Buried, charged, non-ion-paired aspartic acid 76 contributes favorably to the conformational stability of ribonuclease T1.

The side-chain carboxyl of Asp 76 in ribonuclease T1 (RNase T1) is buried, charged, non-ion-paired, and forms three good intramolecular hydrogen bonds (2.63, 2.69, and 2.89 A) and a 2.66 A hydrogen bond to a buried, conserved water molecule. When Asp 76 was replaced by Asn, Ser, and Ala, the conformational stability of the protein decreased by 3.1, 3.2, and 3.7 kcal/mol, respectively. The stability was measured as a function of pH for wild-type RNase T1 and the D76N mutant and showed that the pH dependence below pH 3 was almost entirely due to Asp 76. The pK of Asp 76 is 0.5 in the native state and 3.7 in the denatured state. Thus, the hydrogen bonding of the carboxyl group of Asp 76 contributes more than half of the net stability of RNase T1 at pH 7. In addition, the charged carboxyl of Asp 76 stabilizes structure in the denatured states of RNase T1 that is not present in D76N, D76S, and D76A.

Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1.

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.

A novel ELISA format for the rapid and sensitive detection of staphylococcal enterotoxin A.

Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.

Amino acid sequences of ovomucoid third domains from 27 additional species of birds.

Ovomucoids consist of a single polypeptide chain which is composed of three tandem Kazal domains. Each Kazal domain is an actual or putative protein inhibitor of serine proteinases. Ovomucoid third domains were already isolated and sequenced from 126 species of birds (Laskowski et al., 1987, 1990). This paper adds 27 new species. A number of generalizations are made on the basis of sequences from 153 species. The residues that are in contact with the enzyme in enzyme-inhibitor complexes are strikingly hypervariable. While the primary specificity residue, P1, is the most variable; substitutions occur predominantly among aliphatic, hydrophobic residues. Consensus sequences for an avian ovomucoid third domain, for a b-type Kazal domain (i.e., a COOH terminal domain of multidomain inhibitors) and for a general Kazal domain are given. Finally, the individual new sequences are briefly discussed.

Amino acid sequences of ovomucoid third domain from 25 additional species of birds.

Ovomucoids were isolated from 25 avian species other than the 101 studied in Laskowski et al. (1987, Biochemistry 26, 202-221). These were subjected to limited proteolysis with an appropriate enzyme, and connecting peptide extended ovomucoid third domains were isolated and sequenced to the end in a protein sequencer. Of the 25 new sequences, 13 duplicate ones were already known, and 12 are unique. Probably the most striking findings are a Pro14----Ser14 replacement in weka, an Ala14----Thr15 replacement in Bulwer's pheasant, the discovery of two additional amino acid residues Ile18 and Gly18 at the P1 reactive site position in Kalij pheasant and tawny frogmouth, respectively, and the first finding of a negative (Glu34) rather than positive (Lys34 or Arg34) amino acid residue at the NH2 terminus of the alpha helix in caracara ovomucoid third domain. These results complete the determination of all the sequences of ovomucoid third domains in the four species genus Gallus, in the five species genus Syrmaticus, and in the two species genera Aix and Pavo.