Conformational mapping of the N-terminal segment of surfactant protein B in lipid using 13C-enhanced Fourier transform infrared spectroscopy.

Synthetic peptides based on the N-terminal domain of human surfactant protein B (SP-B1-25; 25 amino acid residues; NH2-FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full-length, 79-residue protein. Here, we used physical techniques to examine the secondary conformation of SP-B1-25 in aqueous, lipid and structure-promoting environments. Circular dichroism and conventional, 12C-Fourier transform infrared (FTIR) spectroscopy each indicated a predominate alpha-helical conformation for SP-B1-25 in phosphate-buffered saline, liposomes of 1-palmitoyl-2-oleoyl phosphatidylglycerol and the structure-promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant beta- and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope-enhanced FTIR spectroscopy was performed with 1-palmitoyl-2-oleoyl phosphatidylglycerol liposomes and a suite of SP-B1-25 peptides labeled with 13C-carbonyl groups at either single or multiple sites. Combining these 13C-enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP-B1-25 in 1-palmitoyl-2-oleoyl phosphatidylglycerol: beta-sheet (residues 1-6), alpha-helix (residues 8-22) and random (residues 23-25) conformations. Analogous structural motifs are observed in the corresponding homologous N-terminal regions of several proteins that also share the 'saposin-like' (i.e. 5-helix bundle) folding pattern of full-length, human SP-B. In future studies, 13C-enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane environments.     

Secondary structure in properdin of the complement cascade and related proteins: a study by Fourier transform infrared spectroscopy

Six structural repeat motifs of 58 amino acids are found in the sequence of both mouse and human properdins. Twelve more examples of the motif are available from the sequences of thrombospondin, the terminal complement components, and the thrombospondin-related anonymous protein. The averaged Robson and Chou-Fasman secondary structure predictions show that there are 57-66% turn and 19-38% beta-sheet structures in the typical repeat motif. The high amount of turn structure is consistent with Gly, Pro, Cys, and Ser being the four most abundant amino acid residues in properdin. Comparisons with sequences found in the circumsporozoite protein from several species of malaria parasites show that their sequences and secondary structures strongly coincide only in a 18-residue segment. Further secondary structure analysis utilized Fourier transform infrared spectroscopy of human properdin in 2H2O buffers. These show a broad amide I band that, after second-derivative and deconvolution calculations, is shown to be composed of several components. Two at 1633 and 1683 cm-1 are strong evidence for beta-sheet structure, although overlap from beta-turns can also contribute. The presence of beta-turn structure is indicated by absorptions at 1662-1675 and 1645 cm-1. The properdin structure contains substantial quantities of beta-sheet and beta-turn structures, which is consistent with the secondary structure predictions and amino acid compositions. The length of the repeat motif is estimated as 3.3-4.3 nm, and an estimated 14-22% of nonexchanged amide protons reside in properdin. This is suggestive of a high degree of solvent accessibility in the structure.     

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.     

Insight into protein structure and protein-ligand recognition by Fourier transform infrared spectroscopy

An overview of the application of Fourier transform infrared spectroscopy for the analysis of the structure of proteins and protein-ligand recognition is given. The principle of the technique and of the spectra analysis is demonstrated. Spectral signal assignments to vibrational modes of the peptide chromophore, amino acid side chains, cofactors and metal ligands are summarized. Several examples for protein-ligand recognition are discussed. A particular focus is heme proteins and, as an example, studies of cytochrome P450 are reviewed. Fourier transform infrared spectroscopy in combination with the various techniques such as time-resolved and low-temperature methods, site-directed mutagenesis and isotope labeling is a helpful approach to studying protein-ligand recognition.     

Temperature-pressure stability of green fluorescent protein: a Fourier transform infrared spectroscopy study

Green fluorescent protein (GFP) is widely used as a marker in molecular and cell biology. For its use in high-pressure microbiology experiments, its fluorescence under pressure was recently investigated. Changes in fluorescence with pressure were found. To find out whether these are related to structural changes, we investigated the pressure stability of wild-type GFP (wtGFP) and three of its red shift mutants (AFP, GFP(mut1), and GFP(mut2)) using Fourier transform infrared spectroscopy. For the wt GFP, GFP(mut1), and GFP(mut2) we found that up to 13-14 kbar the secondary structure remains intact, whereas AFP starts unfolding around 10 kbar. The 3-D structure is held responsible for this high-pressure stability. Previously observed changes in fluorescence at low pressure are rationalized in terms of the pressure-induced elastic effect. Above 6 kbar, loss of fluorescence is due to aggregation. Revisiting the temperature stability of GFP, we found that an intermediate state is populated along the unfolding pathway of wtGFP. At higher temperatures, the unfolding resulted in the formation of aggregates of wtGFP and its mutants.     

