Proteins solution conformation

Raman optical activity is an excellent technique for studying polypeptide and protein structure in aqueous solution since, as mentioned above, their ROA spectra are often dominated by bands originating in the peptide backbone that directly reflect the solution conformation. Furthermore, the special sensitivity of ROA to dynamic aspects of structure makes it a new source of information on order-disorder transitions. 

Having ascertained that low-temperature EPR is nondestructive, we can now address a fundamental follow-up question To what extent does the spectroscopy of a biomolecule at a temperature of, say, 10 K bear relevance to that same molecule s cellular functioning at its ambient temperature of, say, 310 K (=37°C). More specifically, to what aqueous solution conformation temperature, if any, does a frozen solution protein conformation correspond This now is a really hard question to answer, simply because it is difficult to approach experimentally, and consequently, there is... 

Silk type Function Proteins (ratio )a Predicted structure from sequence Amino acid (%Y Structure in solution Conformational change in solutione Fiber degree of crystallinity mf Fiber extensibility (%)e... 

Ragg, E., Tagliavini, F., Malesani, P., Monticelli, L., Bugiani, O., Forloni, G., and Salmona, M. (1999). Determination of solution conformations of PrP106-126, a neurotoxic fragment of prion protein, by 1H NMR and restrained molecular dynamics. Eur.J. Biochem. 266, 1192-1201. 

So-called multidimensional NMR techniques can provide important information about macromolecular conformation. In these cases, the sequence of a protein is aheady known, and establishing covalent connectivity between atoms is not the goal. Rather, one seeks through-space information that can reveal the solution conformation of a protein or other macromolecule. Two-or three-dimensional techniques use pulses of radiation at different nuclear frequencies, and the response of the spin system is then recorded as a free-induction decay (FID). Techniques like COSY and NOESY allow one to deduce the structure of proteins with molecular weights less than 20,000-25,000. 

Hatano, T., Yoshida, T., and Hemingway, R.W., Interaction of flavonoids with peptides and proteins and conformations of dimeric flavonoids in solution. In Plant Polyphenols 2. Chemistry, Biology, Pharmacology, Ecology (eds G.G. Gross, R.W. Hemingway, T. Yoshida, and S.J. Branham), Kluwer Academic/Plenum Publisher, New York, 1999, p. 509. 

Conformation of the Macromolecule. In solution, macromolecules can have a wide variety of shapes or conformations. The simplest is the solid sphere or Einstein sphere. It is a round ball, impermeable to solvent. The ball may be stretched into a prolate ellipsoid like a football or flattened into an oblate ellipsoid like a flying saucer. Many soluble proteins have conformations that approximate ellipsoids. If a prolate ellipsoid is stretched enough, it becomes a rod. Certain virus macromolecules are rodlike. 

Having seen that differences between crystal and solution conformations can exist for small molecules, we now turn to the consideration of proteins. First, we must differentiate between those proteins that crystallize and those that do not. Proteins that crystallize are usually globular, whereas those that do not are often nonglobular and are unlikely to conform to a single conformational family. These may be compared to industrial organic polymers such as those used in the plastics industry. 

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... 

M 35] [protocol see [119]] A protein conformation kinetic study of the small protein ubiquitin was performed both in the continuous and in a stopped-flow mode at low reactant consumption [119], The bifurcation mixer was used prior to an IR flow cell for data monitoring. The change of conformation from native to the A-state was followed when adding methanol under low pH conditions to the protein solution. In the continuous mode, long data acquisition could be made and the reaction time was determined by the flow rate and the volume interconnecting zone between the mixer and IR flow cell, which was small, but not negligible. In the stopped-flow mode, the reaction time resolved was dependent on the time resolution of the FTIR instrument. 

Figure 15.2 Effect of pH on the intrinsic fluorescence of an odorant-binding protein from P diversa, PdivOBP2, with a large conformational transition between pH 6 and pH 5.5. Spectra were obtained on a Shimadzu RF-5301PC spectrofluorophotometer with a 6 pg/ml protein solution in 20 mM buffers, with excitation at 235 nm and emission monitored from 280 to 420 nm. (1) pH 7, (2), pH 6 in sodium phosphate, (3) pH 5.5, (4) pH 5, (5) pH 4.5, and (6) pH 4 in sodium acetate.

Damberger F., Nikonova L., Horst R., Peng G., Leal W. S. and Wuthrich K. (2000) NMR characterization of a pH-dependent equilibrium between two folded solution conformations of the pheromone-binding protein from Bombyx mori. Protein Sci. 9, 1038-1041. 

The simplest experiments are those that focus just on the ligand. These are typically used to determine solution conformations or 3D structures of ligands. Homonuclear1H II ) or 21) NMR experiments are used mainly here. At the other end of the scale, experiments to study the macromolecular binding partner often require labeled protein and multidimensional NMR methods, as indicated on the right-hand side of Fig. 1. Finally, many NMR experiments provide information... 

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