Protein spectral perturbation

Gonzalez, D. S., Sawyer, A., and Ward, W. W. (1997). Spectral perturbations of mutants of recombinant Aequorea victoria green-fluorescent protein (GFP). Photochem. Photobiol. 65 21S. 

Ward WW, Prentice HJ, Roth AF, Cody CW, Reeves SC (1982) Spectral perturbations of the aequoria green-fluorescent protein. Photochem Photobiol 35 803-808... 

It is inappropriate here to discuss details of nmr spectroscopy. However, the full possibilities for structural studies arise from the use of probes that bind to the molecule under study and perturb the nmr spectrum. These probes are generally paramagnetic species, in particular the lanthanide cations. In the nmr spectrum separate signals arise from each nucleus in the molecule, provided that the nucleus possesses a nonzero nuclear spin (e.g., H, 13C, 14N). The extent of the spectral perturbations of a given signal depends on the relative geometries of the paramagnetic species and the nucleus in question. Thus structural parameters can be obtained, in principle, for most atoms (nuclei) in a protein molecule.5... 

The fraction of protein, eluted from Sephadex G-75 which behaves as a single homogeneous protein with respect to its interactions with M2 was also studied with regard to its interaction with monosaccharides. The association constant of the interaction of M2 with intact con A is 19 times greater than that obtained for the interaction of p-nitrophenyl a-D-mannopyranoside with con A. The normalized spectra (Figure 7) of the monosaccharide and disaccharide complexes were found to be very similar suggesting that the interactions between con A and the nitrophenyl group which causes the spectral perturbation are the same for both complexes. 

Study of the ionization of protein groups by measuring the associated spectral changes is commonly called a spectrophotometric titration. Measurements of the (usually) smaller spectral perturbations induced by change of solvent are usually carried out by the difference spectral technique. We have examined both of these methods of study from an interpretative as well as an experimental point of view. 

Chart I (Ramachandran and Witkop, 1959) summarizes these results The two sequences Try-Ala (17-18) and Try-Lys (52-53) are exposed and fully reactive NBS the sequence Try-Thr (151-152) is buried (cf. Herskovits and Laskowski, 1960) in the A-protein, but becomes accessible to NBS in the tryptic hexadecapeptide. In addition to the methods of spectral perturbation of chromophoric groups by polyhydroxy compounds, spectrophotometric titration studies, and ultraviolet difference spectra the reactivity of tryptophan toward selective agents such as NBS may offer a new tool for assessing influences of secondary or tertiary structure in this area. 

Hilty et alP have used spin labels to study protein-lipid interactions in mixed micelles containing dihexanoylphosphatidylcholine and the E. coli outer-membrane protein X (OmpX). As paramagnetic relaxation probes, they used several different nitroxide spin labels attached to the lipid as well as Gd-DOTA, which remains in the aqueous phase. Spectral perturbations were monitored in TROSY and ID H NMR spectra. 

Richards and Logue (368) examined the acid transition of RNase-S in 0.1 M NaCl-0.01 M acetate-tris at 22°. The midpoint for RNase-S was about pH 3.3 and for S-protein about 4.4. The complex with the tetra-methyl ester of S-peptide was intermediate between these values, all being at higher pH than RNase-A. The difference spectra obtained on association of S-peptide and S-protein clearly showed the perturbation of phenylalanine as well as tyrosine residues. Phenylalanine 8 is certainly involved in the spectral shift and perhaps also Phe 120. 

Spectral Properties of Aromatic Side Chains. The CD of proteins arises from signals from aromatic side chains (Phe, Tyr, and Trp) as well as disulfides. As the CD of each of these groups is dependent on the local environment, this leads to the view that perturbation of the near UV indicates disruption of the localized folding pattern. The CD and absorption bands display maxima near 280-290 nm for Trp, near 275-280 nm, and around 250-260 nm for Phe [7, 8],... 

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