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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 4, Iss. 12 — Dec. 1, 2013
  • pp: 2835–2845

Single-molecule force measurement via optical tweezers reveals different kinetic features of two BRaf mutants responsible for cardio-facial-cutaneous (CFC) syndrome

Cheng Wen, Chae-Seok Lim, Anpei Ye, and J. Julius Zhu  »View Author Affiliations

Biomedical Optics Express, Vol. 4, Issue 12, pp. 2835-2845 (2013)

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BRaf (B- Rapid Accelerated Fibrosarcoma) protein is an important serine/threonine-protein kinase. Two domains on BRaf can independently bind its upstream kinase, Ras (Rat Sarcoma) protein. These are the Ras binding domain (RBD) and cysteine-rich-domain (CRD). Herein we use customized optical tweezers to compare the Ras binding process in two pathological mutants of BRaf responsible for CFC syndrome, abbreviated BRaf (A246P) and BRaf (Q257R). The two mutants differ in their kinetics of Ras-binding, though both bind Ras with similar increased overall affinity. BRaf (A246P) exhibits a slightly higher Ras/CRD unbinding force and a significantly higher Ras/RBD unbinding force versus the wild type. The contrary phenomenon is observed in the Q257R mutation. Simulations of the unstressed-off rate, koff(0), yield results in accordance with the changes revealed by the mean unbinding force. Our approach can be applied to rapidly assess other mutated proteins to deduce the effects of mutation on their kinetics compared to wild type proteins and to each other.

© 2013 Optical Society of America

OCIS Codes
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(170.1420) Medical optics and biotechnology : Biology
(170.4520) Medical optics and biotechnology : Optical confinement and manipulation
(350.4855) Other areas of optics : Optical tweezers or optical manipulation

ToC Category:
Optical Traps, Manipulation, and Tracking

Original Manuscript: September 3, 2013
Revised Manuscript: October 9, 2013
Manuscript Accepted: October 23, 2013
Published: November 14, 2013

Cheng Wen, Chae-Seok Lim, Anpei Ye, and J. Julius Zhu, "Single-molecule force measurement via optical tweezers reveals different kinetic features of two BRaf mutants responsible for cardio-facial-cutaneous (CFC) syndrome," Biomed. Opt. Express 4, 2835-2845 (2013)

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