ISSN:0887-3585    

DOI:10.1002/prot.340070102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractA genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild type protein. Small cultures ofEscherichia colicontaining plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of wild‐type protein has a half‐time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 “DTT‐sensitive” mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the naive wild‐type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated a most sites without completely preventing folding Because this genetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding an

ISSN:0887-3585    

DOI:10.1002/prot.340070103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractControlled thrombic digestion of a preparation of components 2 + 3 isolated from the 18.5 kDa bovine myelin basic protein (MBP) yielded a polypeptide that was monophosphorylated on threonine 97 (component 3pT97). This is the first posttranslationally phosphorylated MBP isolated in pure form. We studied the effect of this single phosphate on the conformational adaptability of 18.5 kDa bovine MBP by comparing the circular dichroism (CD) spectrum of component 3pT97 with the spectra of highly purified nonphosphorylated components 1 and 2. The CD spectra of nonphosphorylated component 1 and component 2 [monodeamidated forms(s) of component1] were indistinguishable, while component 3pt97 exhibited a different spectrum. The singly phophorylated MBP component exhibited 13% more ordered conformations than that adopted by nonphosphorylated MBP in dilute aqueous solutions. This was estimated from the CD spectra, and apparently involved about 17 additional amino acid residues in β‐structure(

ISSN:0887-3585    

DOI:10.1002/prot.340070104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractStatistical methodology for the identification and characterization of protein binding sites in a set of unaligned DNA fragments is presented. Each sequence must contain at least one common site. No alignment of the sites is required. Instead, the uncertainty in the location of the sites is handled by employing the missing information principle to develop an “expectation maximization” (EM) algorithm. This approach allows for the simultaneous identification of the sites and characterization of the binding motifs. The reliability of the algorithm increases with the number of fragments, but the computations increase only linearly. The method is illustrated with an example, using known cyclic adenosine monophophate receptor protein (CRP) binding sites. The final motif is utilized in a search for undiscovered CRP binding si

ISSN:0887-3585    

DOI:10.1002/prot.340070105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractStructural and thermodynamic interactions for the binding of trimethoprim and related congeners to the binary complex of diphydrofolate reductase (from chicken) and NADPH are explored using free energy simulation methods. Good agreement between structures from experimental X‐ray refinement and molecular dynamics simulations is found for the complexes. Agreement with thermodyanmic measurements is found as well. Our thermodynamic calculations suggest that entropic contributions and desolvation thermodynamics can play a crucial role in overall bindings, and that extreme care must be taken in the use of simple model building to rationalize or predict protein–drug bind

ISSN:0887-3585    

DOI:10.1002/prot.340070106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe crystal structures of two helical peptides Boc‐Val‐Ala‐Leu‐Aib‐Val‐Ala‐Leu‐OMe (VALU‐7) and Boc‐Val‐Ala‐Leu‐Aib‐Val‐Ala‐Leu‐Aib‐OMe (VALU‐8) have been determined to a resolution of 1.0 and 0.9 Å, respectively. Both the seven and eight residue peptides crystallize with two conformers per asymmetric unit. The VALU‐8 conformers are completely helical and differ only at the C‐terminus by a sign reversal of the ϕ, ψ angles of the last residue. One of the VALUE‐7 conformers occurs as a normal α‐helix, whereas in the other, the N(7)O(3) α‐type hydrogen bond is ruptured by the entry of a water molecule (W) into the helix, which in turn makes hydrogen bonds N(7) ⃛W = 2.97 Å and ⃛O(3) = 2.77 Å. The other side of the water molecule is surrounded by a hydrophobic pocket. These two conformers give a static representation of a step in a possible helix unwinding or folding process. In the value‐8 crystal the helices aggregate in a parallel mode, whereas the aggregation is antiparallel in the VALU‐7 crystal. The crystal parameters are VALUE‐7 crystal. The crystal parameters are VALUE‐7,P21,a= 10.203 (3) Å,b= 19.744 (6) Å,c= 22.561 (6) Å, α = 96.76°,Z= 4, C38, H69N7O10·0.5 H2O,R= 6.65% for 3674 reflections observed>3σ(F): and VALU‐8,P21,a; = 10.596 (4) Å,b= 27.57 (6) Å,c= 17.745 (5) Å, β = 95.7

ISSN:0887-3585    

DOI:10.1002/prot.340070107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractStructures have been determined ofBacillus stearothermophilus“apo” and holo lactate dehydrogenase. The holo‐enzyme had been co‐crystallized with the activator fructose 1,6‐biosphosphate. The “apo” lactate dehydrogenase structure was solved by use of the known apo‐M4dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 Å resolution by means of the noncrystallographic molecular 222 symmetry. TheR‐factor was reduced to 28.7%, using 2.8 Å resolution data, in a restrained least‐squares refnement in which the molecula rsymmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the “apo” enzyme.Further refinement proceeded with the isomorphous holo‐enzyume fromBacillus Stearothermophilus. After removing the noncrystallographic constraints, theR‐factor dropped from 30.3% to a final value of 26.0% with a 0.019 Å and 1.7° r.m.s. deviation from idealized bond length and angles, respectively.Two sulfate ions per subunit were included in the final model of the “apo” ‐from‐one at the substrate binding site and one close to the molecularP‐axis near the location of the fructose 1,6‐bisphosphate activator. The final model of the holo‐enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to theR‐axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6‐bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6‐bisphosphate are close to the sulfate ion near theP‐axis in the “apo” model.This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo‐enzyme showed far greater similarity to the ternary complex of dogfish M4lactate dehydrogenase with incotinamide adenine dinucleotide and oxamate than to apo‐M4dogfish lactate dehydrogenase. The conformations of nicotinamide adenine d

ISSN:0887-3585    

DOI:10.1002/prot.340070108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe mouse hybridoma cell line 40–150 scretes antibodies with high affinity towards the cardiac glycosides digoxin and digitoxin. A spontaneous mutant, 40–150 A2.4, produces and antibody which carries a single residue mutation, Ser → Arg, in its heavy chain (H94) and has an altered specificity. A second order mutant 40–150 A2.4 P.10, produces two antibody molecules, one the same as 40–150 A2.4, the other lacking two residues at the N‐terminus of its H chain, and having a specificity profile approaching that of 40–150 antibody.1The N‐terminus and the position H94 are distant from the antigen‐binding site of the antibody; thus, the structural basic of the specificity changes was not immediately clear. Approximate structures of the 40–150 antibody and its mutants were constructed in the computer, based on atomic coordinates of the homologous mouse antibody McPC 603. Using the program OCNGEN, the torsional space of the polypeptide backbone and side chains around position H94 was uniformly sampled, and the lowest energy conformations were analyzed in detail. The results indicate that when Arg‐H94 is substituted for Ser. Agr‐H94 can hydrogen bond to side chains of Asp‐H101, Arg‐L46, and Asp‐L55. The results in a change in the surface of the combining site which may account for the affinity changes. Deletion of the two N‐terminal residues increases solvent accessibility of Arg‐H94. The solvation may cause a hydrogen bond between Arg‐H94 and Asp‐H101 to be lost, restoring the struc

ISSN:0887-3585    

DOI:10.1002/prot.340070109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

ISSN:0887-3585    

DOI:10.1002/prot.340070101

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY