Protein folding sampling techniques

The introduction and implementation of heteronuclear-based multidimensional techniques have revolutionized the protein NMR field. Large proteins (> 100 residues) are now amenable to detailed NMR studies and structure determination. These techniques, however, necessarily require a scheme by which and isotopes can be incorporated into the protein to yield a uniformly labeled sample. Additional complications, such as extensive covalent post-translational modifications, can seriously limit the ability to efficiently and cost effectively express a protein in isotope enriched media - the c-type cytochromes are an example of such a limitation. In the absence of an effective labeling protocol, one must therefore rely on more traditional proton homonuclear NMR methods. These include two-dimensional (1) and, more recently, three-dimensional H experiments (2,3). Cytochrome c has become a paradigm for protein folding and electron transfer studies because of its stability, solubility and ease of preparation. As a result, several high-resolution X-ray crystal structure models for c-type cytochromes, in both redox states, have emerged. Although only subtle structural differences between redox states have been observed in these... 

To study protein folding theoretically, simulation methods have proved indispensable. The folding transition is ultimately governed by statistical thermodynamics and hence it is paramount to use sampling methods that are able to reproduce the canonical Boltzmann distribution. Common sampling techniques are molecular dynamics (MD), Langevin or Brownian dynamics (BD) and Monte Carlo (MC). 

Synchrotron FT-IRM has also been used in less-traditional ways to study biological systems. For example, a synchrotron IR microscope has been coupled to a rapid-mix flow cell in order to study protein folding [77, 78] and the binding of an antibiotic to a tripeptide [79], on a time scale as short as microseconds. The small spot size of the synchrotron beam permitted a faster time resolution of the technique and the use of smaller volumes of sample. 

The main clinical significance of cerebrospinal fluid (CSF) protein electrophoresis is for the detection of the oligoclonal bands, which are present in multiple sclerosis in the gamma region. Because proteins in CSF occur at much lower concentrations than in serum (100 times less), a 10-20-fold sample concentration is needed. CSF protein separation can be accomplished in less than 10 min with CE versus 2h for AG with the ability to detect oligoclonal banding by this technique. 

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