The structure of the Asn102 mutant of trypsin was determined in order to distinguish whether the reduced activity of the mutant at neutral pH results from an altered active site conformation or from an inability to stabilize a positive charge on the active site histidine. The active site structure of the Asn102 mutant of trypsin is identical to the native enzyme with respect to the specificity pocket, the oxyanion hole, and the orientation of the nucleophilic serine. The observed decrease in rate results from the loss of nucleophilicity of the active site serine. This decreased nucleophilicity may result from stabilization of a His57 tautomer that is unable to accept the serine hydroxyl proton.

The structure of the entire chmmel-fonning bacte1iocin (protein toxin) colicin Iahas been solved to a resolution of2.4A by multiple isomorphous replacement. The fonnation of ion-penneable chmmels in ta.rget cell membrm1es is a general mechanism of cytotoxicity. The process involves secretion of a soluble protein which inserts into the plasma membrm1e of the target cell m1d fon11S a lethal pore. Colicins, Escherichia coli protein toxins, are a well-characterized exmnple of this class of proteins. Colicin Ia crystals, comprised of approximately 80% solvent are in spacegroup C222 1 (a=64.4A, b=l78.6A, c=285.5A). All data sets used in the structure determination were collected fi·om fi·ozen crystals with a synchrotr·on light source (SSRL bean1line 7-1 ). Heavy atom de1ivatives were obtained using mercmial soaks of engineered single-site cysteine mutants.The str1.1cture of the 69kD colicin Ia protein reveals the structural relationships between the three distinct domains which function, respectively, to i) bind to a receptor on the outer membrane of susceptible bacte1ia, ii) tr·anslocate across the outer membrane tlu·ough the receptor, and iii) bind to the inner membrane and fom1 a pore in the presence of the transmembrane voltage. The domains are sepm·ated by an extraordinmily long helical coiled-coil. The cqstal structure of the a-;sembly domain of the cmtilage oligomeric matrix protein (COMP), a pentameric glycoprotein of the thrombospondin family found in cmtilage m1d tendon, was detem1ined at 2.03 A resolution using MIRAS phasing with xenon, (CH3)3Pb(COOCH3)3 and Pr(COOCH3)3 fmther improved by solvent flattenincr and five~fold avera£ing. Self-association ofCOMP, as well as of at le~t two other extracellul~ matrix proteins, thrombosponilins 3 and 4, is achieved through the fonmtion of a five-str·m1ded a-helical bundle which involves 64 N-tem1inal residues (20-83). The complex is fiuther stabilized by the interchain disulphide bonds between cysteines 68 m1d 71. Circular dichroism measurements show that the structme of the assembly domain remains intact even at temperatures above I OOOC. Wlllie the cryst£'11 structures of two-, tlu·ee-and fom-stranded a-helical bm1dles were reported before, that one of tl1e pentai11eric coiled coil is novel. The migins of the extreme thennal stability, the unusual degree of oligomelization and tl1e role of the intemal hydrophobic axial cavity are tl1e questions to be addressed in tl1e cmTent study. The peptides containing 64, 52 or 46 residues were produced by expression in Escherichia coli, but well diffracting crystals were obt£1ined only witl1 t11e 46 residues fragment (Ph a=38.47 A, b=49.47 A, c= 54.98 A and ~= 103.84°). The centr·al pmt of tl1e molecule w11ich includes five heptad repeats (residues 29-65), obeys approximate five-fold symmetry while the remaining residues at theN-and C-tem1ini show significant deviations fi·om tl1at. Str·ong sy1m11etry violations could explain tl1e little success acl1ieved in om earlier attempts to solve t11e structure by tl1e molecuhu·replacem...

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The X-ray crystal structure of diisopropyl fluorophosphate-inhibited bovine trypsin has been refined to a resolution of 1.5 A with the use of a constrained difference Fourier technique. The refinement was carried out almost entirely on a minicomputer with a set of programs written for this study to largely automate the refinement procedure. Use of the minicomputer has allowed the refinement to progress efficiently at an extremely low cost, from a starting R value of 47.2% at 2-7 A resolution to 27-7% at 1-5 A resolution. Two cycles of a least-squares refinement procedure with a first-order gradient minimization have since reduced R to 23-5% at 1.5 A resolution. The refined model and electron density maps are substantially improved over the starting ones. The methods described may be very attractive when computing funds or access to a large computer are limited.

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