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CID fragmentation, H/D exchange and supermetallization of Barnase-Barstar complex.

ABSTRACT: The barnase-barstar complex is one of the most stable protein-protein complexes and has a very wide range of possible applications. Here we report the use of top-down mass spectrometry for the investigation of the structure of this complex, its ionization via ESI, isolation and fragmentation. It was found that the asymmetry of the resulting charge state distributions of the protein monomer product ions increased as the charge state of the precursor ions increased. For the investigation of the 3D structure of the complex, the gas phase H/D exchange reaction was used. In addition, supermetallized ions of the complex with Zn were produced and investigated. It was observed that an increase in the number of metals bound to the complex results in a change in complex stability and the charge distribution between protein fragment. Analysis of the fragmentation pattern of the supermetallized complex [bn-b*?+?5Zn]¹⁰⁺ indicated that this ion is present in different conformations with different charges and Zn distributions. Since Zn cannot migrate, such structures must be formed during ionization.

SUBMITTER: Kostyukevich Y

PROVIDER: S-EPMC5522418 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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CID fragmentation, H/D exchange and supermetallization of Barnase-Barstar complex.

Kostyukevich Yury Y Shulga Aleksej A AA Kononikhin Alexey A Popov Igor I Nikolaev Eugene E Deyev Sergey S

Scientific reports 20170721 1

The barnase-barstar complex is one of the most stable protein-protein complexes and has a very wide range of possible applications. Here we report the use of top-down mass spectrometry for the investigation of the structure of this complex, its ionization via ESI, isolation and fragmentation. It was found that the asymmetry of the resulting charge state distributions of the protein monomer product ions increased as the charge state of the precursor ions increased. For the investigation of the 3D ...[more]

PMID: 28733680

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Project description:Macromolecular crowding within the cell can impact both protein folding and binding. Earlier models of cellular crowding focused on the excluded volume, entropic effect of crowding agents, which generally favors compact protein states. Recently, other effects of crowding have been explored, including enthalpically-related crowder-protein interactions and changes in solvation properties. In this work, we explore the effects of macromolecular crowding on the electrostatic desolvation and solvent-screened interaction components of protein-protein binding. Our simple model enables us to focus exclusively on the electrostatic effects of water depletion on protein binding due to crowding, providing us with the ability to systematically analyze and quantify these potentially intuitive effects. We use the barnase-barstar complex as a model system and randomly placed, uncharged spheres within implicit solvent to model crowding in an aqueous environment. On average, we find that the desolvation free energy penalties incurred by partners upon binding are lowered in a crowded environment and solvent-screened interactions are amplified. At a constant crowder density (fraction of total available volume occupied by crowders), this effect generally increases as the radius of model crowders decreases, but the strength and nature of this trend can depend on the water probe radius used to generate the molecular surface in the continuum model. In general, there is huge variation in desolvation penalties as a function of the random crowder positions. Results with explicit model crowders can be qualitatively similar to those using a lowered "effective" solvent dielectric to account for crowding, although the "best" effective dielectric constant will likely depend on multiple system properties. Taken together, this work systematically demonstrates, quantifies, and analyzes qualitative intuition-based insights into the effects of water depletion due to crowding on the electrostatic component of protein binding, and it provides an initial framework for future analyses.

Dataset Information

CID fragmentation, H/D exchange and supermetallization of Barnase-Barstar complex.

Publications

CID fragmentation, H/D exchange and supermetallization of Barnase-Barstar complex.

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