Three-dimensional solution structure and 13C assignments of barstar using nuclear magnetic resonance spectroscopy

Lubienski, Michael J.; Bycroft, Mark; Freund, Stefan; Fersht, Alan R.

doi:10.1021/bi00196a003

Cited by 113 publications

(160 citation statements)

References 39 publications

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“…The C82A mutant of barstar has activity (3) and thermal stability similar to those of wt barstar (21) with a ∆G°(25°C) of 25 kJ mol -1 and is marginally destabilized to GuHCl denaturation (18) compared to wt barstar. The C82A structure shows significant differences from the solution structure of the protein reported previously (7,22). † 1 Abbreviations: C82A, crystal structure of the Cys82Ala mutant of barstar at 2.8 Å solved in this study; C40A:C82A, barstar double mutant; wt, wild-type barstar; rmsd, root-mean-square deviation; B-factor, crystallographic temperature factor; MC, main chain; SC, side chain; SA, simulated annealing; 1BTA, the uncomplexed NMR structure (7); 1BGS, 2.6 Å structure of barstar C40A:C82A complexed to barnase (9); 1BRS, identical to 1BGS except at a higher resolution of 2.0 Å (8); OS, occluded surface; ∆C p, change in excess heat capacity upon unfolding; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); TNB, 5-thio-2-nitrobenzoic acid; GuHCl, guanidine hydrochloride.…”

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confidence: 68%

“…The NMR structure of free barstar had indicated that there were significant differences in the structures of the bound and free states (7,8,22). These differences were of three types: local changes in the conformations of residues in the binding region, an overall movement of the binding loop away from barnase, and global inward movements of the four helices in going from the bound to the free state.…”

Section: Resultsmentioning

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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contrasting

confidence: 68%

Section: Resultsmentioning

confidence: 99%

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 structure of barstar (21), the inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens, has been solved by NMR spectroscopy in solution (22), and the gross features of its folding pathway have been determined by rapid mixing methods and equilibrium thermodynamics (23). The active barstar mutant C40A/C82A/P27A (pseudo-wild-type barstar), which was used in this study, contains no cysteines which may give rise to crosslinks in the denatured state and only one proline residue.…”

mentioning

confidence: 99%

Submillisecond events in protein folding.

Nölting

Golbik

Fersht

1995

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

138

122

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The pathway of protein folding is now being analyzed at the resolution of individual residues by kinetic measurements on suitably engineered mutants. The kinetic methods generally employed for studying folding are typically limited to the time range of 21 ms because the folding of denatured proteins is usually initiated by mixing them with buffers that favor folding, and the dead time of rapid mixing experiments is about a millisecond. We now show that the study of protein folding may be extended to the microsecond time region by using temperature-jump measurements on the cold-unfolded state of a suitable protein. We are able to detect early events in the folding of mutants of barstar, the polypeptide inhibitor of barnase. A preliminary characterization of the fast phase from spectroscopic and 4D-value analysis indicates that it is a transition between two relatively solventexposed states with little consolidation of structure.The pathway by which a particular protein folds will be resolved experimentally when the structures of all stable, metastable, and transition states adopted by the protein during the process have been characterized structurally and energetically. The only way to analyze the structures of transition states is by kinetics, and the details of their structure and energetics can be gleaned from the kinetics of folding of proteins whose structures have been carefully altered by protein engineering (1-6). This protein engineering procedure has been used to characterize transition states and intermediates on a time scale of .1 ms (1-6), the time resolution of the stopped-flow and other rapid mixing techniques employed so far in these and most other studies (e.g., refs. 7-9). This is too slow to allow detection of the early events that initiate folding. To study faster events, kinetic techniques must be employed that eliminate the time delays arising from rapid mixing techniques. Relaxation methods, by which a preexisting equilibrium between denatured and folded states is rapidly perturbed by a change in physical or chemical conditions, may be used to extend the time range. It is not easy, however, to find methods that can readily cause a denatured state of a protein to renature. Millisecond time resolution has been achieved by using a repetitive pressure-perturbation method (10). Laser flash photolysis has been applied to dissociate CO from CO-bound cytochrome c, with concomitant denaturation (11). However, rapid CO rebinding, binding of non-native ligands, and possibly aggregation prevented the complete transition to the native state (11). A more generally applicable method for studying fast events would be to raise rapidly the temperature of a cold-denatured protein, since cold denaturation is a common phenomenon of globular proteins under suitable experimental conditions (12-15). Temperature jump (T-jump) by electrical discharge (16-20) and laser-induced heating would thus enable microsecond and nanosecond time resolutions, respectively.The structure of barstar (21), the inhibitor of th...

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“…The majority of Barstar's residues are involved in secondary structure, and there is one extended loop between the first two helices which is used for binding with barnase. 15 conformation that has been shown to also be capable of inhibiting barnase activity. 18 Other reports show that there exist intermediates that are 'fast-refolding' unfolded states that differ from 'slow-refolding' unfolded states by cis and trans conformations, respectively, of the Tyr47-Pro48 bond.…”

Section: This Studymentioning

confidence: 99%

A study of Barstar folding events using boundary value simulations

Yunger

2007

Physica A: Statistical Mechanics and its Applications

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This study revolves around a computational algorithm called SDEL (Stochastic Difference Equation in Length) that generates approximate protein folding trajectories on the atomically detailed resolution scale. The protein studied is Barstar-a barnase inhibitor. Because of the protein's interesting structure (four alpha helices, three beta strands) and relatively small size (89 residues), Barstar is an optimal choice for running complete folding trajectories on a computer. 12 pathways were generated with SDEL, starting from a structurally wide selection of unfolded conformations, yet all ending with the native configuration. We tracked hydrogen bonds, dihedral angles, native and non-native contacts, and energetic along these folding pathways. The resulting trajectories show: 1) Barstar follows the Hydrophobic Collapse folding scenario, 2) native α-helices begin forming earlier in the trajectory than the β-sheets, 3) particular residues maintain a propensity for helical structure in their unfolded state, and 4) specific non-native contacts persist during the folding trajectory. Strong correlations were found between the SDEL pathways and data from NMR, CD, and other experimental studies.

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Three-dimensional solution structure and 13C assignments of barstar using nuclear magnetic resonance spectroscopy

Cited by 113 publications

References 39 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^,

Submillisecond events in protein folding.

A study of Barstar folding events using boundary value simulations

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Three-dimensional solution structure and 13C assignments of barstar using nuclear magnetic resonance spectroscopy

Cited by 113 publications

References 39 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,

Submillisecond events in protein folding.

A study of Barstar folding events using boundary value simulations

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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^,

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