D. Michael Byler

[13] Resolution-enhanced fourier transform infrared spectroscopy of enzymes

Publisher Summary This chapter describes the basic principles, techniques, and applications of resolution-enhanced Fourier transform infrared spectroscopy. Infrared spectroscopy constitutes one of the oldest methods for studying the secondary structure of polypeptides and proteins. Polypeptides and proteins exhibit a total of nine characteristic absorption bands in the infrared region. These are usually termed the amide A, B, and amide I-VII bands. The amide I (∼1630-1690 cm -1 ) band is the most useful for protein structure studies by infrared spectroscopy. The use of Fourier transform infrared spectroscopy (FTIR) has led to major improvements in this regard. In principle, FTIR provides several advantages over conventional dispersive techniques: higher (1) resolution, (2) sensitivity, (3) signal-to-noise ratio (S/N), and (4) frequency accuracy. Any one of the first three advantages can be emphasized at the expense of the other two. For protein structure studies, high sensitivity makes it possible to acquire usable infrared spectra of aqueous solutions; such spectra are always notoriously difficult to obtain.

Protein structure by Fourier transform infrared spectroscopy: Second derivative spectra

Second derivative Fourier transform infrared spectra of the proteins ribonuclease A, hemoglobin, and beta-lactoglobulin A (native and denatured) have been obtained in deuterium oxide solution from 1350 to 1800 cm-1. The relationship of the original spectra to their second derivatives is briefly discussed. In the second derivative spectra, clearly resolved peaks are observed which can be associated with the alpha-helix, beta-strands, and turns. No protein spectra with such resolution have heretofore been reported. Tentative assignments are proposed, and the observed peaks are related to the secondary structure of the proteins studied. The data appear to present the first direct spectroscopic evidence of turns in a native protein.

Protein Conformation by Infrared Spectroscopy: Resolution Enhancement by Fourier Self-Deconvolution

Fourier self-deconvolution (FSD) has been employed to enhance the resolution of the infrared spectra of proteins in the solid state and in D2O solution. The feasibility of using diffuse reflectance spectrometry for measuring the infrared spectra of solid proteins has been demonstrated. FSD permits inherently broad absorption bands to be resolved into distinct peaks which can be associated with specific protein secondary structures. Because the areas of the resolved peaks are the same as the areas of the previously unidentifiable components, this new method should enable quantitative estimates of the proportion of each conformation in a protein to be calculated.

Fourier transform infrared study of proteins with parallel β-chains

Abstract Deconvolved and second derivative Fourier transform infrared spectra of the proteins flavodoxin and triosephosphate isomerase have been obtained in the 1600 to 1700 cm −1 (amide I) region. To our knowledge these results provide the first experimental infrared data on proteins with parallel β-chains. Characteristic absorption bands for the parallel β-segments are observed at 1626–1639 cm −1 (strong) and close to 1675 cm −1 (weak). Previous theoretical studies based on hypothetical models with large, regular β-sheets had suggested bands close to 1650 and 1666 cm −1 . Our new assignments were confirmed by band area measurements, which yield conformational information in good agreement with results from X-ray diffraction data. The spectra were compared with corresponding spectra of concanavalin A and carboxypeptidase A. The first contains only antiparallel β-segments, the second “mixed” β-segments, with some strands lying antiparallel and others parallel. None of the observed amide I band frequencies assigned to parallel β-chains occurs in the 1650 cm −1 region associated with helical segments.

Vibrational analysis of amino acids: cysteine, serine, β-chloroalanine

Abstract Normal coordinate calculations were carried out involving a total of seven isotopically substituted analogs of the amino acids cysteine, serine, and β-chloroalanine. Raman spectra were obtained for polycrystalline β-chloroalanine and the ND3 analog. Overlay calculations were employed to obtain 55 force constants which reproduce 206 observed frequencies of seven molecules with an average error of ca. 9 cm−1. The valence force field used was based on local symmetry coordinates. Band assignments were based on the potential energy distribution. About 60% of the normal modes of the seven isotopomers can be called group vibrations by the PED criterion. Most skeletal stretching and bending vibrations are highly mixed and cannot be assigned to individual bond stretching or angle deformation modes.

