Conformational studies proteins

Figure 8 shows a one-dimensional sketch of a small fraction of that energy landscape (bold line) including one conformational substate (minimum) as well as, to the right, one out of the typically huge number of barriers separating this local minimum from other ones. Keeping this picture in mind the conformational dynamics of a protein can be characterized as jumps between these local minima. At the MD time scale below nanoseconds only very low barriers can be overcome, so that the studied protein remains in or close to its initial conformational substate and no predictions of slower conformational transitions can be made. 

Maser, F., Bode,K., Pillai, V. N. R. and Mutter, M. Conformational Studies on Model Peptides. Their Contribution to Synthetic, Structural and Functional Innovations on Proteins. Vol. 65, pp. 177 — 214. 

IV. Advantages and Disadvantages of Using Fluorescence Correlation Spectroscopy to Study Protein Conformational Changes... 

Tanford (1968) reviewed early studies of protein denaturation and concluded that high concentrations of Gdm-HCl and, in some cases, urea are capable of unfolding proteins that lack disulfide cross-links to random coils. This conclusion was largely based on intrinsic viscosity data, but optical rotation and optical rotatory dispersion (ORD) [reviewed by Urnes and Doty (1961) ] were also cited as providing supporting evidence. By these same lines of evidence, heat- and acid-unfolded proteins were held to be less completely unfolded, with some residual secondary and tertiary structure. As noted in Section II, a polypeptide chain can behave hydrodynamically as random coil and yet possess local order. Similarly, the optical rotation and ORD criteria used for a random coil by Tanford and others are not capable of excluding local order in largely unfolded polypeptides and proteins. The ability to measure the ORD, and especially the CD spectra, of unfolded polypeptides and proteins in the far UV provides much more incisive information about the conformation of proteins, folded and unfolded. The CD spectra of many unfolded proteins have been reported, but there have been few systematic studies. 

The use of PIR compounds to study protein interactions is a significant advance over the use of standard homobifunctional crosslinkers. The unique design of the PIR reagent facilitates deconvolution of putative protein interaction complexes through a simplified mass spec analysis. The software can ignore all irrelevant peak data and just focus analysis on the two labeled peptide peaks, which accompany the reporter signal of appropriate mass. This greatly simplifies the bioinformatics of data analysis and provides definitive conformation of protein-protein crosslinks. 

Halverson, K., Fraser, P. E., Kirschner, D. A., and Lansbury, P. T., Jr. (1990). Molecular determinants of amyloid deposition in Alzheimer s disease Conformational studies of synthetic beta-protein fragments. Biochemistry 29, 2639-2644. 

A similar approach was used in the synthesis of the sialyl Lewis X mimetics of type 85 (Scheme 13.22).66 Protein crystallization,67 conformational studies of sLex in solution68 and in bound form to E- and P-selectin69 as well as the study of structure-function relationships70,71 gave information about the functional groups of the sLex-epitope essential for the binding to the selectins. Synthesized mimetics must contain the three essential hydroxyl functions of the fucose. Sialic acid, galactose, and... 

VNR Pillai, M Mutter. Conformational studies of poly(oxyethylene)-bound peptides and protein sequences. Acc Chem Res 14, 122-130, 1981. 

Abstract Conjugated polymers have many unique photophysical properties that make them useful for a variety of applications within the fields of chemistry, molecular biology, and medicine, specifically their ability to produce a conformation-dependant spectral signature reflective of changes in their local environment. This physical property makes conjugated polymers an indispensible tool in the toolbox of fluorescent reporters, and within this chapter, their utilization as molecular probes for studying protein structure and conformation is emphasized. 

The characteristics that discourage the use of RPLC for preparative isolation of bioactive proteins favor its use as an analytical tool for studying protein conformation. Chromatographic profiles can provide information on conformational stability of a protein and the kinetics of folding and unfolding processes. Information about solvent exposure of certain amino acid residues (e.g., tryptophan) as a function of the folding state can be obtained by on-line spectral analysis using diode array UV-vis detection or fluorescence detection. 

