Protein solubility experiments, interpretation

This observed behavior of soy proteins complicates the definition of soy protein solubility, the comparison of solubility data, and the interpretation of solubility experiments. Because the thermodynamic criteria are not met, protein solubility becomes an operationally defined quantity that depends upon the experimental methods of measurement. A number of different operational definitions have been used to measure protein solubility. Each has its own advantages and disadvantages, and limited utility. This plurality, though often desirable, makes it difficult to compare experimental results. 

Changes in a single experimental parameter can simultaneously influence several aspects of a crystallization experiment. For example, temperature changes affect protein solubility, rates of nucleation and growth, and equilibration of the experimental apparatus. The interaction of parameters makes it difficult to design experiments to isolate individual effects and likewise complicates the interpretation of experimental results. 

Another group of experimentors tried to study the preferential utilisation of intact proteins or partial hydrolysates by means of the metabolic trap technique, which consists in measuring the effectiveness with which a labeled free amino acid can compete with larger protein fragments in the formation of new protein. The most positive evidence for the direct utilisation of such fn ments was generally found with embryonic and tumor tissues. Thus Ebert (343) found that when transplants of chick embryo kidney, liver, or spleen, labeled with S -methionine, were made into chick embryos, the radioactivity appeared predominantly in the correspondit organ, and that this transfer of radioactivity could not be inhibited by free methionine. Similarly, Francis and Winnick (344) found that the soluble proteins of a chick embryo extract were utilized in preference to free amino acids by embryonic heart cultures and that, furthermore, p-fluorophenyl-alanine did not inhibit the transfer of phenylalanine from the embryo extract proteins. These experiments, however, are open to many interpretations, especially since functional adoption (or merely adsorption) of the exogenous proteins by the different tissues has not been excluded. 

In a typical free NOESY experiment of a molecule in the absence of any interacting protein, cross-peak volumes are interpreted in terms of a set of interproton distances r that can be used as distance restraints in structure determination procedures, like restrained simulated annealing protocols [44], In a tr-NOESY, i.e. a NOESY measured under exchange-transferred conditions in the presence of a protein - i.e., an excess of soluble ligand is in fast exchange equilibrium with a smaller amount of protein-bound ligand -, these r reflect the interproton distances of the ligand in the bound... 

Whenever possible the basic molecular events of physiological processes are studied in detail in solutions either in parallel with, or as a guide to, experiments on the organized system. For example, the study of the reactions of myosin and the associated proteins of the contractile cycle, by the methods used for the investigation of mechanisms of soluble enzymes (see section S.l), has helped in the planning of experiments and in the interpretation of the events observed in muscle fibres. The most useful methods for kinetic studies on physiological functions are those which can be applied to systems at different levels of organization. We shall return to them at the end of this chapter. 

Ubiquitin has been the subject of a number of experimental studies as it is a small and soluble protein without disulfide linkages. The protein has a well-defined and packed hydrophobic core. There are a number of kinetic folding studies on this system, in addition to a high resolution crystal structure. 2D NMR studies of a partially unfolded form of the protein in 60% methanol indicate that approximately one-half of the molecule contains dismpted but native-like structure, while the other half is unstmctured and/or contains non-native structure. The interpretation of hydrogen-exchange experiments have led to the conclusion that this state is native-like. ... 

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