Fourier Deconvolution of the Amide I Raman Band of Proteins as Related to Conformation

Fourier deconvolution has been employed to enhance the resolution of the amide I Raman band of nine proteins found in milk and/or other foods. The broad band was resolved into several components. The overall shape of the amide I Raman band of proteins was found to be nearly Gaussian or to be composed of Gaussian components. A Gaussian function was therefore used for deconvolution. The results obtained were more detailed than those obtained with the Lorentzian approximation usually employed. The resolved band components were assigned to specific protein conformations. The frequencies and assignments are in good agreement with previous Raman work based on entirely different procedures. The band areas of the resolved components appear to reflect the fraction of any given conformation in a protein. Semiquantitative estimations of protein conformation are in reasonable agreement with data obtained by x-ray diffraction and by infrared methods.

Application of computerized infrared and Raman spectroscopy to conformation studies of casein and other food proteins

SummaryThe ability of modern biotechnology to produce new or modified proteins has outpaced current understanding of the relationship between protein structure and protein function. Resolution-enhanced infrared spectroscopy and Raman spectroscopy are excellent non-destructive techniques for investigating the secondary structure of proteins under a wide variety of conditions. The techniques yield rapid, reliable estimates of the proportion of helical structure, β-strands, and turns of proteins in solution, as gels, or as solids. These methodologies can also detect subtle variations in protein conformation that frequently occur upon change of the biomolecular environment. In particular, it is possible to study structural changes which arise from alterations in pH, ionic strength, nature of solvent, and from interactions with other molecules or ions, such as another protein or Ca2+ ions. The first part of this paper will briefly review various important aspects of the techniques. The subsequent part describes application to structural problems of casein and other food proteins.

Vibrational analysis of l-alanine and deuterated analogs

Abstract Raman spectra of the polycrystalline l -alanine analogs CH 3 CH(NH + 3 )COO − , CH 3 CH(ND + 3 )-COO − , CD 3 CD(NH + 3 )COO − , and CD 3 CD(ND + 3 )COO − have been obtained. A normal coordinate analysis is carried out based on the experimental frequencies of the four isotopic analogs and a 34 parameter valence-type force field defined in terms of local symmetry coordinates. The final refinement, in which five stretching force constants are constrained to fixed values obtained from bond length data, results in an average error of 7 cm −1 (0.9%) for the observed frequencies of the four isotopically substituted molecules. Band assignments are given in terms of the potential energy distribution for local symmetry coordinates. For non-deuterated l -alanine, the vibrations above 1420 cm −1 and below 950 cm −1 may be described as localized group vibrations. By contrast, the eight modes in the middle frequency range, viz. the three skeletal stretching, the COO − symmetric stretching, one NH + 3 rocking, the symmetric CH 3 deformation, and the two methyne CH deformation vibrations, are very strongly coupled to one another. Some decoupling appears to take place in the perdeutero molecule, and all but five modes can be described as localized group vibrations.

Resolution-enhanced FT-IR spectra of soil constituents: humic acid

Fourier deconvolution and second-derivative spectroscopy have been used to enhance the resolution of infrared spectra of humic acid (HA) extracted from dried municipal waste. The results demonstrate the extent to which the use of such resolution enhancement techniques can disclose new valuable information concerning the composition of complex heterogeneous samples. The findings are in good agreement with previously obtained solid-state NMR results.

FT-IR Spectra of Soil Constituents: Fulvic Acid and Fulvic Acid Complex with Ferric Ions

FT-IR spectra and corresponding second-derivative spectra, as well as pertinent difference spectra, of fulvic acid and of fulvic acid complexed with ferric ions have been obtained. The data reveal that the major functional groups present in the fulvic acid studied are carboxyl groupings and CH2 groupings of hydrocarbon chains. The presence of ferric ions results in increased ionization of the COOH groups to form COO− ions, as well as a change of the characteristic COO− stretching frequencies of the carboxylate ions. The data thus indicate that ferric ions in soils interact with carboxylate ions of fulvic acids. The second-derivative spectra permit the identification of certain alkyl and aromatic hydrocarbon vibrational modes which cannot be observed in the original FT-IR spectra.