Near-UV CD spectra

Fig. 41. Near-UV CD spectra of /1-lactamase (A) and apomyoglobin (B) in native state...

Figure 15.14 The effect of ethanol on RNase A structure (a) far-UV and (b) near-UV CD spectra of 0.65mg/mL solutions of RNase A. Curve 1, native RNAse A in PBS curve 2, native RNase A incubated under 100% ethanol for 1 week and then rehydrated in PBS curve 3, RNase A kept in 10% formalin for 1 week curve 4, RNase A fixed in 10% formalin, incubated under 100% ethanol for 1 week, and then rehydrated in PBS. See Fowler et al.12 for details.

Figure B3.5.5 Near-UV CD spectra. (A) Bovine a -casein peptide under a variety of conditions (data from Alaimo et al., 1999). Peptide concentration 0.631 mg/ml in 2 mM PIPES, 4 mM KCI, pH 6.75 scan rate 40 sec/nm path length 10 mm bandwidth 1.5 nm. The loss of aromatic dichroism with increasing temperature indicates denaturation, which is, however, not complete at 70°C or in 6 M guanidine hydrochloride. The shift in maximum wavelength indicates loss of tryptophan asymmetry, but less so of tyrosine. (B) Seed coat soybean peroxidase under native and denaturing conditions (data from Kamal and Behere, 2002). Protein concentration 15 pM and path length 10 mm. The negative aromatic band centered around 280 nm and the Soret band around 410 nm both disappear at 90°C, indicating the loss of net conformational asymmetry of the aromatic and heme chromophores.

Figure B3.5.10 The near-UV CD spectra of cathepsin D. The solid line represents the porcine enzyme and the dashed line represents the bovine enzyme, each cleaved into two chains which remain associated the dotted line represents the bovine intact enzyme. The spectra were recorded using 10-mm- and 20-mm-path-length cells, with enzyme concentrations of 0.8 to 0.4 g/liter. The far-U V CD and fluorescence spectra of the cleaved and intact enzymes are not significantly different (Pain et at., 1985).

Distillation method of TBARS determination in lipids, 550-551,559-560 (tables) Disulfide bonds, near-UV CD spectra, 235 (figs.), 236-237 Dithiothreitol (DTT) for protein electrophoresis, 158 to reduce samples for IAEDANS labeling, 203, 205... 

Use of Near UV CD Spectra to Probe the Globular Structure of Proteins... 

While the far UV region has received the most attention in the study of protein CD, there are intrinsic chromophores in proteins which give nse to signals in the near U V (A. = 250-350 nm). These include the side chains of aromatic amino acids (tryptophan, Trp, tyrosine, Tyr, and phenylalanine, Phe) and the disulfide moiety of cystine. The exact position of these bands depends on the extent of exposure to the solvent, the solvent polarity and pH, and their proximity to other groups. The analysis of the near UV CD spectra of proteins has been reviewed [7, 8],... 

Aromatic Side Chains. The usual dogma is that while far UV CD spectra of proteins reflect the secondary structure of proteins, the near UV CD spectra indicate changes in tertiary structure. This viewpoint arises from the fact that near UV CD spectra arise from aromatic groups in a fixed geometry relative to the peptide backbone and surrounding chromophores. Loss of tertiary structure would disrupt this ordering and lead to a diminished or altered near UV CD spectrum. 

Use of Near UV CD Spectra to Probe Aggregation in Polypeptides. Near UV CD spectroscopy can be employed to monitor the aggregation of a polypeptide species. Upon association, the aromatic groups will be in different environments than in the isolated monomers and the CD may change dramatically, especially in small peptides. This can be seen for insulin, where the near UV CD intensity of a monomeric form is only [e] - -150 deg cm2 dmoH. Yet, the hexameric form displays a signal nearly twice as intense [110-112],... 

Figure 3. Near UV CD spectra of wild type BPTI and two mutants, F22L and F33L.

Figure 4. Concentration dependence of the near UV CD spectra of leupralide acetate dissolved in a 4 1 ethanol/water (v/v) mixture.

