No abstract

Specific protein-ligand interactions are critical for cellular function, and most proteins select their partners with sharp discrimination. However, the oligopeptide-binding protein of Salmonella typhimurium (OppA) binds peptides of two to five amino acid residues without regard to sequence. The crystal structure of OppA reveals a three-domain organization, unlike other periplasmic binding proteins. In OppA-peptide complexes, the ligands are completely enclosed in the protein interior, a mode of binding that normally imposes tight specificity. The protein fulfills the hydrogen bonding and electrostatic potential of the ligand main chain and accommodates the peptide side chains in voluminous hydrated cavities.

Oligodeoxynucleotide-directed mutagenesis has been used on the gene of tyrosyl-tRNA synthetase from Bacillus stearothermophilus to produce mutant enzymes altered at the adenosine 5'-triphosphate (ATP) binding site. Deliberate attempts were made to alter rather than destroy enzymic activity so that kinetic measurements may be made to identify the subtle roles of the enzyme-substrate interactions in catalysis. Cys-35, the -SH group of which is involved in binding the 3'-OH of the ribose ring of ATP, has been mutated to a serine residue [Winter, G., Fersht, A. R., Wilkinson, A. J., Zoller, M., & Smith, M. (1982) Nature (London) 299, 756-758] or glycine residue. The mutant enzymes are less active than the wild type, and the reduction in activity can be attributed to a decrease in the value of kcat and an increase in KM. Thus, the interaction energy of the side chain of Cys-35 with the substrate is not fully realized in the enzyme-substrate complex but is used preferentially to stabilize the transition state. Relative to its absence in the Gly-35 mutant, the side chain of Cys-35 is calculated to stabilize the transition state for pyrophosphate exchange by 1.2 kcal/mol and the transition state for aminoacylation by 1.0 kcal/mol.

Factors governing the stability of sperm whale, pig, and human metmyoglobin were examined by (1) measuring guanidinium chloride induced unfolding of apoglobins containing 22 replacements at positions 29(B10), 43(CD1), 64(E7), 68(E11), and 107(G8), (2) determining the rates of hemin loss from the recombinant holoproteins, and (3) estimating constitutive expression levels of the corresponding genes in Escherichia coli TB-1 cells. The denaturant titrations were analyzed in terms of a two-step unfolding reaction, N(native apoprotein)-->I(intermediate)-->U(unfolded), in which the intermediate is visualized by an increase in tryptophan fluorescence emission. Two key conclusions were reached. First, high rates of hemin loss are not necessarily correlated with unstable globin structures and vice versa. In general, both rates of hemin loss and the equilibrium constants for apoprotein unfolding must be determined in order to understand the overall stability of heme proteins and to predict the efficiency of their expression. Second, polar residues in the distal pocket cause marked decreases in the overall stability of apomyoglobin. Removal of hemin from V68N and L29N sperm whale myoglobins produces the molten globular I state at pH 7, 25 degrees C, without addition of denaturant. In contrast, the H64L and H64F mutations produce apoproteins which are 10-30 times more stable than wild-type apoglobin. The latter results show that protein stability is sacrificed in order to have the distal histidine (H64) present to increase O2 affinity and inhibit autooxidation.

The epidermal growth factors (EGFs) are powerful mitogens for a wide variety of cells in culture; human EGF (hEGF), known as urogastrone, also inhibits gastric acid secretion in vivo. The transforming growth factors (TGF-alpha) are related to the EGF family both in sequence and activity and EGF-like sequences are often observed in a wide range of functionally unrelated proteins. Attempts to examine the structure of EGF by diffraction methods have not yet succeeded because of difficulties with crystallization. We report here a three-dimensional structure of a biologically active derivative (residues 1-48) of the 53-residue human EGF. An analysis of high resolution 1H nuclear magnetic resonance (NMR) spectra was used together with a combination of distance geometry, restrained energy minimization and restrained molecular dynamics methods. The three-dimensional structure provides a basis for understanding the properties of EGFs and for predicting the structures of homologous sequences in other proteins.