Aqueous solution infrared spectroscopy proteins

Very few of the infrared studies of proteins have been carried out on aqueous solutions of the proteins. Except for the work of Koenig and Tabb (1), the few aqueous IR studies have been on single proteins. Correspondingly, most of the assignments of the backbone vibrations (the so-called Amide I, II, III, etc. vibrations) have been based on either Raman spectra of aqueous solutions (2) or on infrared spectra of proteins in the solid state (3). Where infrared solution spectra have been obtained, it has mostly been on D2O solutions (4) - not H2O solutions. Since these Amide I, II, III, etc. vibrations involve motion of the protein backbone, they are sensitive to the secondary structure of the protein and thus valid assignments are necessary in order to use infrared spectroscopy for determining the conformations of proteins. 

The secondary structure of proteins may also be assessed using vibrational spectroscopy, fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy both provide information on the secondary structure of proteins. The bulk of the literature using vibrational spectroscopy to study protein structure has involved the use of FTIR. Water produces vibrational bands that interfere with the bands associated with proteins. For this reason, most of the FTIR literature focuses on the use of this technique to assess structure in the solid state or in the presence of non-aqueous environments. Recently, differential FTIR has been used in which a water background is subtracted from the FTIR spectrum. This workaround is limited to solutions containing relatively high protein concentrations. 

Fourier-transform infrared (FTIR) spectroscopy is particularly useful for probing the structures of membrane proteins [3, 23]. This technique can be used to study the secondary structures of proteins, both in their native environment as well as after reconstitution into model membranes. Myelin basic protein (MBP) is a major protein of the nervous system and has been studied by using FTIR spectroscopy in both aqueous solution and after reconstitution in myelin lipids [24]. The amide I band of MBP in D2O solution (deconvolved and curve-fitted) is... 

The combination of infrared reflection absorption spectroscopy and the Fourier transform technique has proven to be useful for structural studies of mono-molecular protein and amino acid films formed on metal surfaces. All protein films investigated exhibit a distinct blue shift of the Amide I frequency upon adsorption on metal surfaces, compared to the same band in aqueous solution. The magnitude of this blue shift seems to be larger for proteins with dominating )8-structure (32 cm" ) than for those with a large amount of a-helix or disordered structures (ca. 20 cm" ). It is concluded from reference spectra of... 

In the solid state, chitin mainly occurs as two allomorphs, the a- and (3-forms, which are differentiated by infrared and solid state NMR spectroscopies and X-ray diffraction [3,4]. a-chitin is the most abundant, occurring in krill, lobsters and crab tendons, shrimp and crab shells. It also results from recrystallization of (3-chitin in solution treated with strong aqueous HCl (over 7N) and washed with water because the a-form is thermodynamically more stable than (3-chitin [5]. (3-chitin is found in association with proteins in squid pens [3, 6] or in the tubes synthesized by pogonophoran and vestimetiferan worms [7,8]. In the solid state, the chains are parallel in (3-chitin and antiparallel in a-chitin. Their crystalline structures were reviewed in different papers [1, 4, 9-13]. Chitin is insoluble in all the usual solvents while taking into account their crystalline forms (a or (3) because the reactivity of p-chitin is larger than that of the a-isomorph, which is important for enzymatic and chemical transformations of chitin [14]. 

Raman spectroscopy is a vibrational spectroscopic technique which can be a useful probe of protein structure, since both intensity and frequency of vibrational motions of the amino acid side chains or polypeptide backbone are sensitive to chemical changes and the microenvironment around the functional groups. Thus, it can monitor changes related to tertiary structure as well as secondary structure of proteins. An important advantage of this technique is its versatility in application to samples which may be in solution or solid, clear or turbid, in aqueous or organic solvent. Since the concentration of proteins typically found in food systems is high, the classical dispersive method based on visible laser Raman spectroscopy, as well as the newer technique known as Fourier-transform Raman spectroscopy which utilizes near-infrared excitation, are more suitable to study food proteins (Li-Chan et aL, 1994). In contrast the technique based on ultraviolet excitation, known as resonance Raman spectroscopy, is more commonly used to study dilute protein solutions. 

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