摘要:

AbstractCultured mouse myeloma cells were transfected with expression vectors encoding the aspartyl proteinase, human renin. The full construct, encoding the renin precursor prorenin, allows transfected cellsto secrete the enzymically inactive pro‐protein. Activity is detectable only following trypsin treatment which mimics the physiological activation step. Accordingly, it appears that myeloma cells do not contain detectable levels of an appropriate activating proteinase. However, when these cells are transfected with a construct from which the pro‐peptide coding sequence has been deleted, they secrete an apparently fully active enzyme which is indistinguishable from mature renin. We conclude that expression of the pro‐peptide is not necessary to allow correct folding of the molecule and its passage through the secretory pa

ISSN:0887-3585    

DOI:10.1002/prot.340050402

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

年代:1989

数据来源: WILEY

摘要:

AbstractSingle crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 × 0.3 × 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which in enzymatically active upon dissolution. These crystals belong to the monoclinic space groupP21and have unit cell parametersa=114.5 Å,b=139.6 Å,c=125.7 Å, β=98.1° Self‐rotation function studies indicate that there are three molecules per asymmetricunit. The crystals diffract to at least 3.0 Å resolution. These are the first crystals of a DNA‐dependent RNA polymerase suitable for high‐resolution X‐ray structur

ISSN:0887-3585    

DOI:10.1002/prot.340050403

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe crystal structure of a fluorescein–Fab (4‐4‐20) complex was determined at 2.7 Å resolution by molecularreplacement methods. The starting model was the refined 2.7 Å structure of unliganded Fab from an autoantibody (BV04‐01) with specificity for single‐stranded DNA. In the 4‐4‐20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol‐arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2‐3 orders of magnitude in the 4‐4‐20 molecules relative to other members of an idiotypic family of monoclonalantifluorescyl antibodies. The phenyl carboxyl group of fluorescein appearsto be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2‐methyl‐2,4‐pentanediol (MPD), trapped in the interface of the variable domainsjust below the fluorescein binding site, may be partly responsible for the decrease in a

ISSN:0887-3585    

DOI:10.1002/prot.340050404

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe prokaryotic protein HU, integration host factor (IHF) fromEscherichia coliand transcription factor 1 (TF1) from bacteriophage SPO1 are closely related molecules. Biochemical results suggest that the role of these proteins is to bind and bend DNA. From the high‐resolution structure of HU, we propose a model for thisinteraction with DNA. Crucial amino acid differences between the proteins can be rationalized in terms of their different specific function

ISSN:0887-3585    

DOI:10.1002/prot.340050405

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe crystal structure of the 80,000 Da FeS enzyme aconitase has been solved and refined at 2.1 Å resolution. The protein contains four domains; the first three from the N‐terminus are closely associated around the [3Fe–4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Associationof the larger C‐terminal domain with the first three domains createsan extensive cleft leading to the FeS cluster. Residues from all four domains contribute to the active site region, which is defined by the FeS cluster and a bound SO 42−ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well asseveral bound water molecules. Three of these side chains reside on a threeturn 310helix in the first domain. The SO 42−ion is bound 9.3 Å from the center of the [3Fe–4S] cluster by the side chains of 2 Arg and 1 Gln rsidues. Each of 3 His side chains in the putative active site is paired with

ISSN:0887-3585    

DOI:10.1002/prot.340050406

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

年代:1989

数据来源: WILEY

摘要:

AbstractElectrostatic calculations have been carried out on a number of structural conformers of tuna cytochromecConformers were generated using molecular dynamics simulations with a range of solvent simulating, macroscopic dielectric formalisms, and one solvent model that explicitly included solvent water molecules. Structures generated using the lowest dielectric models were relatively tight, with‐side chains collapsed on the surface, while those from the higher dielectric modelshad more internal and external fluidity, with surface side chains exploring a fuller range of conformational space. The average structure generated with the explicitly solvated model corresponded most closely with the crystal structure. Individual pKvalues, overall titration curves, and electrostatic potential surfaces were calculated for average structures and along each simulation. Differences between structural conformers within each simulation give rise to substantial changes in calculated local electrostatic interactions, resulting in pKvalue fluctuations for individual sites in the protein that very by 0.3–2.0 pKunits from the calculated time average. These variations are due to the thermal side chain reorientations that produce fluctuations in charge site separations. Properties like overall titration curves and pH dependent stability are not as sensitive to side chain fluctuations within a simulation, but there are substantial effects between simulation due to markeddifferences in average side chain behavior. These findings underscore the importance of proper dielectric formalism in molecular dynamics simulations when used to generate alternate solution structures from a crystal structure, and suggest that conformers significantly removed from the averagestructure have altered electrostatic properties that may prove important inepisodic protein properties such as cataly

