Bruce Furie

An immunological approach to the conformational equilibrium of staphylococcal nuclease

Abstract The conformational equilibrium constant, K conf , of staphylococcal nuclease, describing the equilibrium between the native conformation and non-native or disordered conformations, has been estimated using an immunologic method and an interpretive model. Using goat antisera prepared toward a conformationally disordered nuclease fragment (99–149), antibodies specific for the disordered form of the helix-rich sequence 99 to 126, anti-(99–126) R , were isolated by sequential immunoabsorption. Anti-(99–126) R forms soluble 7 S complexes with fragment (99–149), but this interaction may be inhibited by a large excess of nuclease. By using fragment (99–149) preferentially carbamylated at the α-amino terminus with KN 14 CO and rabbit anti-goat immunoglobulin to distinguish between antibody-bound and free fragment (99–149), an assay for the quantitation of the degree of inhibition of anti-(99–126) R . (99–149) complex formation by nuclease was developed. Using a formal analysis based on the hypothesis that nuclease is in a conformational equilibrium between a folded and unfolded form and that anti-(99–126) R binds effectively only to the unfolded form, the K conf of nuclease was estimated to be 2900. In the presence of the ligands Ca(II), or Ca(II) and thymidine-3′,5′-diphosphate, K conf values of 6500 and 30,000 to 50,000 were estimated, respectively. The K conf of nuclease at 4 °C and 39 °C was 3900 and 400, respectively.

PART II. COMPUTER‐ASSISTED MACROMOLECULAR STRUCTURE GENERATION: EXTENSION OF EXISTING INFORMATION: On the Construction of Computer Models of Proteins by the Extension of Crystallographic Structures

One of the central beliefs of molecular biology is that the sequence of amino acids determines the structure of a p r ~ t e i n . ~ ~ Yet our scientific culture has still to discover many of the rules that specify and govern this relationship. The results of X-ray crystallographic determination of specific proteins have led to an understanding of the taxonomy of protein architecture^.^ The protein sequence fileS which had been growing rather slowly because each amino acid sequence had to be determined by chemical techniques is now growing exponentially because amino acid sequences can be derived from nucleic acid sequencese6 Protein extension attempts to shorten the delay be-

Application of nuclear magnetic resonance spectroscopy to proteins.

The active sites of enzymes can be studied in great detail using nuclear magnetic resonance spectroscopy. The determination of pKa values of active site histidine residues in bovine pancreatic ribonuclease and the characterization of the binding of peptide hormones to carrier proteins are two such examples. The study of the active site of staphylococcal nuclease is another example and is presented in detail in this paper. The structure of 35-thymidine diphosphate bound in the active site of staphylococcal nuclease has been studied by measuring the relaxation rate enhancement of substrate analog nuclei by a paramagnetic metal ion. The lanthanide ion, Gd(III), was substituted for Ca(II) in the formation of the ternary complex of nuclease: Gd(III) : 35-thymidine diphosphate. Measurements were made of the transverse relaxation rates of protons and the longitudinal and transverse relaxation rates of the phosphorus nuclei of bound nucleotide. Internuclear distances between the metal ion and atoms of the 35-thymidine diphosphate nucleotide were determined from these data by using the Solomon-Bloembergen equation. In general, these distances corresponded closely to those determined by previous X-ray crystallography of the thymidine diphosphate complex. These internuclear distances were also used with a computer program and graphics display to solve for metal : nucleotide geometries which were consistent with the experimental data. A geometry similar to the structure of the metal : nucleotide complex bound to nuclease determined by X-ray analysis was one of the solutions to this computer modeling process. For staphylococcal nuclease the NMR and X-ray methods yield compatible high resolution information about the structure of the active site.

New Perspectives in P-Selectin Biology

P-selectin is a cell adhesion molecule that is an integral membrane protein of the alpha granules of resting platelets 1,2 and the Weibel-Palade bodies of endothelial cells3,4. Stimulation of these cells results in translocation of P-selectin to the plasma membrane where it serves as a receptor for leukocytes5,6. P-selectin is a member of the selectin family of adhesion molecules. It shares a common domain structure with other family members, E-selectin and L-selectin. These proteins contain a lectin domain, an EGF domain, a variable number of concensus repeats, a transmembrane domain, and a cytoplasmic tail7,8,9,10