| 1. |
- Abdulkarim, Farhad, et al.
(författare)
-
Homologous recombination between the tuf genes of Salmonella typhimurium
- 1996
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 260:4, s. 506-522
-
Tidskriftsartikel (refereegranskat)abstract
- The genes coding for the translation factor EF-Tu, tufA and tufB are separated by over 700 kb on the circular chromosome of Salmonella typhimurium. The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication. Sequence comparisons between S. typhimurium and Escherichiacoli suggest that within each species the two tuf genes are evolving inconcert. Here we show that each of the S. typhimurium tuf genes cantransfer genetic information to the other. In our genetic system thetransfers are seen as non-reciprocal, i.e. as gene conversion events.However, the mechanism of recombination could be reciprocal, with sisterchromosome segregation and selection leading to the isolation of aparticular class of recombinant. The amount of sequence informationtransferred in individual recombination events varies, but can be close tothe entire length of the gene. The recombination is RecABCD-dependent,and is opposed by MutSHLU mismatch repair. In the wild-type, this typeof recombination occurs at a rate that is two or three orders of magnitudegreater than the nucleotide substitution rate. The rate of recombinationdiffers by six orders of magnitude between a recA and a mutS strain.Mismatch repair reduces the rate of this recombination 1000-fold. The rateof recombination also differs by one order of magnitude depending onwhich tuf gene is donating the sequence selected for. We discuss threeclasses of model that could, in principle, account for the sequencetransfers: (1) tuf mRNA mediated recombination; (2) non-allelic reciprocalrecombination involving sister chromosomes; (3) non-allelic geneconversion involving sister chromosomes, initiated by a double-strandbreak close to one tuf gene. Although the mechanism remains to bedetermined, the effect on the bacterial cells is tuf gene sequencehomogenisation. This recombination phenomenon can account for theconcerted evolution of the tuf genes.
|
|
| 2. |
|
|
| 3. |
|
|
| 4. |
- Dalby, Paul A, et al.
(författare)
-
Folding intermediates of wild-type and mutants of barnase. I. use of @f-value analysis and m-values to probe the cooperative nature of the folding pre-equilibrium
- 1998
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 276:3, s. 625-646
-
Tidskriftsartikel (refereegranskat)abstract
- It is difficult to determine whether transient folding intermediates have a cooperative (or first-order) folding transition without measuring their rates of formation directly. An intermediate I could be formed by a second-order transition from a denatured state D that is progressively changed into I as conditions are changed. We have not been able to monitor the rate of formation of the folding intermediate of barnase directly, but have analysed its reactivity and the equilibrium constant for its formation over a combination of wide ranges of temperature, concentration of denaturant and structural variation. Phase diagrams have been constructed for wild-type and 16 mutant proteins to map out the nature of the energy landscape of the denatured state. The free energy of unfolding of I, @DGD-I, changes with [urea] according to a highly cooperative transition. Further, mD-I(=@d@DGD-I/@d[urea]) for wild-type and several mutants is relatively insensitive to temperature, as would be expected for an intermediate that is formed cooperatively, rather than one that melts out according to a second-order transition. The @f-values for the formation of I change abruptly through the folding transitions rather than have the smooth changes expected for a second-order transition. There is a subset of mutants for which both mD-I and @f-value analysis indicate that a second intermediate becomes populated close to the melting temperatures of the native proteins. The folding intermediate of barnase is, thus, a relatively discrete and compact entity which is formed cooperatively.
|
|
| 5. |
- Dauter, Zbigniew, et al.
(författare)
-
Crystal Structure of dUTPase from Equine Infectious Anaemia Virus; Active Site Metal Binding in a Substrate Analogue Complex
- 1999
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 285:2, s. 655-673
-
Tidskriftsartikel (refereegranskat)abstract
- The X-ray structures of dUTPase from equine infectious anaemia virus (EIAV) in unliganded and complexed forms have been determined to 1.9 and 2.0 A resolution, respectively. The structures were solved by molecular replacement using Escherichia coli dUTPase as search model. The exploitation of a relatively novel refinement approach for the initial model, combining maximum likelihood refinement with stereochemically unrestrained updating of the model, proved to be of crucial importance and should be of general relevance.EIAV dUTPase is a homotrimer where each subunit folds into a twisted antiparallel @b-barrel with the N and C-terminal portions interacting with adjacent subunits. The C-terminal 14 and 17 amino acid residues are disordered in the crystal structure of the unliganded and complexed enzyme, respectively. Interactions along the 3-fold axis include a water-containing volume (size 207 A3) which has no contact with bulk solvent.It has earlier been shown that a divalent metal ion is essential for catalysis. For the first time, a putative binding site for such a metal ion, in this case Sr2+, is established. The positions of the inhibitor (the non-hydrolysable substrate analogue dUDP) and the metal ion in the complex are consistent with the location of the active centre established for trimeric dUTPase structures, in which subunit interfaces form three surface clefts lined with evolutionary conserved residues. However, a detailed comparison of the active sites of the EIAV and E. coli enzymes reveals some structural differences. The viral enzyme undergoes a small conformational change in the uracil-binding @b-hairpin structure upon dUDP binding not observed in the other known dUTPase structures.
