One of my major reasons for attending BPS this year was to expand my knowledge in a field that isn’t very important at all for the work that I do in my day to day. My work involves designing molecules that can alter protein function and hopefully “drug” an interaction or protein conformation that is useful therapeutically. The readouts for whether we are successful are pragmatic ones — we look at cell viability, downstream effects, preservation or desolation of certain cellular pathways as needed. What we generally don’t concern ourself with is confirming with mechanistic insight how exactly the molecules we make do what they do. So I decided to go learn more about biophysical techniques for looking at protein dynamics and allostery — the best place to do that was BPS.
Well I’ve learned a lot, and have a lot more to learn from all the papers and techniques that others have suggested I look into. One of the most fascinating examples of a study hoping to shed light on protein dynamics of therapeutic importance was presented yesterday by James Munro. Professor Munro used single molecule FRET to monitor conformational changes in the HIV envelope protein gp120 as it interacted with receptors on the host cell surface. Only one envelope protein on each HIV virion was dually labeled, with a FRET donor at one relatively “fixed” location, and an acceptor at one of three locations on nearby loops of gp120. FRET is a very powerful technique, and smFRET is even better since it gives conformational trajectories that can give valuable information about the kinetics being observed. However with two labels, the FRET readout is one dimensional — only one coordinate is generated, with points along a line of FRET efficiency indicating the distance between two points on a protein surface. Can something as complex as HIV envelope binding and entry be observed usefully along a single coordinate?
The answer, as published in Science last year, is yes. With the choice of a coordinate indicating the distance between the V1/V1 loop region and the V5 loop in the outer domain, Munro and his coworkers were able to observe three distinct conformations accessed by the envelope protein: a highly occupied low-FRET “ground” state indicating prefusion envelope protein, a high-FRET state indicating the envelope protein bound to its receptor CD4, and an intermediate-FRET state indicating binding to both CD4 and the HIV coreceptor. Many experiments with both laboratory and clinically-derived HIV strains with a variety of ligands confirmed this result. The smFRET kinetics also supported this view, as fitting the traces to a three-state Markov model showed many transitions from unbound to CD4-bound, and transitions from CD4-bound to CD4+Coreceptor-bound, but very rarely transitions from unbound directly to the doubly-bound conformation.
This choice of coordinate was not a lucky guess, it was guided by existing low-resolution structures of the envelope protein during membrane fusion, and even so, likely was the result of many grad student/postdoc-years of trial and error. What this study does show is that even complicated and dynamic processes like HIV membrane fusion can often be monitored and deep information gleaned from a very clever choice of one coordinate.
I’ve often spend time choosing a coordinate to succinctly show the transitions in a molecular dynamics system of interest, and seeing someone not only choose the right coordinate, but get a working smFRET experiment working along it for such a cool system was a lot of fun.