Dataset Information

Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins.

ABSTRACT: Flexible polypeptide linkers composed of glycine and serine are important components of engineered multidomain proteins. We have previously shown that the conformational properties of Gly-Gly-Ser repeat linkers can be quantitatively understood by comparing experimentally determined Förster resonance energy transfer (FRET) efficiencies of ECFP-linker-EYFP proteins to theoretical FRET efficiencies calculated using wormlike chain and Gaussian chain models. Here we extend this analysis to include linkers with different glycine contents. We determined the FRET efficiencies of ECFP-linker-EYFP proteins with linkers ranging in length from 25 to 73 amino acids and with glycine contents of 33.3% (GSSGSS), 16.7% (GSSSSSS), and 0% (SSSSSSS). The FRET efficiency decreased with an increasing linker length and was overall lower for linkers with less glycine. Modeling the linkers using the WLC model revealed that the experimentally observed FRET efficiencies were consistent with persistence lengths of 4.5, 4.8, and 6.2 Å for the GSSGSS, GSSSSS, and SSSSSS linkers, respectively. The observed increase in linker stiffness with reduced glycine content is much less pronounced than that predicted by a classical model developed by Flory and co-workers. We discuss possible reasons for this discrepancy as well as implications for using the stiffer linkers to control the effective concentrations of connected domains in engineered multidomain proteins.

SUBMITTER: van Rosmalen M

PROVIDER: S-EPMC6150656 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins.

van Rosmalen Martijn M Krom Mike M Merkx Maarten M

Biochemistry 20171207 50

Flexible polypeptide linkers composed of glycine and serine are important components of engineered multidomain proteins. We have previously shown that the conformational properties of Gly-Gly-Ser repeat linkers can be quantitatively understood by comparing experimentally determined Förster resonance energy transfer (FRET) efficiencies of ECFP-linker-EYFP proteins to theoretical FRET efficiencies calculated using wormlike chain and Gaussian chain models. Here we extend this analysis to include li ...[more]

PMID: 29168376

Similar Datasets

Project description:The emerging complexity of large macromolecules has led to challenges in their full scale theoretical description and computer simulation. Multiscale multiphysics and multidomain models have been introduced to reduce the number of degrees of freedom while maintaining modeling accuracy and achieving computational efficiency. A total energy functional is constructed to put energies for polar and nonpolar solvation, chemical potential, fluid flow, molecular mechanics, and elastic dynamics on an equal footing. The variational principle is utilized to derive coupled governing equations for the above mentioned multiphysical descriptions. Among these governing equations is the Poisson-Boltzmann equation which describes continuum electrostatics with atomic charges. The present work introduces the theory of continuum elasticity with atomic rigidity (CEWAR). The essence of CEWAR is to formulate the shear modulus as a continuous function of atomic rigidity. As a result, the dynamics complexity of a macromolecular system is separated from its static complexity so that the more time-consuming dynamics is handled with continuum elasticity theory, while the less time-consuming static analysis is pursued with atomic approaches. We propose a simple method, flexibility-rigidity index (FRI), to analyze macromolecular flexibility and rigidity in atomic detail. The construction of FRI relies on the fundamental assumption that protein functions, such as flexibility, rigidity, and energy, are entirely determined by the structure of the protein and its environment, although the structure is in turn determined by all the interactions. As such, the FRI measures the topological connectivity of protein atoms or residues and characterizes the geometric compactness of the protein structure. As a consequence, the FRI does not resort to the interaction Hamiltonian and bypasses matrix diagonalization, which underpins most other flexibility analysis methods. FRI's computational complexity is of O(N(2)) at most, where N is the number of atoms or residues, in contrast to O(N(3)) for Hamiltonian based methods. We demonstrate that the proposed FRI gives rise to accurate prediction of protein B-Factor for a set of 263 proteins. We show that a parameter free FRI is able to achieve about 95% accuracy of the parameter optimized FRI. An interpolation algorithm is developed to construct continuous atomic flexibility functions for visualization and use with CEWAR.

Project description:Sequence-selective chemical modification of DNA by synthetic ligands has been a long-standing challenge in the field of chemistry. Even when the ligand consists of a sequence-specific DNA binding domain and reactive group, sequence-selective reactions by these ligands are often accompanied by off-target reactions. A basic principle to design DNA modifiers that react at specific sites exclusively governed by DNA sequence recognition remains to be established. We have previously reported selective DNA modification by a self-ligating protein tag conjugated with a DNA-binding domain, termed as a modular adaptor, and orthogonal application of modular adaptors by relying on the chemoselectivity of the protein tag. The sequence-specific crosslinking reaction by the modular adaptor is thought to proceed in two steps: the first step involves the formation of a DNA-protein complex, while in the second step, a proximity-driven intermolecular crosslinking occurs. According to this scheme, the specific crosslinking reaction of a modular adaptor would be driven by the DNA recognition process only when the dissociation rate of the DNA complex is much higher than the rate constant for the alkylation reaction. In this study, as a proof of principle, a set of combinations for modular adaptors and their substrates were utilized to evaluate the reactions. Three types of modular adaptors consisting of a single type of self-ligating tag and three types of DNA binding proteins fulfill the kinetic requirements for the reaction of the self-ligating tag with a substrate and the dissociation of the DNA-protein complex. These modular adaptors actually undergo sequence-specific crosslinking reactions exclusively driven by the recognition of a specific DNA sequence. The design principle of sequence-specific modular adaptors based on the kinetic aspects of complex formation and chemical modification is applicable for developing recognition-driven selective modifiers for proteins and other biological macromolecules.

Dataset Information

Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins.

Publications

Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets