Circular dichroism, native state

The nature of the unfolded state in denaturant and how it relates to the denatured state under native conditions in the bilayer is a major issue in all denaturation experiments. Thermodynamic arguments from the two-stage model suggest that the relevant denatured state has lost its tertiary structure and maintained the transmembrane helix secondary structure. As noted above, CD spectra on thermally denatured bacteriorhodopsin suggest that the denatured protein maintains most of its helical secondary structure. The extent to which tertiary structure is disrupted is unclear, however. It is possible that some stable interhelical interactions are maintained even at high temperature. The helical secondary structure content is also maintained in SDS micelles, and near-UV circular dichroism (CD) spectra suggest substantial loss or... 

To add to the dilemma, phosphorylated protein has been shown to be structurally similar, but not identical, to the native protein by circular dichroism (Buelt et al., 1992). If indeed a major conformational change accompanies modification by the protein kinase, it could only happen in some temporal dynamic state. Finally, it is interesting that the tyrosyl-phosphorylated protein is readily recognized by antiphosphotyrosine monoclonal antibodies. Moreover, the phospho-ALBP is readily de-... 

To understand the function of membrane-active peptides, it is important to know the structure and orientation of the peptide in the membrane. As is evident from Figure 18.1, it is possible to distinguish between, for example, carpet and pore mechanisms of action by determining the peptide s orientation in the membrane. Various techniques, such as electron spin resonance (ESR) [35], infrared (IR) spectroscopy [36-38], circular dichroism (CD) [35, 39,40], and solid-state NMR (SSNMR) [4-7] are used to investigate membrane-active peptides in a quasi-native lipid bilayer environment. In the following sections, methods to determine peptide structure and orientation are presented. 

Fig. 16A-D. Mechanical switching in rotaxanes. A Rotaxanes may exist in isomeric states by the movement of the ring component between dissymmetric sites on the string component. B A redox- or pH-switchable [2]rotaxane. While the cyclophane complexes the native benzidine site (spectrum, curve a), the reduced or protonated benzidine repels the cyclophane, causing it to move to the dioxybiphenylene site (spectrum, curve b). C An azobenzene-based switchable [2]rotaxane. The cyclodextrin ring complexes the azobenzene site in the trans-state, but it is repelled from the ds-azobenzene. The state of the system is measurable by circular dichroism (plot). D A pH-switchable rotaxane. When the amine on the string component is protonated, it complexes the crown ether ring by hydrogen-bonding interactions (40a). When the amine is deprotonated, however, the ring component moves to the bipyridinium unit, where it is complexed by n donor-acceptor interactions (40b). The plots in B and C are adapted from [67] and [69], respectively, with permission...

Among the cosolvents, the effects of DMSO on proteins are particularly interesting and diverse. It can play a role as a stabilizer, an activator, a denaturant, an inhibitor, and also as a cryoprotector. Denaturation of proteins induced by DMSO as well as other cosolvents occurs at threshold concentrations where the tertiary and even the secondary structures of proteins are highly disrupted [23]. UV circular dichroism spectral study shows that from 20-25% (v/v) ( 6% mole fraction) DMSO concentration, lysozyme proceeds gradually from its native to the partially unfolded state. This indicates a broad structural transition which is essentially completed by 50% (v/v) (-18% mole fraction) DMSO concentration [24]. 

Here we present experimental evidence that the monomeric bacteriochlorophyll is required for triplet energy transfer from the primary donor to the carotenoid in photosynthetic bacterial reaction centers. Our approach is to use sodium borohydride to extract the monomeric bacteriochlorophyll from the reaction centers of the carotenoidless mutant Rb. sphaeroides R26 [3, 4]. The borohydride treated reaction centers are then reconstituted with the carotenoid, spheroidene [5], and the ability of the reaction center complex to carry out the primary donor-to-carotenoid triplet transfer reaction was examined by transient optical spectroscopy. Steady state optical absorption and circular dichroism (CD) measurements demonstrate diat spheroidene reconstituted into borohydride-treated Rb, sphaeroides R26 reaction centers is bound in a single site, in the same environment and with the same structure as spheroidene reconstituted into native Rb. sphaeroides R26 reaction centers. It is shown herein that the primary donor-to-carotenoid triplet transfer reaction is inhibited in the absence of the accessory bacteriochlorophyll. 

In the present article, the absorption properties of the two native proteins, as well as the ESR and circular dichroism (CD) parameters, will be analyzed, at density functional theory (DFT) and time-dependent DFT (TD-DFT) level. The use of hybrid QM/MM approach will allow us to take into account the role of the macromolecular environment [32-37]. The evolution of the spectroscopic parameters with mutations will be also considered. The use of natural transition orbitals (NTOs) [26, 38] techniques will allow us to perform a systematic analysis of the nature and electronic properties of the excited states. 

Borohydride, 69, 71 Carotenoid, binding site, 6f, 221 CD (Circular Dichroism), 324, 384 Charge-transfer states, 109, 139, 230, 231f, 410 Chemically modified reaction centers, see Pigments Chloroflexus (Cf.) auranliacus, see Native reaction centers Conformations of reaction centers, see Protein Crystals, 4, 45, 46, 53, 59, 75, 203... 

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