Circular dichroism protein structure determination

Of the visible spectroscopic techniques, CD spectroscopy has seen the most rapid and dramatic growth. The far-UV circular dichroism spectrum of a protein is a direct reflection of its secondary structure [71]. An asymmetrical molecule, such as a protein macromolecule, exhibits circular dichroism because it absorbs circularly polarized light of one rotation differently from circularly polarized light of the other rotation. Therefore, the technique is useful in determining changes in secondary structure as a function of stability, thermal treatment, or freeze-thaw. 

Determination of protein secondary structure has long been a major application of optical spectroscopic studies of biopolymers (Fasman, 1996 Havel, 1996 Mantsch and Chapman, 1996). These efforts have primarily sought to determine the average fractional amount of overall secondary structure, typically represented as helix and sheet contributions, which comprise the extended, coherent structural elements in well-structured proteins. In some cases further interpretations in terms of turns and specific helix and sheet segment types have developed. Only more limited applications of optical spectra to determination of tertiary structure have appeared, and these normally have used fluorescence or near-UV electronic circular dichroism (ECD) of aromatic residues to sense a change in the fold (Haas, 1995 Woody and Dunker, 1996). 

Aside from the direct techniques of X-ray or electron diffraction, the major possible routes to knowledge of three-dimensional protein structure are prediction from the amino acid sequence and analysis of spectroscopic measurements such as circular dichroism, laser Raman spectroscopy, and nuclear magnetic resonance. With the large data base now available of known three-dimensional protein structures, all of these approaches are making considerable progress, and it seems possible that within a few years some combination of noncrystallo-graphic techniques may be capable of correctly determining new protein structures. Because the problem is inherently quite difficult, it will undoubtedly be essential to make the best possible use of all hints available from the known structures. 

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. 

Although X-ray crystallography, NMR, and circular dichroism are extremely valuable techniques for determining the structure of crystalline proteins or proteins in solution, they cannot be used to study proteins adsorbed on surfaces. Vibrational spectroscopy (infrared and Raman) appears to be the best approach available for bridging the gap between adsorbed proteins and proteins in solution. 

Bioactive macromolecules like peptides, proteins, and nucleic acids have been successfully embedded in planar LbL films. An important question is the retention of the bioactivity of the film-embedded biomolecules. The structural properties and stability of the LbL films formed from synthesized polypeptides of various amino acid sequences were recently reported [50]. The authors showed that control over the amino acid sequence enables control over non-covalent interpolypeptide interaction in the film, which determines the film properties. Haynie and coworkers showed by circular dichroism spectroscopy that the extent of adsorption of poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) in the LbL films scales with the extent of secondary structure of the polypeptides in solution [51]. Boulmedais demonstrated that the secondary structure of the film composed of these polypeptides is the same as the peptide structure in the complex formed in solution [52], as found by Fourier transform IR spectroscopy (FUR). 

In general, when one wants to determine if global and/or local structural modifications have occurred within a protein, circular dichroism experiments are performed. Also, one can record the fluorescence excitation spectrum of the protein. If perturbations occur within the protein, one should observe excitation spectra that differ from one state to another. One should not forget to correct the recorded spectra for the inner filter effect. 

Optical chirality of molecules is a characteristic measure of stereo-chemical property of biological, pharmaceutical, and metal coordination compounds. Choral structures of amino acids, proteins, DNAs, and various drugs in solutions have been determined from the measurement of circular dichroism (CD). However, small amount of molecules at the liquid-liquid interfaces has never been measured before CLM/CD method [19] and SHG/CD method have been reported [20],... 

The study by Determan et al. [224] focuses on the effects of polymer degradation products on the primary, secondary, and tertiary structure of TT, OVA, and lysozyme after incubation for 0 or 20 days in the presence of ester (lactic acid and glycolic acid) and anhydride [sebacic acid and l,6-bis(p-carboxyphenoxy)hexane] monomers. The structure and antigenicity or enzymatic activity of each protein in the presence of each monomer was quantified. SDS-PAGE, circular dichroism, and fluorescence spectroscopy were used to assess/evaluate the primary, secondary, and tertiary structures of the proteins, respectively. ELISA was used to measure changes in the antigenicity of TT and OVA and a fluorescence-based assay was used to determine the enzymatic activity of lysozyme. TT toxoid was found to be the most stable in the presence of anhydride monomers, while OVA was most stable in the... 

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