Sampling protocols protein folding

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... 

If the gels will be stained with Coomassie brilliant blue (see Support Protocol 1), a starting sample protein concentration of 10 to 20 mg/ml should be used. This will be diluted by the 2x SDS sample buffer to give 5 to 10pg/fil. For complex mixtures, 50 fig protein (5 to 10 fil treated sample) per lane is recommended. For highly purified proteins, 0.5 to 5 fig per lane is usually adequate. Silver staining (see Support Protocol 2) requires 10- to 100-fold less protein per lane. 

Separations in Protocol 9 are comparable to those of SDS-PAGE but each sample run is less than 20 min and more accurate quantitation can be achieved. Limits of protein impurity detection in CGE systems with absorbance detection at a low UV wavelengths are approximately 0.1% (1000 ppm). This is about 10-fold lower than the detection limit of SDS-PAGE. 

Sample concentration is another way to increase the sensitivity of a method. Besides liquid-liquid and solid-phase extraction protocols, which result in more concentrated solutions in addition to sample cleanup, several electrokinetic concentration techniques can be applied including sample stacking and field-amplified sample injection/stacking. Over 1000-fold sensitivity enhancement compared to hydrodynamic sample injection without stacking has been reported. When acetonitrile has been used for the removal of proteins from plasma, sample injections of up to 50% of the capillary volume become possible. Stacking techniques are also available for MEKC. 

Another interesting methodological approach represents the use of NMR data to characterize and optimize the production of bicelles to study protein structure in lipid environments without the need for isotopic enrichment for solution NMR and to follow lipid-peptide interactions, as described by Al-Soufi et al and Yamamoto et al As an example, Morrison and Henzler-Wildman described a reconstitution protocol for the small multidrug resistance transporter, EmrE, which is highly sensitive to its environment, into isotropic bicelles with improved sample stability and expanded lipid composition profile. Furthermore, Lyukmanova et u/. characterized lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state and compared their properties with detergent micelles, bicelles and liposomes. 

---