ISSN:0887-3585    

DOI:10.1002/prot.340050407

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

年代:1989

数据来源: WILEY

摘要:

AbstractDetailed analysis of the CuZn superoxide dismutase (SOD) structure provides new results concerning the significance and molecular basis for sequence conservation, intron‐exon boundary locations, gene duplication, and Greek key β‐barrel evolution. Using 15 aligned sequences, including a new mouse sequence, specific roles have been assigned to all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of uperoxide dismutation. The β‐strands have no sequence insertions and deletions, whereas insertions occur within the loops connecting the β‐strands and at both termini. Thus, the β‐barrel with only four invariant residues is apparently over determined, but dependent on multiple cooperative side chain interactions. The regions encoded by exon I, a proposed nucleation site for protein folding, and exon III, the Zn loop involved in stability and catalysis, are the major structural subdomains not included in the internal twofold axis of symmetry passing near the catalytic Cu ion. This provides strong confirmatory evidence for gene evolution by duplication and fusion followed by the addition of these two exons. The proposed evolutionary pathway explains the structural versatility ofthe Greek key β‐barrel through functional specialization and subdomain insertions in new loop connections, and provides a rationale for the size of the pr

ISSN:0887-3585    

DOI:10.1002/prot.340050408

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe relation between protein secondary structure and internal motions was examined by using molecular dynamics to calculate positional fluctuations of individual helix, β‐sheet, and loop structural elements in free and substrate‐bound hen egg‐white lysozyme. The time development of the fluctuations revealed a general correspondence between structure and dynamics; the fluctuations of the helices and β‐sheets converged within the 101 psec period of the simulation and were lower than average in magnitude, while the fluctuations of theloop regions were not converged and were mostly larger than average in magnitude. Notable exceptions to this pattern occurred in the substrate‐bound simulation. A loop region (residues 101–107) of the active site cleft had significantly reduced motion due to interactions withthe substrate. Moreover, part of a loop and a 310helix (residues of 67–88) not in contact with the substrate showeda marked increase in fluctuations. That these differences in dynamics of free and substrate‐bound lysozyme did not result simply from sampling errors was established by an analysis of the variations in the fluctuationsof the two halves of the 101 psec simulation of free lysozyme. Concerted transitions of four to five mainchain ϕ and ψ angles between dihedral wells were shown to be responsible for large coordinate shifts in the loops. These transitions displaced six or fewer residues and took place eitherabruptly, in 1 psec or less, or with a diffusive character over 5–10 psec. Displacements of rigid secondary structures involved longer timescale motions in bound lysozyme; a 0.5 Å rms change in the position of a helix occurred over the 55 psec simulation period. This helix reorientation within the protein appears to be a response to substrate binding. There was little correlation between the solvent accessible surface areaand the dynamics of the differe

ISSN:0887-3585    

DOI:10.1002/prot.340050409

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

年代:1989

数据来源: WILEY

摘要:

AbstractA new approach is introduced for analyzing and ultimately predicting protein structures, defined at the level ofCαcoordinates. We analyze hexamers (oligopeptides of six amino acid residues) and show that their structure tends to concentrate in specific clusters rather than vary continuously. Thus, we can use a limited set ofstandard structural building blocks taken from these clusters as representatives of the repertoire of observed hexamers. We demonstrate that protein structures can be approximated by concatenating such building blocks. We have identified about 100 building blocks by applying clustering algorithms, and have shown that they can “replace” about 76% ofall hexamers in well‐refined known proteins with an error of less than 1 Å, and can be joined together to cover 99% of the residues. After replacing each hexamer by a standard building block with similar conformation, we can approximately reconstruct the actual structure by smoothly joining the overlapping building blocks into a full protein. The reconstructed structures show, in most cases, high resemblance to the original structure, although using a limited number of building blocks and local criteria of concatenating them is not likely to produce a very precise global match. Since these building blocks reflect, in many cases, some sequence dependency, it may be possible to use the results of this study as a basis for a protein structure prediction pr

ISSN:0887-3585    

DOI:10.1002/prot.340050410

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

年代:1989

数据来源: WILEY

ISSN:0887-3585    

DOI:10.1002/prot.340050401

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

年代:1989

数据来源: WILEY