Fourier transform infrared dichroism with

The availability of the purified transporter in large quantity has enabled investigation of its secondary structure by biophysical techniques. Comparison of the circular dichroism (CD) spectrum of the transporter in lipid vesicles with the CD spectra of water-soluble proteins of known structure indicated the presence of approximately 82% a-helix, 10% ) -turns and 8% other random coil structure [97]. No / -sheet structure was detected either in this study or in a study of the protein by the same group using polarized Fourier transform infrared (FTIR) spectroscopy [98]. In our laboratory FTIR spectroscopy of the transporter has similarly revealed that... 

IR dichroism has also been particularly helpful in this regard. Of predominant interest is the orientation factor S=( 1/2)(3—1) (see Chapter 8), which can be obtained experimentally from the ratio of absorbances of a chosen peak parallel and perpendicular to the direction in which an elastomer is stretched [5,249]. One representation of such results is the effect of network chain length on the reduced orientation factor [S]=S/(72—2 1), where X is the elongation. A comparison is made among typical theoretical results in which the affine model assumes the chain dimensions to change linearly with the imposed macroscopic strain, and the phantom model allows for junction fluctuations that make the relationship nonlinear. The experimental results were found to be close to the phantom relationship. Combined techniques, such as Fourier-transform infrared (FTIR) spectroscopy combined with rheometry (see Chapter 8), are also of increasing interest [250]. 

Even though these approaches are powerful methods for determining functional sites on proteins, they are limited if not coupled with some form of structural determination. As Figure 2 illustrates, molecular biology and synthetic peptide/antibody approaches are not only interdependent, they are tied in with structural determination. Structural determination methods can take many forms, from the classic x-ray crystallography and NMR for three-dimensional determination, to two-dimensional methods such as circular dichroism and Fourier Transformed Infrared Spectroscopy, to predictive methods and modeling. A structural analysis is crucial to the interpretation of experimental results obtained from mutational and synthetic peptide/antibody techniques. 

Secondary structure is most easily assessed by spectroscopic methods, in particular circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). These techniques can be used to determine the content of a-helices, /3-sheets and, with... 

Low-resolution optical spectroscopic measurements established that the PrPc—>PrPSc conversion is accompanied by a major decrease in a-helical content and an increase in (3-sheet structure [101-104]. However, the secondary structure of PrPSc remains controversial, with the estimated content of a-helical structure ranging from 0%, as assessed by circular dichroism spectroscopy [104], to 0-21%, as inferred from Fourier-transform infrared measurements [101-103, 105]. These low resolution spectroscopic measurements are subject to considerable uncertainty thus, caution should be exercised when using global secondary structure estimates for constructing specific high-resolution structural models of PrPSc. 

A study with the hydrophobic signal peptide of coli lambda phage in phospholipid monolayers, showed a preference for pC helical conformation when the peptide was inserted into the lipid phase (18), However, interaction with the lipid surface without insertion induced the peptide to adopt the -structure ), These observations, obtained with circular dichroism and Fourier transform-infrared (FT-IR) data, provide the first direct evidence for interconversions between various conformational... 

The stmctural and conformational analysis of proteins adsorbed to solid surfaces is difficult because most common analytical methods are not compatible with the presence of the interacting solids. With recent developments in instrumentation and techniques, our understanding of protein adsorption behavior has improved considerably [4, 14]. The most commonly used techniques include attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), radiolabeling techniques, immunofluorescence enzyme-linked immunosorbent assay (ELISA), ellipsometry, circular dichroism (CD) spectroscopy, surface plasmon resonance (SPR), and amide HX with nuclear magnetic resonance (NMR). Atomic force microscopy (AFM) and scanning... 

Infrared dichroism was studied using a Fourier-transform infrared spectrometer equipped with a polarizer. The absorbances of the bands at 841 and 973 cm were measured using a linearly polarized IRbeam. ... 

Despite the sequence identity of PrP and PrP from the same species, they display quite different physical properties. Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy reveal that PrP - is essentially a-helical and devoid of P-pleated sheet structure, whereas the P-pleated sheet content of PrP is -40%, while PrP27-30 contains more than 50 % p-pleated sheet. Procedures that destroy the infectivity of PrP27-30 and PrP (e. g. purification by SDS-polyacrylamide gel electrophoresis or treatment with alkali) also substantially reduce the P-pleated sheet content. It therefore appears that the difference between the normal and disease-causing isoforms of PrP is entirely conformational and related to the respective contents of a-helical structure and P-pleated sheet structure. 

Rusling and his collaborators have obtained reversible voltammetric responses of cyt. P-450cam. hemoglobin, Mb, cyt. c, and chlorella ferredoxin cast in bquid crystal films or composites of liquid crystal and Nafion [79-81] on gold electrodes. The structure of the cast films was characterized by low angle x-ray diffraction. The structures of Mb and cyt. P-450cam hi the cast film were characterized by UV-vis. Electron Spin Resonance (ESR), and reflectance Fourier Transform Infrared (FT-IR) spectroscopic techniques, and are shown to be similar to the native conformations. Visible linear dichroism and ESR anisotropy showed that Mb is specifically oriented in the static films. The orientation of the heme plane of Mb averaged 60° with respect to the normal to the film plane, and distributions are rather broad. 

Because neither PrP" nor PrP " " could be crystallized, it was not possible to establish the 3D structure of these proteins by X-ray crystallography To circumvent this xmfavorable situation, Prusiner used circular dichroism and Fourier transformed infrared (FTIR) spectroscopy, both techniques that allow determination of the respective percentages of the secondary structures (a, (3, and turns) in a peptide or a protein. Applied to PrP and PrP samples, these spectroscopic approaches definitely demonstrated that both isoforms have a distinct 3D structure. In a seminal article published in 1993, the Prusiner s group established that PrP is chiefly an a-helical protein (43% of a-helix structure) with few -structures (3%). In contrast, PrP has less a-helix (30%) but has gained a high percentage of -structures (43%). 

This process was confirmed by both circular dichroism and Fourier transform infrared spectroscopy, suggesting tirat a-synuclein has a slightly collapsed conformation, with 14% of its secondary structure corresponding to tums. In line with these data, the determination of the hydrodynamic dimensions of a-synuclein revealed that the protein was indeed... 

In this chapter, a sensitive method for measurement by continuous-scan Fourier-transform infrared (FT-IR) spectrometry called polarization-modulation spectrometry is introduced this is a useful method for measuring, with high signal-to-noise ratio, not only the reflection-absorption spectra of thin films adsorbed onto metal substrates but also other spectra such as vibrational circular dichroism (VCD) spectra. Polarization-modulation spectrometry is a type of double-modulation FT-IR spectrometry [1], In this chapter, descriptions of double-modulation spectrometry are given first, then polarization-modulation spectrometry is discussed, and then its application to the measurement of reflection-absorption spectra of thin films on metal substrates is discussed. 

A benefit of peptide synthesis is that one can easily fine tune amino acid content (i.e. primary structure) of peptides to customize final molecular conformations, gelation times, or other molecular interactions. This allows the creation of an array of peptides with minute differences in sequences but vast differences in final properties. Characterization is important to understanding the secondary structures formed by individual peptides and, consequently, the higher order structures that are formed during gelation. To confirm and characterize second order stracture, two spectroscopic methods, circular dichroism spectroscopy (CD) and Fourier-transform infrared spectroscopy (FUR) are frequently used. Both methods examine light absorption... 

The vast introduction of the more sophisticated Fourier transform infrared (FT-IR) spectrometers for spectral studies made the CBS method senseless in terms of its further application. In the FT-IR analyses, the linearly polarized measurements are conducted by sequential photometrization of a sample for both positions of the polarizer. The linear dichroism is differentially registered by subtraction of the two spectra, which is implanented promptly and easily by the modem sophisticated FT-IR spectrophotometers, equipped with computerized systans. 

We present the basic concepts and methods for the measurement of infrared and Raman vibrational optical activity (VOA). These two forms of VOA are referred to as infrared vibrational circular dichroism (VCD) and Raman optical activity (ROA), respectively The principal aim of the article is to provide detailed descriptions of the instrumentation and measurement methods associated with VCD and ROA in general, and Fourier transform VCD and multichannel CCD ROA, in particular. Although VCD and ROA are closely related spectroscopic techniques, the instrumentation and measurement techniques differ markedly. These two forms of VOA will be compared and the reasons behinds their differences, now and in the future, will be explored. 

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