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Published on: September 14, 2014
Functional characterization and conformational analysis of the Herpesvirus saimiri Tip-C484 protein
Jennifer L Mitchell1, Ronald P Trible, Lori A Emert-Sedlak
1Department of Chemistry, University of New Mexico, Albuquerque, NM 87131, USA.
Abstract:
Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific member of the Src family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen-exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured, so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel-filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of activating Lck kinase activity strongly and was multiply phosphorylated by Lck. Hydrogen-exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogen atoms became deuterated after only 10 s of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium-labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that, although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.
Insights
Herpesvirus saimiri tyrosine kinase interacting protein (Tip) is largely unstructured but functionally active. This protein binds and activates Lck kinase, crucial for HVS-induced oncogenesis.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Herpesvirus saimiri (HVS) utilizes tyrosine kinase interacting protein (Tip) to activate Lck, a lymphoid-specific Src family kinase.
- The Tip-Lck interaction is critical for HVS-mediated cellular transformation and oncogenesis.
- Lack of structural data for Tip necessitates investigation into its conformation and function.
Purpose of the Study:
- To determine the structural conformation of the Herpesvirus saimiri Tip protein (residues 1-187).
- To confirm the functionality of recombinant Tip in binding and activating Lck kinase.
- To elucidate the relationship between Tip's structure and its biological activity.
Main Methods:
- Hydrogen-exchange mass spectrometry (HX-MS) to probe protein structure in solution.
- Circular dichroism (CD) and gel-filtration analysis to assess protein conformation.
- In vitro and in vivo binding and kinase assays to evaluate Tip-Lck interaction and Lck activation.
Main Results:
- Circular dichroism and gel-filtration indicated Tip is an extended, unstructured protein.
- Recombinant Tip demonstrated robust binding to Lck and strong activation of Lck kinase activity.
- HX-MS revealed rapid deuterium exchange across most of the Tip protein, confirming its largely unstructured nature in solution.
- Minor regions of protection from exchange were observed upon deuterium labeling.
Conclusions:
- Despite being largely unstructured, recombinant Herpesvirus saimiri Tip protein is functional.
- Tip effectively binds and activates its target Lck kinase.
- The study provides insights into the structural basis of Tip-mediated Lck activation and HVS oncogenesis.
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