Author:
Okwei Elleansar,Ganguly Soumya,Darling Heather L.,Harp Joel M.,Gulsevin Alican,Coin Irene,Mchaourab Hassane,Ledwitch Kaitlyn,Kuenze Georg,Meiler Jens
Abstract
SummarySite-directed spin labeling of proteins via non-canonical amino acids (ncAAs) is a non-traditional method for the measurement of pseudocontact shifts (PCSs) by nuclear magnetic resonance (NMR) spectroscopy. PCSs provide long-range distance and orientational information between a paramagnetic center and protein nuclei that can be used as restraints for computational structural modeling techniques. Here, we present the first experimental structure of an ncAA chemically linked to a lanthanide tag conjugated to the protein, T4-Lysozyme (T4L). T4L was crystallized with a cyclen-based C3 tag coordinated to the paramagnetic ion terbium (Tb3+). The paramagnetic C3-lanthanide tag generated PCSs measured at four different ncAA sites. We show that the addition of these restraints improves structure prediction protocols for T4L using the RosettaNMR framework. Generated models provide insight into T4L conformational flexibility sampled in solution. This integrative modeling protocol is readily transferable to larger proteins. Methods to predict protein structures are advancing into an exciting arena such that reliable experimental data will play important roles for evaluating the biophysical relevance of predicted structural models. Our contribution here caters to the growing interest in using ncAAs for a range of biophysical studies, and these methods can be readily transferred to larger protein systems of interest.
Publisher
Cold Spring Harbor Laboratory
Cited by
2 articles.
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