1993
DOI: 10.1016/0014-5793(93)80319-p
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Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen‐deuterium exchange

Abstract: The extracellular ribonuclease from Bucrllus amylohquifacwns, barnase. forms a tightly-bound one-to-one complex with its intracellular inhibitor barstar. The barstar binding site on barnase was charactemed by comparing the differences m the chemical shift and hydrogen-deuterium exchange rates between free and bound barnase Chemical shift assignments of bamase in the complex with barstar were determined from 3D NOESY-HMQC and TOCSY-HMQC spectra of a complex that had been prepared with uniformly 'SN-labelled bar… Show more

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Cited by 40 publications
(26 citation statements)
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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%
“…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%
“…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%
“…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%