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1 H-Detected Solid-State NMR Studies of Water-Inaccessible Proteins In?Vitro and In?Situ.

ABSTRACT: 1 H detection can significantly improve solid-state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for 1 H?detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water-inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high-resolution 1 H-detected solid-state NMR (ssNMR) studies of water-inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K+ channel KcsA in liposomes and in?situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K+ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.

SUBMITTER: Medeiros-Silva J

PROVIDER: S-EPMC5113794 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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1 H-Detected Solid-State NMR Studies of Water-Inaccessible Proteins In Vitro and In Situ.

Medeiros-Silva João J Mance Deni D Daniëls Mark M Jekhmane Shehrazade S Houben Klaartje K Baldus Marc M Weingarth Markus M

Angewandte Chemie (International ed. in English) 20160927 43

1 H detection can significantly improve solid-state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for 1 H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water-inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high-resolution 1 H-detected solid-state NMR (ssNM ...[more]

PMID: 27671832

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Project description:The mechanical properties of Bombyx mori silk fibroin (SF), such as elasticity and tensile strength, change remarkably upon hydration. However, the microscopic interaction with water is not currently well understood on a molecular level. In this work, the dynamics of water molecules interacting with SF was studied by 2H solution NMR relaxation and exchange measurements. Additionally, the conformations of hydrated [3-13C]Ala-, [3-13C]Ser-, and [3-13C]Tyr-SF fibers and films were investigated by 13C DD/MAS NMR. Using an inverse Laplace transform algorithm, we were able to identify four distinct components in the relaxation times for water in SF fiber. Namely, A: bulk water outside the fiber, B: water molecules trapped weakly on the surface of the fiber, C: bound water molecules located in the inner surface of the fiber, and D: bound water molecules located in the inner part of the fiber were distinguishable. In addition, four components were also observed for water in the SF film immersed in methanol for 30s, while only two components for the film immersed in methanol for 24h. The effects of hydration on the conformation of Ser and Tyr residues in the site-specific crystalline and non-crystalline domains of 13C selectively labeled SF, respectively, could be determined independently. Our measurements provide new insight relating the characteristics of water and the hydration structure of silk, which are relevant in light of current interest in the design of novel silk-based biomaterials.Statements of significanceThe mechanical properties of Bombyx mori silk fibroin (SF) change remarkably upon hydration. However, the microscopic interaction between SF and water is not currently well understood on a molecular level. We were able to identify four distinct components in the relaxation times for water in SF fiber by 2H solution NMR relaxation and exchange measurements. In addition, the effects of hydration on the conformation of Ser and Tyr residues in the site-specific crystalline and non-crystalline domains of 13C selectively labeled SF, respectively, could be determined independently. Thus, our measurements provide new insight relating the characteristics of water and the hydration structure of silk, which are relevant in light of current interest in the design of novel silk-based biomaterials.

Dataset Information

1 H-Detected Solid-State NMR Studies of Water-Inaccessible Proteins In?Vitro and In?Situ.

Publications

<sup>1</sup> H-Detected Solid-State NMR Studies of Water-Inaccessible Proteins In Vitro and In Situ.

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