Far-UV CD spectra

As a prelude to our binding studies, the secondary structure of aPNA itself was examined using CD spectroscopy [52]. The first aPNA to be studied was the tail-to-tail bl dimer, [Ac-Cys-Gly-Ser -Asp-Ala-Glu-Ser -Ala-Ala-Lys-Ser -Ala-Ala-Glu-Ser -Ala-Aib-Ala-Ser -Lys-Gly-NH2]2- The far-UV CD spectra of this aPNA in water at 30 °C showed the double minimum at 220 nm (n-n transition) and 206 nm (n-n transition) as well as the maximum at 193 nm (n-n transition), characteristic of a peptide a-hehx. Upon increasing the temperature, the intensity of the minimum at 200 nm decreased indicating a transition from a-helix to random stracture. An isodichroic point at 202 nm was suggestive of a temperature-depen-dent a-helix to random coil transition. The helical content of this T5(bl)-dimer at 20°C in water was estimated to be 26% [40]. 

Fig. 27. Far-UV CD spectra of six proteins in various states. Native state at 10°C at...

Fig. 40. Far-UV CD spectra of /Flactamase from Bacillus cereus (A), horse apomyo-globin (B), and horse ferricytochrome c (C) as a function of HC1 concentration. Protein concentrations were 10 fiM. The numbers refer to the HC1 concentration (mM). The spectra of the native state (A), the A state induced by KC1, pH ss 2), (O) and GdmCl-unfolded state (4-5 M GdmCl, 25 mM phosphate buffer, pH 7.0) ( ) are shown for comparison. From Goto et al (1990a). 1990, with permission of the authors.

Figure B3.5.12 Effect of mutations detected by CD. The far-UV CD spectra (A) show that the secondary structure of p-lactamase PC1 (solid line) from Staphylococcus aureus is essentially unaffected by point mutations P2 (Thr 140—>lle dashed line) and P54 (Asp 146->Asn dotted line). The crystallographic structure of P54 (Herzberget al., 1991) confirms that, apart from a loop region, the main body of the molecule that contains the thirteen tyrosine residues is very closely similar to that in the wild-type enzyme. The intensity of the tyrosine ellipticity (B) is, however, markedly decreased in each of the mutants, the lower thermodynamic stabilities of which support the interpretation of increased dynamics (Craig et al., 1985).

Figure B3.5.13 A convincing artifact. In an attempt to study the conformational consequences of adding an acceptor, D-glutamine, to a DD-peptidase, far-UV CD spectra were recorded for the enzyme in the presence of increasing concentrations of glutamine a = 0 mM, b = 3 mM, c = 7 mM, d = 10 mM, e = 20 mM, f = 30 mM, g = 50 mM, and h = 95 mM. The enzyme concentration was 0.1 mg/ml, equivalent to 10 3 M peptide bond, in 10 mM sodium phosphate pH 7.2. A 2-mm cell path length was used.

Several examples of near- and far-UV CD spectra are given in the figures included in this unit. With current instruments the final spectra exhibit low noise and—provided that the instrument and sample parameters have been optimized—should be true and reproducible within the limits of protein-in-buffer absorbance of < 1. The most frequent source of nonreproducibility of intensity in spectra is the difficulty of determining the protein concentration, particularly of larger proteins that scatter more and of those with a greater tendency to aggregate. 

FAB-MS. see Fast atom bombardment mass spectrometry FAMES, see Fatty acid methyl esters Far-UV CD spectra, see also Circular dichroism... 

Figure 15.6 Far-UV CD spectra of the odorant-binding proteins from the scarab beetle P. diversa (1) PdivOBPI and (2) PdivOBP2. The spectra of the pure proteins were recorded in 20 mM sodium phosphate buffer, pH 6.8, at 0.4 mg/ml on a JASCO J-720 spectropolarimeter at 25°C in a 0.1 mm pathlength cell.