Contributions of Aromatic Side Chains to the Far UV CD of Proteins. Numerous theoretical studies of the effects of aromatic groups on both the far and near UV CD spectra of proteins have been conducted by Hooker and co-workers [143-154], While the calculations on larger proteins were limited in scope, they do provide the only comprehensive attempt to include these chromophores into CD calculations (see below). Other researchers have attempted coupled-oscillator calculations on proteins such as insulin [155, 156], to assess the effects of tertiary structure on near UV CD spectra. More recent work by Woody and co-workers expanded the matrix method to include more elaborate descriptions of... 

To check for the presence of an intermediate in a protein folding process, the temperatures at which the secondary structure (Tsec) and the tertiary structure (Ttert) of the folded conformation are half-formed can be compared. If both coincide, the protein loses the tertiary and the secondary structures simultaneously, and only a native conformation with secondary and tertiary structures ordered or an unfolded conformation with both structural levels unordered describe the process. If significant differences are observed in the crossing temperatures of concentration profiles, a new, intermediate third species with the secondary structure ordered and the tertiary unordered may be needed to explain the shift in the appearance of the tertiary and secondary structures. The difference of almost 20°C found between Tsec and Ttert in the above two experiments seems to guarantee the presence of an intermediate conformation in the folding of a-apolactalbumin, but only the multivariate resolution analysis of the suitable measurements (far-UV and near-UV CD spectra) together can confirm this hypothesis and model the appearance of the intermediate conformation. 

Circular dichroism spectra - Spectra were recorded on a Jasco J-600 spectropolarimeter at room temperature. Far UV CD spectra (190 to 260 nm) of 7.5 nM peptide calmodulin complex in 25 mM Tris, 100 mM KCl and 1 mM CaClj were measured in a 0.1 cm path length cuvette. Near UV CD spectra (250 to 340 nm) of 20 /iM peptideicalmodulin complex in the same buffer were measured in a 1 cm path length cuvette. 

Figure 1 - A) Fluorescence spectra of WFF and FFW peptides, free and bound to wildtype calmodulin, [peptide] = 200 nM, [CaM] = 200 nM in 25 mM Tris (pH 7.5), 100 mM KCl, and 1 mM CaCl2. B) Near UV CD spectra of 20 M wildtype calmodulin alone and in (1 1) complex with WFF and FFW jjeptide (Ae is per mole calmodulin).

Fig. 10.1 (a) Variation in the secondary structure components calculated from far UV CD spectra of CALB in water (A), n-hexane (B), [EMIM][Tf2N] (O, and [BTMA][Tf2N] (D) square a helix, circle P sheet, triangle random coil) (b) Alteration in near-UV CD spectra of CaLB in water (7), n-hexane (2), [EMIM][Tf2N] 3 and [BTMA][Tf2N] 4) after 24 h incubation at 50°C (Reproduced from Ref. [42], with kind permission of The American Chemical Society)... 

Figure 4 Near-UV CD spectra of YPDC. Spectrum 1. YPDC with 0.1 mmol ThDP, 2.0 mmol r MgC. Spectra 2,3,4,5 after addition of 2-17.5 mmol acetylphosphinate. The spectra show presence of 1, 4 -iminophosphinolactyl-ThDP at 302 nm and of the Michaelis complex at 328 nm. Inset dependence of 1, 4 -iminophosphinolactyl-ThDP formation at 302 nm on acetylphosphinate.

No differences are detected in the far-UV or near-UV CD spectra of recombinant and native Sac7d. However, the CD of Sso7d differs significantly from that of Sac7d (Fig. 4), presumably because of a truncated and more disordered C-terminal a helix. The near-UV CD of protein is an induced optical activity of the aromatic side chains due to the asymmetric environment of the folded protein. Therefore, the near-UV CD is often used as an indicator of the protein tertiary structure. The far-UV CD is dominated by contributions ftom the peptide bond and, thus, is a measure of the protein secondary structure. 

CD is one of the most widely used techniques for determining the structure of proteins. The far-UV CD spectra mainly reflect the secondary structure of a protein. The near-UV CD spectra, derived from the aromatic residues of protein and its disulfide bonds in the asymmetric environment, are often used to reveal the tertiary structure. 

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