Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen‐deuterium exchange

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

doi:10.1016/0014-5793(93)80319-p

Cited by 40 publications

(26 citation statements)

References 25 publications

(18 reference statements)

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“…The barnase-barstar binding is overall both enthalpy-and entropy-driven, although the enthalpy term is about 4 times higher than the entropic one at pH 7.0 at 25 "C. This high negative enthalpy binding value correlates very well with our present knowledge of the structural information of the complex (Hartley, 1993;Jones et al, 1993;Guillet et al, 1993;Buckle et al, 1994). Thus, the barstar-barnase interface is formed by highly polar and charged surfaces, forming a network of polar interactions with 14 hydrogen bonds and several electrostatic bridges, interactions characterized by a few kilocalories per mole [cf.…”

Section: Discussionsupporting

confidence: 78%

A Calorimetric Study of the Thermal Stability of Barstar and Its Interaction with Barnase

Martínez¹,

Filimonov²,

Mateo³

et al. 1995

Biochemistry

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The temperature-induced unfolding of single, double, and triple mutants of barstar, the specific intracellular protein inhibitor of barnase from Bacillus amyloliquefaciens, has been studied by high-sensitivity differential scanning calorimetry. The thermal unfolding of barstar mutants, where at least one of the two cysteine residues in the molecule had been replaced by alanine, follows a two-state mechanism at neutral and alkaline pH. The unfolding enthalpy and heat capacity changes are slightly lower than those accepted for highly compact, small, globular proteins. We have found that at pH 2.5, where barstar seems to be in a molten globule state, the protein has a heat capacity between that of the native and the unfolded states and shows some tendency for association. Scanning calorimetry experiments were also extended to the barstar--barnase complex in the neutral and alkaline pH range. The binding constants obtained from DSC studies are similar to those already obtained from other (kinetic) studies. The interaction of barstar and barnase was also investigated by isothermal calorimetry in various buffers within the pH range 6.0-10.0 and a temperature range of 15-35 degrees C. The favorable enthalpy contribution to the binding is about 4 times higher than the entropic one at 25 degrees C. The overall data analysis of the combined calorimetric results has led to the thermodynamic characterization of barstar unfolding and the interaction of barstar and barnase over a wide range of temperatures.

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Section: Discussionsupporting

confidence: 78%

A Calorimetric Study of the Thermal Stability of Barstar and Its Interaction with Barnase

Martínez¹,

Filimonov²,

Mateo³

et al. 1995

Biochemistry

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“…The chemical shifts of the backbone amides were assigned by comparison with previously assigned spectra. 55 Each sample was shimmed and tuned before the start of data acquisition. The length of the 908 pulse was determined by adjusting the p1 value until the transformed and phased 1 H water peak showed equal areas above and below the baseline.…”

Section: Nuclear Magnetic Resonance Experimentsmentioning

confidence: 99%

The Kinetic Pathway of Folding of Barnase

Khan¹,

Chuang²,

Gianni³

et al. 2003

Journal of Molecular Biology

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“…Barnase was purified from cultures of Escherichia coli (BL21) containing the plasmid pMT410 (9) as described previously (10,11). Separate samples were produced of uniformly 15N-labeled and uniformly 15N, '3C-labeled protein.…”

Section: Methodsmentioning

confidence: 99%

Toward solving the folding pathway of barnase: the complete backbone 13C, 15N, and 1H NMR assignments of its pH-denatured state.

Arcus

Vuilleumier

Freund

et al. 1994

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

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The structures of the major folding intermediate, the transition state for folding, and the folded state of barnase have been previously characterized. We now add a further step toward a complete picture ofthe folding of barnase by reporting the backbone 15N, 13C, and 'H NMR assignments for barnase unfolded at pH 1.8 resonances and has very recently been applied to the assignment and structural characterization of the urea-and guanidinium chloride-denatured states of the FK506-binding protein (8).Barnase has a cooperative and reversible unfolding transition between pH 2.8 and pH 2.0 at 300C (M. Oliveberg, S.V., and A.R.F., unpublished results). Here we report the sequence-specific NMR assignment of barnase in the pHdenatured form, which constitutes the first major step in the characterization of this unfolded state by NMR. Magnetization transfer methods at the transition midpoint (pH = 2.4) provided sequence-specific assignments for 30%o of the protein, and these assignments were confirmed and augmented, and the full assignment was completed by using threedimensional (3D) backbone triple-resonance experiments. MATERIALS AND METHODSBarnase was purified from cultures of Escherichia coli (BL21) containing the plasmid pMT410 (9) as described previously (10,11). Separate samples were produced of uniformly 15N-labeled and uniformly 15N, '3C-labeled protein. NMR samples at pH 2.9 and pH 1.8 were prepared by dissolving lyophilized protein in 0.5 ml of 90% H20/10% 2H20 (vol/vol) with appropriate concentrations of HCl. A sample at pH 2.4 additionally contained 50 mM glycine-d5 buffer. The final concentrations for samples of uniformly 15N-labeled barnase were 2.4 mM and for 15N, 13C-labeled barnase, 1.4 mM.All NMR spectra were recorded at 300C on a Bruker AMX500 spectrometer fitted with a fourth channel. Spectra were processed by using the Felix software package (Biosym Technologies, San Diego). The '5N and 'H amide resonances for native barnase at pH 2.9 were assigned by reference to the full barnase assign-

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Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen‐deuterium exchange

Cited by 40 publications

References 25 publications

A Calorimetric Study of the Thermal Stability of Barstar and Its Interaction with Barnase

A Calorimetric Study of the Thermal Stability of Barstar and Its Interaction with Barnase

The Kinetic Pathway of Folding of Barnase

Toward solving the folding pathway of barnase: the complete backbone 13C, 15N, and 1H NMR assignments of its pH-denatured state.

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