A variable light domain fluorogen activating protein homodimerizes to activate dimethylindole red

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Imaging

Background:

• Novel fluorescent tools like fluorogen activating proteins (FAPs) are crucial for real-time biological imaging.
• FAPs are single-chain variable fragments (scFvs) that activate non-fluorescent dyes upon binding.
• Previous work identified V(H)-V(L) M8 as a FAP, with activation localized to the M8V(L) domain.

Purpose of the Study:

• To elucidate the structural basis of fluorogen activation by the M8V(L) domain.
• To understand how M8V(L) activates the fluorogen dimethylindole red (DIR).
• To rationally design improved FAPs with enhanced affinity and quantum yield.

Main Methods:

• Nuclear magnetic resonance (NMR) spectroscopy
• X-ray diffraction
• Directed evolution experiments

Main Results:

• The M8V(L) domain forms noncovalent, antiparallel homodimers.
• These homodimers are the active species that bind and activate DIR.
• Activation occurs through the restriction of internal rotation of the bound DIR dye.
• Designed tandem covalent homodimers of M8V(L) showed high affinity for DIR and good quantum yields.

Conclusions:

• M8V(L) homodimerization is essential for fluorogen activation.
• Structural insights enable the rational design of enhanced FAPs for biological imaging.
• The developed FAPs offer improved performance for tracking protein dynamics.