Low-temperature Fourier transform infrared spectroscopy of photoactive yellow protein

The photocycle intermediates of photoactive yellow protein (PYP) were characterized by low-temperature Fourier transform infrared spectroscopy. The difference FTIR spectra of PYP(B), PYP(H), PYP(L), and PYP(M) minus PYP were measured under the irradiation condition determined by UV-visible spectroscopy. Although the chromophore bands of PYP(B) were weak, intense sharp bands complementary to the 1163-cm(-1) band of PYP, which show the chromophore is deprotonated, were observed at 1168-1169 cm(-1) for PYP(H) and PYP(L), indicating that the proton at Glu46 is not transferred before formation of PYP(M). Free trans-p-coumaric acid had a 1294-cm(-1) band, which was shifted to 1288 cm(-1) in the cis form. All the difference FTIR spectra obtained had the pair of bands corresponding to them, indicating that all the intermediates have the chromophore in the cis configuration. The characteristic vibrational modes at 1020-960 cm(-1) distinguished the intermediates. Because these modes were shifted by deuterium-labeling at the ethylene bond of the chromophore while labeling at the phenol part had no effect, they were attributed to the ethylene bond region. Hence, structural differences among the intermediates are present in this region. Bands at about 1730 cm(-1), which show that Glu46 is protonated, were observed for all intermediates except for PYP(M). Because the frequency of this mode was constant in PYP(B), PYP(H), and PYP(L), the environment of Glu46 is conserved in these intermediates. The photocycle of PYP would therefore proceed by changing the structure of the twisted ethylene bond of the chromophore.     

Secondary structure changes stabilize the reactive-centre cleaved form of SERPINs. A study by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy.

Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals.

Aquaporins are integral membrane proteins found in diverse animal and plant tissues that mediate the permeability of plasma membranes to water molecules. Projection maps of two-dimensional crystals of aquaporin-1 (AQP1) reconstituted in lipid membranes suggested the presence of six to eight transmembrane helices in the protein. However, data from other sequence and spectroscopic analyses indicate that this protein may adopt a porin-like beta-barrel fold. In this paper, we use Fourier transform infrared spectroscopy to characterize the secondary structure of highly purified native and proteolyzed AQP1 reconstituted in membrane crystalline arrays and compare it to bacteriorhodopsin. For this analysis the fractional secondary structure contents have been determined by using several different algorithms. In addition, a neural network-based evaluation of the Fourier transform infrared spectra in terms of numbers of secondary structure segments and their interconnections [sij] has been performed. The following conclusions were reached: 1) AQP1 is a highly helical protein (42-48% alpha-helix) with little or no beta-sheet content. 2) The alpha-helices have a transmembrane orientation, but are more tilted (21 degrees or 27 degrees, depending on the considered refractive index) than the bacteriorhodopsin helices. 3) The helices in AQP1 undergo limited hydrogen/deuterium exchange and thus are not readily accessible to solvent. Our data support the AQP1 structural model derived from sequence prediction and epitope insertion experiments: AQP1 is a protein with at least six closely associated alpha-helices that span the lipid membrane.     

In situ study by polarization modulated Fourier transform infrared spectroscopy of the structure and orientation of lipids and amphipathic peptides at the air-water interface.

Free amphipathic peptides and peptides bound to dimyristoylphosphatidylcholine (DMPC) were studied directly at the air/water interface using polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). Such differential reflectivity measurements proved to be a sensitive and efficient technique to investigate in situ the respective conformations and orientations of lipid and peptide molecules in pure and mixed films. Data obtained for melittin, a natural hemolytic peptide, are compared to those of L15K7, an ideally amphipathic synthetic peptide constituted by only apolar Leu and polar Lys residues. For pure peptidic films, the intensity, shape, and position of the amide I and II bands indicate that the L15K7 peptide adopts a totally alpha-helical structure, whereas the structure of melittin is mainly alpha-helical and presents some unordered domains. The L15K7 alpha-helix axis is oriented essentially parallel to the air-water interface plane; it differs for melittin. When injected into the subphase, L15K7 and melittin insert into preformed expanded DMPC monolayers and can be detected by PMIRRAS, even at low peptide content (> 50 DMPC molecules per peptide). In such conditions, peptides have the same secondary structure and orientation as in pure peptidic films.     

Application of Fourier transform infrared spectroscopy to studies of aqueous protein solutions.