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
- Ekiel, I, et al.
(författare)
-
NMR structural studies of human cystatin C dimers and monomers
- 1997
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 271:2, s. 266-277
-
Tidskriftsartikel (refereegranskat)abstract
- Human cystatin C undergoes dimerization before unfolding. Dimerization leads to a complete loss of its activity as a cysteine proteinase inhibitor. A similar process of dimerization has been observed in cells, and may be related to the amyloid formation seen for the L68Q variant of the protein. Dimerization is barrier controlled, and no dimer/monomer interconversion can be observed at physiological conditions. As a consequence, very stable, “trapped” dimers can be easily separated from monomers. A study of the structural aspects of cystatin C dimer formation was undertaken using NMR spectroscopy. The monomer/dimer model was verified by (pulse field gradient NMR) self-diffusion molecular mass measurements. Complete backbone resonance assignments and secondary structure determination were obtained for the monomer using data from triple resonance experiments performed on 13C/15N doubly labeled protein. A marked similarity of the cystatin C secondary structure to that of chicken cystatin was observed. Using uniformly and amino-acid-specific 15N-enriched protein, backbone NH signals were assigned for cystatin C in its dimeric state. Comparison of 1H-15N correlation NMR spectra of the monomer and dimer shows that the three-dimensional structure remains unchanged in the dimer and that only local perturbations occur. These are localized to the amino acid residues comprising the cysteine proteinase binding site. Such a mode of dimerization readily explains the complete loss of the inhibitory activity in the dimer. The NMR results also demonstrate that the dimer is symmetric.
|
|
| 9. |
- Friedrich, M V, et al.
(författare)
-
Structural basis of glycosaminoglycan modification and of heterotypic interactions of perlecan domain V
- 1999
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 294:1, s. 259-270
-
Tidskriftsartikel (refereegranskat)abstract
- The C-terminal perlecan domain V of about 90 kDa consists of laminin-type G domain modules (LG) (25 kDa) and epidermal growth factor-like modules (EG) (4 kDa) in the tandem arrangement LG1-EG1-EG2-LG2-EG3-EG4-LG3. Several shorter fragments have been prepared by recombinant production in mammalian cells and used to map the single glycosaminoglycan (GAG) substitution site and the binding of several carbohydrate and protein ligands. This identified a Ser3511 residue located in a short link region between EG4 and LG3 as being involved in GAG attachment. Electron microscopy provided evidence that the same substitution exists in tissue forms of perlecan. Heparan sulphate attached to this site was shown to bind to the alpha1LG4 module of laminin-1, indicating a role in basement membrane assembly and cell-matrix interactions. This site is also close to an Asn-Asp bond which is readily cleaved by an endogenous protease that depends on the presence of Asp and the LG2 module. A weak heparin binding site was shown to include the EG2 module, which contains five basic residues. Binding to sulphatides and the alpha-dystroglycan receptor was much stronger and required at least two LG modules. However, single LG modules appear to be sufficient for the interaction with the laminin-nidogen complex, while EG3-4 and some flanking regions are apparently involved in fibulin-2 binding. These observations indicate that a complex modular structure is required for domain V in order to provide a rich repertoire of potential biological functions.
|
|
| 10. |
- Johnson, Christopher M, et al.
(författare)
-
Thermodynamics of Denaturation of Mutants of Barnase with Disulfide Crosslinks
- 1997
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 268:1, s. 198-208
-
Tidskriftsartikel (refereegranskat)abstract
- We have measured the effects of disulfide crosslinks on the thermodynamics of denaturation of three mutants of barnase that contain cystine and the corresponding single and double cysteine mutants. At first sight, the data are consistent with the hypothesis that disulfide crosslinks stabilise proteins through entropic destabilisation of the denatured state, but the decreases in the entropy of denaturation are larger than predicted and are accompanied by decreases in the enthalpy of denaturation. These effects are not a unique feature of the disulfide crosslink and are observed in a range of non-crosslinked mutants of barnase as part of a general enthalpy-entropy compensation phenomenon. Similarly, effects on the heat capacity change for denaturation (@DCpd), determined from the slope of the enthalpy of denaturation versus temperature, are not confined to mutants with disulfide crosslinks. The value of @DCpd is lower in four stabilised mutants than in wild-type barnase, irrespective of the presence of a disulfide crosslink, while the @DCpd remains unchanged in a destabilised mutant containing a disulfide. The variation in @DCpd may result from an inherent temperature-dependence of @DCpd, since it is measured for each mutant over a different temperature range. The thermodynamics of denaturation of the disulfide mutant with a crosslink between positions 70 and 92 change anomalously with pH but in a similar way to that of the D93N mutant of barnase, which lacks the D93-R69 salt-bridge present in the wild-type. This finding confirms initial observations in the X-ray structure of this disulfide mutant that the salt-bridge has been disrupted by the introduced crosslink.
|
|