Abstract In recent years NMR methods have been developed that enable the observation of proteins inside living bacterial cells. Because of the sensitivity of the chemical shift to environmental changes these in-ceU NMR experiments can be used to study protein conformation, molecular interaction or dynamics in a protein s natmal surrounding. Detection of proteins in the bacterial cytoplasm relies on labeling of the protein of interest with NMR active isotopes. This review describes different labeling techniques based on either imiform i5n or labeling as well as amino acid specific labeling schemes. In addition potential applications of these in-cell NMR experiments and their limitations are discussed. 

Protein-Ligand Interactions Exchange processes and determination of ligand conformation and protein-ligand contacts, 238, 657 nuclear magnetic studies of protein-peptide complexes,... 

B. Penke, G. Toth, G. Varadi, Analogue and conformational studies on peptides, hormones and other biologically active peptides. Amino Acids Pept. Proteins 34 (2003) 55-148. 

The recognition step, the first event in carbohydrate protein interactions, is dependent on the overall conformation of the oUgosac aride in aqueous solution. Therefore, it is important to determine whether the conformational characteristics of the natural compounds are reflected in the substrate analogues. The most usual methods to achieve this goal is the combination of NMR spectroscopy, molecular mechanics calculations and X-ray data. On this basis, only few conformational studies of thiodisaccharides were reported. 

The possibility to carry out conformational studies of peptides at low concentrations and in the presence of complex biological systems represents a major advantage of fluorescence spectroscopy over other techniques. Fluorescence quantum yield or lifetime determinations, anisotropy measurements and singlet-singlet resonance energy transfer experiments can be used to study the interaction of peptides with lipid micelles, membranes, proteins, or receptors. These fluorescence techniques can be used to determine binding parameters and to elucidate conformational aspects of the interaction of the peptide with a particular macro-molecular system. The limited scope of this chapter does not permit a comprehensive review of the numerous studies of this kind that have been carried and only a few general aspects are briefly discussed here. Fluorescence studies of peptide interactions with macromolecular systems published prior to 1984 have been reviewed. 

In order to be exploitable for extraction and purification of proteins/enzymes, RMs should exhibit two characteristic features. First, they should be capable of solubilizing proteins selectively. This protein uptake is referred to as forward extraction. Second, they should be able to release these proteins into aqueous phase so that a quantitative recovery of the purified protein can be obtained, which is referred to as back extraction. A schematic representation of protein solubilization in RMs from aqueous phase is shown in Fig. 2. In a number of recent publications, extraction and purification of proteins (both forward and back extraction) has been demonstrated using various reverse micellar systems [44,46-48]. In Table 2, exclusively various enzymes/proteins that are extracted using RMs as well as the stability and conformational studies of various enzymes in RMs are summarized. The studies revealed that the extraction process is generally controlled by various factors such as concentration and type of surfactant, pH and ionic strength of the aqueous phase, concentration and type of CO-surfactants, salts, charge of the protein, temperature, water content, size and shape of reverse micelles, etc. By manipulating these parameters selective sepa-... 

Rigid model compounds (71NL457 83H817) of the type 146 and 147 were employed to study the effect of dipole interactions between C=0 groups on the conformation of proteins. The lactone C=0 group in 147 confers a marked preferential orientation of the N-acyl group, whereas in 146, two conformers are observed in nearly equal amounts, although the equilibrium is solvent dependent. 

NMR analysis allows characterization of proteins to an atomic level. The most frequently used nuclei on protein NMR are 41, 2H, 13C, 15N, and 170 with proton NMR (Jefson, 1988). The use of NMR methods for protein sequence and conformational studies was limited to the small proteins or peptides because high magnetic fields were required but not widely available to study larger molecules and it was very time consuming with the capability of instruments in the past. 

Most interesting applications of intramolecular energy transfer between nonconjugated chromophores are found in the conformational studies of biomolecules like nucleic acids and proteins. The experiments on rotational depolarization of emission from intrinsic fluorescent groups on externally attached fluorescent probes, have resulted in a vast store of knowledge which has helped to enrich the subject of photobiology. 

The physical environment within a crystal, of course, is not identical to that in solution or in a living cell. A crystal imposes a space and time average on the structure deduced from its analysis, and x-ray diffraction studies provide little information about molecular motion within the protein. The conformation of proteins in a crystal could in principle also be affected by nonphysiological factors such as incidental... 

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