Conformational properties of Porcine Somatotropin: A Biological Application of 1H NMR
Dziadziola, James K., Jr.
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The biological importance of NMR in the modern laboratory grows in significance everyday. For example, numerous studies have shown the applicability of 13C NMR to metabolic studies on yeast, various microorganisms, hepatocytes, organ extracts, as well as, organs in vivo (1). Such studies tend to employ 13c enriched substrates to increase the sensitivity of the experiment. Protein conformation studies have been performed using both 1H ( 2 , 3 ) and 13c NMR techniques ( 4 ) • The advantages of 1H NMR over 13c NMR are the greater sensitivity of NMR due to its larger gyromagnetic ratio 0 ( 5 ) and greater natural abundance 1 of the H isotope (6)• This paper presents the results of a 1H NMR spectroscopic study of pituitary porcine growth hormone (ppST) which demonstrates the biological applicability of NMR techniques. Pituitary porcine growth hormone or somatotropin is a globular protein consisting of 191 amino acid residues; the primary sequences of ppST and pituitary bovine somatotropin (pbST) to which ppST is similar are shown in figure 1 for comparison (7). The structures of all residues discussed in this report can be seen in figure 2. There is a great deal of interest in the conformational properties of somatotropins. Ideally these studies will provide more information concerning the solution structure of ppST which may help determine the structure/function relationship between hormone and membrane receptor. The primary sequences of pituitary porcine somatotropin and pituitary bovine somatotropin differ by only 18 of 191 residues (figure 1) (7). Somatotropin derived from other species, still retain a high degree of homology (7). It is believed that with such strong similarities of primary sequence that the secondary and tertiary structures may well be similar (8)• Currently, porcine somatotropin can be produced by recombinant DNA techniques (rpST) in Escherichia coli (7). In an effort to target future research involving recombinant processes the conformation of ppST is also being studied to determine the location of the active areas of the protein, so that only those areas of the protein need be produced. Although bovine somatotropin has been crystallized from natural sources (9), no high resolution X-ray studies have been performed due to the poor quality of the crystals. In general, growth hormones are difficult to crystallize without amending the primary sequence (8), due to heterogeneity of these proteins (9). NMR can be used to study the conformational properties, as well as the intermediate conformational folding patterns, without having to obtain crystals of the protein. These circumstances and advantages make NMR techniques applicable to growth hormone conformation studies and unique tools in protein intermediate folding studies. Since little work has been done in determining the conformational properties of ppST, one goal of the study and the subject of this paper was to characterize the solution properties of ppST including any folding intermediates. Studies in protein folding patterns are important in determining the system by which activator hormones recognize specific proteins of cell receptors. The conformational relationship of structure to function is a mechanism which is not yet well understood. Folding studies such as this one are important initial steps into discovering that process.