, suggesting a role for carbohydrate in protein stabilization.