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Title: Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod

Abstract

X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 – Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.

Authors:
; ; ; ORCiD logo
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1418060
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Structural Biology; Journal Volume: 200; Journal Issue: 3
Country of Publication:
United States
Language:
ENGLISH
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Andreas, Michael P., Ajay, Gautam, Gellings, Jaclyn A., and Rayment, Ivan. Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod. United States: N. p., 2017. Web. doi:10.1016/j.jsb.2017.07.006.
Andreas, Michael P., Ajay, Gautam, Gellings, Jaclyn A., & Rayment, Ivan. Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod. United States. doi:10.1016/j.jsb.2017.07.006.
Andreas, Michael P., Ajay, Gautam, Gellings, Jaclyn A., and Rayment, Ivan. Fri . "Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod". United States. doi:10.1016/j.jsb.2017.07.006.
@article{osti_1418060,
title = {Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod},
author = {Andreas, Michael P. and Ajay, Gautam and Gellings, Jaclyn A. and Rayment, Ivan},
abstractNote = {X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 – Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.},
doi = {10.1016/j.jsb.2017.07.006},
journal = {Journal of Structural Biology},
number = 3,
volume = 200,
place = {United States},
year = {Fri Dec 01 00:00:00 EST 2017},
month = {Fri Dec 01 00:00:00 EST 2017}
}