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Engineering encodable lanthanide-binding tags into loop regions of proteins.

ABSTRACT: Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1? (IL1?) and varied the length of the spacer between the LBT and the protein (denoted 1?3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1?-S1 and IL1?-L3 constructs and for the remaining constructs by comparing the 1H?15N heteronuclear single-quantum coherence NMR spectra with that of the wild-type IL1?. Additionally, binding of LBT-loop IL1? proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1? structure. The paramagnetic NMR spectra of loop-LBT mutant IL1?-R2 were assigned and the ?? tensor components were calculated on the basis of RDCs and pseudocontact shifts. A structural model of the IL1?-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modeling.

SUBMITTER: Barthelmes K

PROVIDER: S-EPMC3043167 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Engineering encodable lanthanide-binding tags into loop regions of proteins.

Barthelmes Katja K Reynolds Anne M AM Peisach Ezra E Jonker Hendrik R A HR DeNunzio Nicholas J NJ Allen Karen N KN Imperiali Barbara B Schwalbe Harald H

Journal of the American Chemical Society 20110201 4

Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β (IL1β) and varied the length of the spacer between the LBT and the protein (denoted 1−3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No sign ...[more]

PMID: 21182275

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Project description:Many disordered proteins function via binding to a structured partner and undergo a disorder-to-order transition. The coupled folding and binding can confer several functional advantages such as the precise control of binding specificity without increased affinity. Additionally, the inherent flexibility allows the binding site to adopt various conformations and to bind to multiple partners. These features explain the prevalence of such binding elements in signaling and regulatory processes. In this work, we report ANCHOR, a method for the prediction of disordered binding regions. ANCHOR relies on the pairwise energy estimation approach that is the basis of IUPred, a previous general disorder prediction method. In order to predict disordered binding regions, we seek to identify segments that are in disordered regions, cannot form enough favorable intrachain interactions to fold on their own, and are likely to gain stabilizing energy by interacting with a globular protein partner. The performance of ANCHOR was found to be largely independent from the amino acid composition and adopted secondary structure. Longer binding sites generally were predicted to be segmented, in agreement with available experimentally characterized examples. Scanning several hundred proteomes showed that the occurrence of disordered binding sites increased with the complexity of the organisms even compared to disordered regions in general. Furthermore, the length distribution of binding sites was different from disordered protein regions in general and was dominated by shorter segments. These results underline the importance of disordered proteins and protein segments in establishing new binding regions. Due to their specific biophysical properties, disordered binding sites generally carry a robust sequence signal, and this signal is efficiently captured by our method. Through its generality, ANCHOR opens new ways to study the essential functional sites of disordered proteins.

Dataset Information

Engineering encodable lanthanide-binding tags into loop regions of proteins.

Publications

Engineering encodable lanthanide-binding tags into loop regions of proteins.

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