A Variable Light Domain Fluorogen Activating Protein Homodimerizes To Activate Dimethylindole Red
- Scripps
Novel fluorescent tools such as green fluorescent protein analogues and fluorogen activating proteins (FAPs) are useful in biological imaging for tracking protein dynamics in real time with a low fluorescence background. FAPs are single-chain variable fragments (scFvs) selected from a yeast surface display library that produce fluorescence upon binding a specific dye or fluorogen that is normally not fluorescent when present in solution. FAPs generally consist of human immunoglobulin variable heavy (V{sub H}) and variable light (V{sub L}) domains covalently attached via a glycine- and serine-rich linker. Previously, we determined that the yeast surface clone, V{sub H}-V{sub L} M8, could bind and activate the fluorogen dimethylindole red (DIR) but that the fluorogen activation properties were localized to the M8V{sub L} domain. We report here that both nuclear magnetic resonance and X-ray diffraction methods indicate the M8V{sub L} forms noncovalent, antiparallel homodimers that are the fluorogen activating species. The M8V{sub L} homodimers activate DIR by restriction of internal rotation of the bound dye. These structural results, together with directed evolution experiments with both V{sub H}-V{sub L} M8 and M8V{sub L}, led us to rationally design tandem, covalent homodimers of M8V{sub L} domains joined by a flexible linker that have a high affinity for DIR and good quantum yields.
- Research Organization:
- Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Organization:
- NIHHHMI
- OSTI ID:
- 1044409
- Journal Information:
- Biochemistry (Eaton), Vol. 51, Issue 12; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- ENGLISH
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