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Validation of Computer Simulations of Protein FoldingValidation of Computer Simulations | Computational Methods Protein folding takes place in millionth of a second (10-6 seconds or microseconds). As one imagine to get experimental data to validate computational methods in protein folding is not an easy task. Folded states of proteins can be routinely validated by x-ray diffraction or Nuclear Magnetic Resonance (NMR) data. Protein Data Bank (PDB) files are usually derived from x-ray diffraction and NMR analysis. Fortunately experimental methods exist to validate the results of computer simulations of protein folding. Some amino acids fluoresce. Because it is a non-invasive technique, fluorescence does not interfere with a sample. The excitation light levels required to generate a fluorescence signal are low, reducing the effects of photo-bleaching, and living tissue can be investigated with no adverse effects on its natural physiological behavior. Of the 20 amino acids usually found in proteins, the aromatic phenylalanine, tyrosine and tryptophane are the only ones of sufficient fluorescence intensity to be measured directly in solution. The fluorescence of proteins due to these residues is a highly specific and sensitive tool for studying structure and conformation. The fluorescence of the aromatic residues varies in somewhat unpredictable manner in various proteins. Comparing to the unfolded state, the quantum yield may be either increased or decreased by the folding. Accordingly, a folded protein can have either greater or less fluorescence than the unfolded form. The intensity of fluorescence is not very informative in itself. The magnitude of intensity, however, can serve as a probe of perturbations of the folded state. The wavelength of the emitted light is a better indication of the environment of the fluorophore. Tryptophan residues that are exposed to water, have maximal fluorescence at a wavelength of about 340-350 nm, whereas totally buried residues fluoresce at about 330 nm. Proteins in an aqueous solution are placed in the "sample cell". The proteins are unfolded by cooling to low temperatures (cold denaturation). Folding is then reinitiated by laser-jumping the temperature of the aqueous protein sample in a few nanoseconds. The temperature-jump 'instantaneously' changes the aqueous environment from one that favors the unfolded protein conformation to one favoring the folded conformation, thus initiating refolding. Alternatively, temperature-jump initiated unfolding may also be studied. A UV beam probes the protein generating fluorescence. A photomultiplier tube detects the light generated by fluorescence and the intensity of the light depends on the folding state of the protein. As the protein folds, the hydrophobic tryptophane amino acids gets buried inside the protein structure and their fluorescence gets masked. Protein folding can be monitored in real time by collecting laser induced fluorescence from the protein's tryptophan residues once every 14 ns. From intensity and wavelength studies the folding rate of proteins can be measured experimentally.
Key: Image is courtesy of Gruebele Group: http://www.scs.uiuc.edu/mgweb/protein_folding_research.shtml From studies like the ones described above, folding rate of proteins can be measured experimentally. As the graph below indicates there is a strong correlation between prediction by computer simulations of protein foldings generated by folding@home and experimental results.
Author: Tug Sezen References:
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