Assignment of the backbone <sup>1</sup>H and <sup>15</sup>N NMR resonances and secondary structure characterization of barstar

Lubienski, Michael J.; Bycroft, Mark; Jones, David N. M.; Fersht, Alan R.

doi:10.1016/0014-5793(93)80489-h

Cited by 23 publications

(31 citation statements)

References 22 publications

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“…The positive entropic contribution has previously been suggested to be due to both an increase in flexibility of the molecule and a release of water molecules upon complex formation (12). Earlier measurements of hydrogen exchange rates by NMR (22,42,43) have suggested that free barstar is a flexible molecule. The C82A structure does not show any evidence for increased flexibility of barstar upon binding to barnase.…”

Section: Resultsmentioning

confidence: 98%

“…The average side-chain rmsd for a similar comparison yields values of 1.06 and 1.11 Å, respectively. The main-chain residues of barstar (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)) that lie at the binding interface with barnase do not show significant deviations in comparison to the structure in the complex, except for Gly 43 ( Figures 2B, 3A, and 4A). Taking into consideration the resolution of this structure as well as the quality of the electron density map, the side-chain residues in the binding site which show significant changes in C82A when compared to 1BRS are Y29, D35, and D39 ( Figure 4A).…”

Section: Resultsmentioning

confidence: 99%

“…Model bias in the binding region (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44) was minimized by rebuilding the region after SA σ Aweighted omit map calculations in X-PLOR. The last stage of refinement used all reflections and included constant temperature torsion angle refinement (500 K), positional refinement, and B-factor refinement with an NCS weight of 100 kcal mol -1 Å -1 .…”

Section: Methodsmentioning

confidence: 99%

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Discrepancies between the NMR and X-ray Structures of Uncomplexed Barstar: Analysis Suggests That Packing Densities of Protein Structures Determined by NMR Are Unreliable^,

et al. 1998

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The crystal structure of the C82A mutant of barstar, the intracellular inhibitor of the Bacillus amyloliquefaciens ribonuclease barnase, has been solved to a resolution of 2.8 Å. The molecule crystallizes in the space group I4 1 with a dimer in the asymmetric unit. An identical barstar dimer is also found in the crystal structure of the barnase-barstar complex. This structure of uncomplexed barstar is compared to the structure of barstar bound to barnase and also to the structure of barstar solved using NMR. The free structure is similar to the bound state, and there are no significant main-chain differences in the 27-44 region involved in barstar binding to barnase. The C82A structure shows significant differences from the average NMR structure, both overall and in the binding region. In contrast to the crystal structure, the NMR structure shows an unusually high packing value based on the occluded surface algorithm, indicating errors in the packing of the structure. We show that the NMR structures of homologous proteins generally show large differences in packing value, while the crystal structures of such proteins have very similar packing values, suggesting that protein packing density is not well determined by NMR.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Discrepancies between the NMR and X-ray Structures of Uncomplexed Barstar: Analysis Suggests That Packing Densities of Protein Structures Determined by NMR Are Unreliable^,

et al. 1998

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“…By contrast, for His-2f3, no amide *The sequences of the chimeric proteins are as described in Table 1 with appended tags MRGSHHHHGSR (N terminus) and AQAEA (C terminus). † The conformational stability ⌬G at a temperature T was calculated by using the Gibbs-Helmholtz equation ⌬G(T) ϭ ⌬H m(1 Ϫ T͞Tm) Ϫ ⌬Cp [(Tm Ϫ T) ϩ ln(T͞T m)], while inferring the midpoint of thermal unfolding (Tm) and the enthalpy change for unfolding (⌬H m) at the Tm from the denaturation curve (30) and assuming for ⌬C p (the difference in heat capacity between unfolded and folded conformation at constant pressure) a value of 12 cal per residue (40). ‡ The His-Csp͞2 protein, which comprises the N-terminal half of CspA (LQSGK-MTGIV KWFNADKGFG FITPDDGSKD VFVHFSAW) plus the terminal tags, was found in neither the soluble nor the insoluble fraction of the cytoplasm and is presumed to have been degraded within the cell.…”

Section: Resultsmentioning

confidence: 99%

“…For selections, 10 10 colony-forming units (cfu) of phage were treated with 200 nM trypsin (specific for Arg or Lys in the P 1 position) and 384 nM thermolysin (specific for aliphatic side chains in the P 1Ј position) in TBS-Ca buffer (25 mM Tris⅐HCl͞137 mM NaCl͞1 mM CaCl 2 , pH 7.4) for 10 min at 10°C. After proteolysis, phage was captured for 1 h with biotinylated C40A,C82A double-mutant of barstar (29,30) immobilized on streptavidin-coated microtiter plate wells in 3% Marvel in PBS. Wells were washed 20 times with PBS (and once with 50 mM DTT in PBS for 5 min to elute phage containing proteolyzed p3 fusions held together solely by disulfide bridges).…”

Section: Methodsmentioning

confidence: 99%

Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

Riechmann

Winter

2000

Proc. Natl. Acad. Sci. U.S.A.

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It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and͞or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a ␤-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the proteaseresistant chimeric polypeptides comprised genomic segments in their natural reading frames. Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding. Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself. These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures in vitro.

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Two structural subdomains of barstar detected by rapid mixing NMR measurement of amide hydrogen exchange

Bhuyan

Udgaonkar

1998

Proteins

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Equilibrium amide hydrogen exchange studies of barstar have been carried out at pH 6.7, 32 degrees C using one- and two-dimensional nuclear magnetic resonance. An unusually large fraction of the backbone amide hydrogens of barstar exchange too fast to be measured, and the exchange rates of only fifteen slow-exchanging amide sites including indole amides of two tryptophans could be measured in the presence of 0 to 1.8 M guanidine hydrochloride (GdnHCl). Measurement of exchange occurring in tens of seconds in the unfolding transition region was possible by the use of a fast stopped-flow mixing method. The observed exchange rates have been simulated in the EX2 limit according to a two-process model that incorporates two exchange-competent states: a transiently unfolded state (U*) in which many amide hydrogens are completely accessible to solvent-exchange, and a near-native locally unfolded state (N*), in which only one or a few amide hydrogens are completely accessible to solvent-exchange. The two-process model appears to account for the observed exchange behavior over the entire range of GdnHCl concentrations studied. For several measurable slow-exchanging amide hydrogens, the free energies of production of exchange-competent states from the exchange-incompetent native state are significantly higher than the free-energy of production of the equilibrium unfolded state from the native state, when the latter is determined from circular dichroism- or fluorescence-monitored equilibrium unfolding curves. The result implies that U*, which forms transiently in the strongly native-like conditions used for the hydrogen exchange studies, is higher in energy than the equilibrium-unfolded state. The higher energy of this transiently unfolded exchange-competent state can be attributed to either proline isomerization or to the presence of residual structure. On the basis of the free energies of production of exchange-competent states, the measured amide sites of barstar appear to define two structural subdomains--a three-helix unit and a two-beta-strand unit in the core of the protein.

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Assignment of the backbone ¹H and ¹⁵N NMR resonances and secondary structure characterization of barstar

Cited by 23 publications

References 22 publications

Discrepancies between the NMR and X-ray Structures of Uncomplexed Barstar: Analysis Suggests That Packing Densities of Protein Structures Determined by NMR Are Unreliable^,

Discrepancies between the NMR and X-ray Structures of Uncomplexed Barstar: Analysis Suggests That Packing Densities of Protein Structures Determined by NMR Are Unreliable^,

Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

Two structural subdomains of barstar detected by rapid mixing NMR measurement of amide hydrogen exchange

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Assignment of the backbone 1H and 15N NMR resonances and secondary structure characterization of barstar

Cited by 23 publications

References 22 publications

Discrepancies between the NMR and X-ray Structures of Uncomplexed Barstar: Analysis Suggests That Packing Densities of Protein Structures Determined by NMR Are Unreliable,

Discrepancies between the NMR and X-ray Structures of Uncomplexed Barstar: Analysis Suggests That Packing Densities of Protein Structures Determined by NMR Are Unreliable,

Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

Two structural subdomains of barstar detected by rapid mixing NMR measurement of amide hydrogen exchange

Contact Info

Product

Resources

About

Assignment of the backbone ¹H and ¹⁵N NMR resonances and secondary structure characterization of barstar

Discrepancies between the NMR and X-ray Structures of Uncomplexed Barstar: Analysis Suggests That Packing Densities of Protein Structures Determined by NMR Are Unreliable^,

Discrepancies between the NMR and X-ray Structures of Uncomplexed Barstar: Analysis Suggests That Packing Densities of Protein Structures Determined by NMR Are Unreliable^,