Protein mobility, native

The set of buffers compiled by McLellan provides the simplest way to carry out the electrophoresis of proteins in their native state.25 McLellan s buffers range from pH 3.8 to pH 10.2, all with relatively low conductivity (Table 8.1). By using different buffers from the set it is possible to compare the effect of pH changes on protein mobility while maintaining similar electrical conditions. This is demonstrated... 

Figure 8.5 Effect of pH on protein mobility. Hemoglobin A (pi 7.1) and Hemoglobin C (pi 7.4) were electrophoresed in eight of the McLellan native, continuous buffer systems (Table 8.1). The diagram is drawn to scale. Migration is from top to bottom as shown by the vertical arrows. Bands marked A or C indicate the positions of the two hemoglobin variants in each gel representation. The polarities of the voltages applied to the electrophoresis cell are indicated by + and - signs above and below the vertical arrows. Run times are shown below the arrows. Note the polarity change between the gel at pH 7.4 and the one at pH 8.2. This reflects the pis of the two proteins (and was accomplished by reversing the leads of the electrophoresis cell at the power supply).

One of the most important of these applications is determining the molecular weights of native proteins. Under restrictive electrophoresis conditions, that is where the gel porosity affects protein mobility, there is a linear relationship between... 

Native GPI-anchored proteins diffuse more rapidly in supported lipid bilayers than transmembrane proteins, presumably because the lipid tail of the GPI anchor does not extend completely through the lipid bilayer [111]. To investigate the relationship of GPI-anchor structure to the mobility on the membrane, the glycan core of GPI anchor was substituted with no (87), one (88) or two mannosyl units (89). These GPI anchored protein analogues were incorporated into supported lipid bilayers. The diffusion properties of GFP-2, GFP-3 and GFP-4 in supported lipid bilayers were investigated by FCS. From these FCS measurements, the characteristic correlation times (td) and the diffusion coefficient (D), a physical measure of protein mobility, were obtained. GFP-4, which contains two monosaccharides in... 

The conformational plasticity supported by mobile regions within native proteins, partially denatured protein states such as molten globules, and natively unfolded proteins underlies many of the conformational (protein misfolding) diseases (Carrell and Lomas, 1997 Dobson et al., 2001). Many of these diseases involve amyloid fibril formation, as in amyloidosis from mutant human lysozymes, neurodegenerative diseases such as Parkinson s and Alzheimer s due to the hbrillogenic propensities of a -synuclein and tau, and the prion encephalopathies such as scrapie, BSE, and new variant Creutzfeldt-Jacob disease (CJD) where amyloid fibril formation is triggered by exposure to the amyloid form of the prion protein. In addition, aggregation of serine protease inhibitors such as a j-antitrypsin is responsible for diseases such as emphysema and cirrhosis. 

This chapter has reviewed the application of ROA to studies of unfolded proteins, an area of much current interest central to fundamental protein science and also to practical problems in areas as diverse as medicine and food science. Because the many discrete structure-sensitive bands present in protein ROA spectra, the technique provides a fresh perspective on the structure and behavior of unfolded proteins, and of unfolded sequences in proteins such as A-gliadin and prions which contain distinct structured and unstructured domains. It also provides new insight into the complexity of order in molten globule and reduced protein states, and of the more mobile sequences in fully folded proteins such as /1-lactoglobulin. With the promise of commercial ROA instruments becoming available in the near future, ROA should find many applications in protein science. Since many gene sequences code for natively unfolded proteins in addition to those coding for proteins with well-defined tertiary folds, both of which are equally accessible to ROA studies, ROA should find wide application in structural proteomics. 

Protein stability is just the difference in free energy between the correctly folded structure of a protein and the unfolded, denatured form. In the denatured form, the protein is unfolded, side chains and the peptide backbone are exposed to water, and the protein is conformationally mobile (moving around between a lot of different, random structures). The more stable the protein, the larger the free energy difference between the unfolded form and the native structure. 

Recent results show large variations in intramolecular rotations of tryptophan residues in proteins on the nanosecond time scale, ranging from complete absence of mobility to motions of considerable angular amplitudes. Among native proteins with internal tryptophan residues, wide angular amplitude rotations were observed only in studies of azurin,(28 29) where the correlation time of the rapid component was x = 0.51 ns.(28) The existence of... 

Figure 4.2 Protein transformations in reversed-phase chromatography for a two-state model. The native folded state can exist in either the mobile phase (Fm) or the stationary phase (Fs), as can the unfolded state (Um, Us). The equilibrium constants (k) for interconversions of the four species are indicated. (Reproduced from X.M. Lu, K. Benedek, and B.L. Karger, J. Chromatogr., 359 19 [1986]. With permission from Elsevier Science.)...

When chromatographic resolution of species based on modifications located at the protein surface is desired, it may be advisable to use conditions that favor retention of native conformation.17 Here, the standard acidic conditions described in the preceding text may be inappropriate, and mobile phases buffered near neutrality may be required. Buffers based on ammonium acetate, ammonium bicarbonate, and triethylammonium phosphate may prove more useful in resolving polypeptide variants with differing posttranslational modifications, amino acid substitutions, or oxidation and deamidation products. The addition of more hydro-phobic ion-pairing agents may be needed to obtain polypeptide retention, and a variety of alkyl sulfonates and alkyl amines have been described for specific applications.17... 

An example of the effect of pore size on the separation of a set of native proteins is shown in Figure 8.4. The 4%T, 2.67%C gel shown on the left is essentially nonsieving. Proteins in the artificial sample migrate in the gel more or less on the basis of their free mobility. The 8%T, 2.67%C gel on the right sieves the proteins shown and demonstrates the combined effects of charge and size on protein separation. The relative positions of some proteins are shifted in the sieving gel as compared to the nonsieving one. 

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