Modern protein Fourier transform infrared (FT-IR) spectroscopy has proven to be a versatile and sensitive technique, applicable to many aspects of protein characterization. The major practical drawback for the FT-IR spectroscopy of proteins is the large absorbance band of water, which overlaps the amide I resonances. D2O is often substituted for H2O in infrared experiments. Removal of water from protein samples can be complicated and tedious and potentially lead to denaturation, aggregation, or sample loss. Solvent removal by dialysis is difficult for suspensions and sols. A new method called the D2O dilution technique (Ddt) is described which simplifies the sample preparation step and improves the solvent subtraction. The effect of the D2O concentration on the IR spectrum of aqueous solutions of several model proteins was studied. Dilution of aqueous samples with D2O yields good quality spectra. The Ddt has been evaluated for quantitative analysis using standard proteins and its applicability to solutions and suspensions of a genetically engineered malaria antigen is demonstrated. Use of resolution-enhancement techniques with spectra in mixed solvents has also been investigated.     

Characterization of an RNA bulge structure by Fourier transform infrared spectroscopy

There may be several advantages associated with an antisense oligonucleotide that induces a bulged structure into its RNA target molecule. Many structures of RNA bulges are elucidated from single-stranded RNA models. However, a two-component system is the minimum requirement for a realistic antisense model. We have used Fourier transform infrared spectroscopy to investigate a single-stranded RNA oligonucleotide with known NMR solution structure, constructed to model a five nucleotide bulge, and its two-component oligonucleotide counterpart. The infrared spectra show A-helical base-paired stems and non-base-paired loops in both systems. The nucleosides are mainly in an anti-conformation. Both N-type and S-type of sugar puckers can be inferred from the infrared region sensitive to sugar conformations. The S-type of sugar pucker is likely to be associated with the nucleotides in the bulge. The FTIR results display an overall structural similarity between the two model systems.     

Secondary structure of the hydrophobic myelin protein in a lipid environment as determined by Fourier-transform infrared spectrometry

The secondary structure of a hydrophobic myelin protein (lipophilin), reconstituted with dimyristoylphosphatidylcholine or dimyristoylphosphatidylglycerol, was investigated by Fourier-transform infrared spectroscopy. Protein infrared spectra in the amide I region were analyzed quantitatively using resolution enhancement and band fitting procedures. Lipophilin in a phospholipid environment adopts a highly ordered secondary structure which at room temperature consists predominantly of alpha-helix (approximately 55%) and beta-type conformations (36%). The secondary structure of the protein is not affected by the lipid gel to liquid crystalline phase transition. Heating of the lipid-protein complex above approximately 35 degrees C results in a gradual decrease in alpha-helical content, accompanied by an increase in the amount of beta-structures. Lipophilin dissolved in 2-chloroethanol is, compared to the protein in a lipid environment, richer in the alpha-helical conformation but still contains a sizable amount of beta-structure.     

A study of the structure of human complement component factor H by Fourier transform infrared spectroscopy and secondary structure averaging methods

Fourier transform infrared spectroscopy was used to investigate the secondary structure of human complement component factor H in H2O and 2H2O buffers. The spectra show a broad amide I band which after second-derivative calculations is shown to be composed of three components at 1645, 1663, and 1685 cm-1 in H2O and at 1638, 1661, and 1680 cm-1 in 2H2O. The frequencies of these components are consistent with the existence of an extensive antiparallel beta-strand secondary structure. The exchange properties of the amide protons of factor H as measured in 2H2O buffers are rapid and lead to an estimate of NH proton nonexchange that is comparable with those for small globular proteins. Human factor H is constructed from a linear sequence of 20 short consensus repeats with a mean of 61 residues in each one. To investigate the secondary structure further, secondary structure predictions were carried out on the basis of an alignment scheme for 101 sequences for these repeats as found in human factor H and 12 other proteins. These predictions were averaged in order to improve the reliability of the calculations. Both the Robson and the Chou-Fasman methods indicate significant beta-structural contents. Residues 21-51 in the 61-residue repeat show a clear prediction of four strands of beta-structure and four beta-turns. A structural model based on antiparallel beta-strands in the secondary structure is proposed and discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secondary structure of food proteins by Fourier transform spectroscopy in the mid-infrared region

Fourier transform spectroscopy in the mid-infrared (400–5,000 cm⁻¹) (FT-IR) is being recognized as a powerful tool for analyzing chemical composition of food, with special concern to molecular architecture of food proteins. Unlike other spectroscopic techniques, it provides high-quality spectra with very small amount of protein, in various environments irrespective of the molecular mass. The fraction of peptide bonds in α-helical, β-pleated sheet, turns and aperiodic conformations can be accurately estimated by analysis of the amide I band (1,600–1,700 cm⁻¹) in the mid-IR region. In addition, FT-IR measurement of secondary structure highlights the mechanism of protein aggregation and stability, making this technique of strategic importance in the food proteomic field. Examples of applications of FT-IR spectroscopy in the study of structural features of food proteins critical of nutritional and technological performance are discussed.     