Figure 3 Self-assembling properties of Pirl. (a) Far-UV CD spectra as a function of peptide concentration in water at pH = 2. (b) Theoretical concentration dependence of the average number (m) of peptides per single tape (dotted line) and in ribbons (dash-dot line). Minimum number of peptides in tapes is two and in ribbons is four. The predicted lengths of tapes and ribbons are in agreement with the observed lengths in the TEM pictures for the same peptide concentration (Aggeli et al., 2001b).

Figure 1. Far UV CD spectra of homopolymers which adopt an a-helical conformation poly(Glu-OMe) in hexafluoroisopropanol, — poly(Lys), . (From refs. 14 and 87)...

Early attempts used data obtained from homopolypeptides, such as poly(Lys), for their basis spectra [87, 88]. In the past fifteen years, approaches using data from globular proteins have emerged [18, 89-101]. Basically, a data base comprised of proteins with known secondary structure compositions is assembled and far UV CD spectra recorded. The choice of the proteins to be included is critical and various combinations have been examined. Mathematical matrix methods can be used to extract basis spectra which represent the contributions from the various secondary structures. Typically, four or five basis spectra can be obtained (corresponding to a helices, jj sheets, p turns, and random coil structures). In some approaches, such as those developed by Johnson and co-workers [11, 12, 51, 52, 102], separate basis spectra can be obtained for parallel and antiparallel p sheets. These basis spectra are then linearly combined to reconstruct the CD spectrum of the protein of interest The proportion of the basis spectra used to provide the best fit to the spectrum corresponds to the percentage of that secondary structure in the protein of interest Complete details of the mathematical algorithms that have been employed can be found elsewhere [10, 12, 17, 89, 103]. 

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. 

Fig. 14.3. Secondary structural changes of prion protein, (32-microglobulin and a-synuclein upon UV exposure under their respective amyloid-forming conditions. Far UV CD spectra of (a) prion protein in 20 mM sodium phosphate buffer (pH 6.8) containing 100 mM NaCl, 3M urea and 1M GdmCl. Inset shows the Far UV CD spectrum of native prion protein (b) (32-microglobuhn in 50 mM citrate buffer (pH 2.5) containing 100mM KC1 and (c) a-synuclein in 20 mM 11EPES- NaOl I buffer (pH 7.0) containing 100 mM NaCl and 0.5 mM SDS. In each panel, curves 1 and 2 show the far UV CD spectra of the protein before and after exposure to UV light, respectively. Panel 3a reproduced from [3]...

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 2 - Far UV CD spectra of wildtype and four mutant calmodulins A) alone and B) in (1 1) complex with WFF peptide (Ac is per mole calmodulin).

As shown in Fig. 2B, the far UV CD spectra of the mutant calmodulins in complex with the WFF peptide more closely resemble that of the wildtype calmodulin WFF peptide complex than do the corresponding spectra in the absence of peptide (Fig. 2A). Quantitation of this effect shows that in addition to induction of a-helix in the bound peptide, the mutant calmodulins have recovered at least some of the "native" helical structure. 

Figure 1. Comparison of the far-UV CD spectra of Pal-HI and HI. All spectra were recorded with samples dissolved in 5 mM Tris-HCl, pH 7.5, and placed in a cuvette of 0.01 cm path length. Panel A shows Pal-HI (dotted line) and HI (solid line), both at 0.1 mg/mL. Pand B shows Zn(II)-Pal-HI hexamer (solid line), Zn(II)-HI hexamer (dotted line), Zn(II)-Pal-HI hexamer in 10 mM phenol (dashed-dotted line) and Zn(II)-HI hexamer in 10 mM phenol (dashed line), all at 1.7 mg/mL and with the mole ratio of Zn protein at 0.33.

Figure 4. (A) [left] Far UV CD spectra of the different KGF samples. 1 -KGF control without cation exchange HPLC step. 2 -KGF with Met 28 and Met 60 oxidized. 3 -KGF with Met 28, Met 60, and Met 160 oxidized.

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)... 

Electric Field Effect on CD of Bacteriorhodopsin. Figure 3 shows the effect of valinomycin on the far UV CD spectra of the bacteriorhodopsin in lipid vesicles. In the presence of a potential gradient, irrespec-... 

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