Spectroscopy protein conformation

Because membrane filtration is the only currently acceptable method of sterilizing protein pharmaceuticals, the adsorption and inactivation of proteins on membranes is of particular concern during formulation development. Pitt [56] examined nonspecific protein binding of polymeric microporous membranes typically used in sterilization by membrane filtration. Nitrocellulose and nylon membranes had extremely high protein adsorption, followed by polysulfone, cellulose diacetate, and hydrophilic polyvinylidene fluoride membranes. In a subsequent study by Truskey et al. [46], protein conformational changes after filtration were observed by CD spectroscopy, particularly with nylon and polysulfone membrane filters. The conformational changes were related to the tendency of the membrane to adsorb the protein, although the precise mechanism was unclear. 

K. Wuthrich, Six years of protein structure determination by NMR spectroscopy What have we learned in Protein Conformation. Ciba Foundation Symposium 161, John Wiley Sons, Chichester, 1991, pp. 136-149. 

Fluorescence correlation spectroscopy (FCS) measures rates of diffusion, chemical reaction, and other dynamic processes of fluorescent molecules. These rates are deduced from measurements of fluorescence fluctuations that arise as molecules with specific fluorescence properties enter or leave an open sample volume by diffusion, by undergoing a chemical reaction, or by other transport or reaction processes. Studies of unfolded proteins benefit from the fact that FCS can provide information about rates of protein conformational change both by a direct readout from conformation-dependent fluorescence changes and by changes in diffusion coefficient. 

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

Lehrer, S. S. (1997). Intramolecular pyrene excimer fluorescence A probe of proximity and protein conformational change. Fluorescence Spectroscopy 278, 286-295. 

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... 

Raman spectroscopy is a related vibrational spectroscopic method. It has a different mechanism and therefore can provide complementary information to infrared absorption for the peptide protein conformational structure determination and some multicomponent qualitative and/or quantitative analysis (Alix et al. 1985). 

In both the far- and near-UV regions, CD spectra can be used empirically as fingerprints of a particular protein, with the spectrum resulting from the aromatic residues being rather more specific and hence diagnostic. The far-UV spectra, however, can provide information about the protein conformation in terms of its secondary structure. As for fluorescence spectroscopy or any spectroscopic method, the sample needs to be chemically pure and homogeneous. 

Observations were made of lipid-protein phases in which the structure is determined mainly by the protein. Raman spectroscopy is a useful method for structure analysis of such phases. The structures described above were analyzed successfully by an x-ray diffraction technique. Lipid-protein complexes, however, are often amorphous, and alternative methods to study their structures are therefore needed. It was demonstrated that Raman spectroscopy can be used to obtain structural information about lipid-protein interaction (16, 17). It is thus possible to determine the conformation as well as the type of environment of the lipid molecules. With the protein, interpretation is more complicated. It is usually possible to determine whether the complex has the same protein conformation as the component used in the preparation, or, if a change occurs, it may be possible to correlate it with denaturation of the pure protein. For complexes formed by long-chain alkyl phosphates and insu-... 

Resonance spectroscopies have an important role in determining electronic configurations and protein conformations. High resolution proton magnetic resonance may provide information on conformations in solu-... 

R.K. Dukor, Protein conformational studies using vibrational spectroscopy Comparison of techniques. Paper presented at 232nd ACS national meeting, biophysical and biomolecular symposium New and emerging techniques for protein characterization, Hilton, San Francisco, 10-14 September 2006... 

Wilkes et al (22.23) coupled calorimetric, dynamic-mechanical and x-ray diffraction techniques to demonstrate crystallization of the lipids was completely reversible in neonatal rat stratum corneum, and only partially reversible in human stratum corneum. Melting regions near 40°C and from 70 to 90°C corresponded to the thermal transitions noted in the calorimetric studies for both species. The crystalline nature of the lipids did not appear to be dependent on the presence of water. X-ray diffraction and infrared spectroscopy studies (23.28-34) have also shown a to p conformational changes occurred in keratin and stratum corneum protein components with hydration or exposure to increased temperatures. Oertel (28) has reported pretreatment with dimethylsulfox-ide, hexylmethylsulfoxide and decylmethylsulfoxide resulted in the formation of p-sheet protein conformations in vitro in human... 

Permeability changes in full-thickness skin have been associated with temperature or solvent pretreatment. The molecular basis of these permeability changes has been attributed to lipid melting or protein conformational changes. The current studies have probed the role of lipid fluidity in the permeability of lipophilic solutes, and examined the effects of temperature on the physical nature of the stratum corneum by differential scanning calorimetry and thermal perturbation infrared spectroscopy. Combining molecular level studies that probe the physical nature of the stratum corneum with permeability results, a correlation between flux of lipophilic solutes and nature of the stratum corneum barrier emerges. 

Krimm S, Bandekar J. Vibrational spectroscopy and conformation of peptides, polypeptides and proteins. Adv Protein Chem 1986 38 181-365. 

Therefore, IR spectroscopy provides structural information about peptide and protein conformations, which are stabilized by the hydrogen bonding of amide groups. 

The simplicity of performing this protein secondary structure analysis makes CD spectroscopy useM in monitoring protein conformational changes between wild-type and mutant proteins, as well as in identifying changes in the protein secondary structure upon addition of a bound cofactor such as a metal ion. Several recent examples of applying CD spectroscopy for these purposes are now described. 

Photoreceptor Pigments. There have been several reviews on the structures, photochemistry, and functioning of the retinal-protein photoreceptor pigments involved in the processes of visionand in the purple membrane of Halobacteria (bacteriorhodopsin). ° ° In addition to the papers quoted earlier on the spectroscopy of these pigments, many other reports have appeareddealing with rhodopsin and intermediates in its photocycle, especially photochemistry, chromophore-protein conformation and binding, and reaction kinetics. Similar studies on bacteriorhodopsin have also been described." "-"" ... 

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