See y’all there!

Many questions (what benefits of protein dynamics for the host fungus?) and implications (better/new opto or thermo proteins). Overall, intra-protein network motifs provide a new way to think about compact, dynamic computation on the protein level.

Indeed, 1 A.A. mutation from each of these hotspots dramatically extended the pulsatory response (>18 hrs vs 10s of min), suggesting evolutionary tuning of dynamics.

Remarkably, sequence analysis found these *exact same hotspots* had co-evolved in BcLOV4 homologs, implying function.

Structural studies (limited proteolysis, HDX-MS by the Gardner lab) identified temperature-sensitive hot-spots in DUF.

What about the temperature-sensitive inhibitory Node C? We mapped this to the mysterious “Domain of Unknown Function” (DUF). We still call it DUF even though function is now known :P.

We then mapped the network nodes: The light-sensing LOV domain (A) regulates a charged clustering region (B), which drives PM binding. Aside: this inspired an entire toolset for single-protein translocation to various compartments. Preprint soon!

The inter-domain IFFL idea started as a hunch, but models made several non-intuitive predictions that we then confirmed experimentally through mutations that altered the energetics of LOV domain interactions.

Answer: smaller-scale networks of interactions between the protein’s domains. Light response (A) quickly activates a region for plasma membrane (PM) binding (B), but also enables a temp.-sensitive domain (C) to inhibit the PM-binding region more slowly.

Step-to-pulse response – adaptation – is common in all kinds of networks, often through an incoherent feed-forward loop (IFFL). Node A quickly activates Node B but also —more slowly— Node C, which inhibits Node B. Result: a pulse of B. But how can a single protein do it w/o a larger network?

Why does BcLOV4 turn itself off despite constant light stimulation, and why does this depend on temperature? The answer could make better optogenetic and thermogenetic tools, and itself was a fascinating puzzle.

How can an individual protein show adaptation? The light- and temp-sensitive BcLOV4 gives a pulse of translocation during a step input of light. Answer in our preprint: *intra-molecular* feed-forward regulation. bit.ly/3Rg9veD
A @bugajlab.bsky.social collab with @nmrkaygee.bsky.social 🧵