Protein secondary structure from Fourier transform infrared spectroscopy: a data base analysis

An infrared (ir) method to determine the secondary structure of proteins in solution using the amide I region of the spectrum has been devised. The method is based on the circular dichroism (CD) matrix method for secondary structure analysis given by Compton and Johnson (L. A. Compton and W. C. Johnson, 1986, Anal. Biochem. 155, 155-167). The infrared data matrix was constructed from the normalized Fourier transform infrared spectra from 1700 to 1600 cm-1 of 17 commercially available proteins. The secondary structure matrix was constructed from the X-ray data of the seventeen proteins with secondary structure elements of helix, beta-sheet, beta-turn, and other (random). The CD and ir methods were compared by analyzing the proteins of the CD and ir databases as unknowns. Both methods produce similar results compared to structures obtained by X-ray crystallographic means with the CD slightly better for helix conformation, and the ir slightly better for beta-sheet. The relatively good ir analysis for concanavalin A and alpha-chymotrypsin indicate that the ir method is less affected by the presence of aromatic groups. The concentration of the protein and the cell path length need not be known for the ir analysis since the spectra can be normalized to the total ir intensity in the amide I region. The ir spectra for helix, beta-sheet, beta-turn, and other, as extracted from the data-base, agree with the literature band assignments. The ir data matrix and the inverse matrix necessary to analyze unknown proteins are presented.     

Determination of molecular order in supported lipid membranes by internal reflection Fourier transform infrared spectroscopy.

When polarized internal reflection infrared spectroscopy is used to determine molecular order in supported lipid membranes, the results are critically dependent on the accuracy of assumptions made about the evanescent electric field amplitudes in the membrane. In this work, we examine several expressions used for calculating evanescent electric field amplitudes in supported lipid monolayers and bilayers, and test their validity by measuring the infrared dichroism of poly-gamma-benzyl-L-glutamate and poly-beta-benzyl-L-aspartate under conditions in which their molecular order is known. Our results indicate that treating such systems as a simple single interface between two semi-infinite bulk phases is more accurate than the commonly employed thin-film approximation. This implies that earlier conclusions about molecular order in supported lipid membranes may require substantial revision.     

Localization and orientation of functional water molecules in bacteriorhodopsin as revealed by polarized Fourier transform infrared spectroscopy.

Linear dichroic difference Fourier transform infrared spectra upon formation of the M photointermediate were recorded with oriented purple membranes. The purpose was to determine the angle of the directions of the dipole moments of 1) the water molecule whose O-H stretching vibration appears at 3643 cm-1 for the unphotolyzed state and 3671 cm-1 for the M intermediate, and 2) the C=O bond of protonated Asp85 in the M intermediate. The angle of 36 degrees we find for the C=O of the protonated Asp85 in the M intermediate is not markedly different from 26 degrees for unprotonated Asp85 in the model based on cryoelectron diffraction, indicating the absence of gross orientation changes in Asp85 upon its protonation. The O-H band at 3671 cm-1 of a water molecule in the M intermediate, although its position has not determined, is fixed almost parallel to the membrane plane. For the unphotolyzed state the angle of the water O-H to the membrane normal was determined to be 60 degrees. On the basis of these data and the structural model, we place the water molecule in the unphotolyzed state at a position where it forms hydrogen bonds with the Schiff base, Asp85, Asp212, and Trp86.     

The orientation of beta-sheets in porin. A polarized Fourier transform infrared spectroscopic investigation.

The orientation of the protein secondary structures in porin is investigated by Fourier transform infrared (FTIR) linear dichroism of oriented multilayers of porin reconstituted in lipid vesicles. The FTIR absorbance spectrum shows the amide I band at 1,631 cm-1 and several shoulders around 1,675 cm-1 and at 1,696 cm-1 indicative of antiparallel beta-sheets. The amide II is centered around 1,530 cm-1. The main dichroic signals peak at 1,738, 1,698, 1,660, 1,634, and 1,531 cm-1. The small magnitude of the 1,634 cm-1 and 1,531 cm-1 positive dichroism bands demonstrates that the transition moments of the amide I and amide II vibrations are on the average tilted at 47 degrees +/- 3 degrees from the membrane normal. This indicates that the plane of the beta-sheets is approximately perpendicular to the bilayer. From these IR dichroism results and previously reported diffuse x-ray data which revealed that a substantial number of beta-strands are nearly perpendicular to the membrane, a model for the packing of beta-strands in porin is proposed which satisfies both IR and x-ray requirements. In this model, the porin monomer consists of at least two beta-sheet domains, both with their plane perpendicular to the membrane. One sheet has its strands direction lying nearly parallel to the membrane normal while the other sheet has its strands inclined at a small angle away from the membrane plane.