Last week we wrote about NMR, one
of the most popular fragment-finding methods. This week we turn to a less common
technique: weak affinity chromatography, or WAC. As we’ve written previously, WAC
involves immobilizing a protein of interest in a chromatography column and
flowing a ligand-containing solution through the column. If the ligand
interacts with the protein, its elution time will be delayed in proportion to its
affinity. In a new (open-access) Molecules paper, Claire Demesmay and collaborators
at Universite Claude Bernard Lyon and Ecole Supérieure de Biotechnologie de
Strasbourg extend the technique to membrane proteins.
Membrane proteins are themselves
tricky to study, since removing them from their membranes often denatures them.
One trick is to use nanodiscs, which are tiny lipid bilayer islands surrounded
by proteins that keep them soluble in water. These scaffolding proteins can
also be biotinylated so that the nanondiscs can be attached to streptavidin,
which itself can be linked to a surface or matrix. Each nanodisc holds one or
at most a few membrane proteins.
When we first wrote about WAC in
2011 the technique used standard HPLC columns, which required non-negligible
amounts of protein. Here, the technique has been miniaturized to use glass
capillaries with volumes of less than 1 microliter, requiring only a few tens
of picomoles of protein. The researchers fill the capillaries with a
bio-compatible polymer, functionalize it with streptavidin, and then capture
biotinylated nanodiscs containing the membrane protein of interest.
A long-recognized challenge with
WAC is nonspecific binding of the fragments to the column or matrix. Here, the
researchers chose a filling (or monolith) that is more hydrophilic (for
aficionados, they picked poly(DHPMA-co-MBA)) and found it superior to the
previous polymer both with regards to capacity and non-specific binding.
Another challenge with WAC is
detecting low-affinity binders: because interactions with the protein are weak,
the shift in retention time is harder to detect. One solution is to pack more
protein in the column, and the researchers develop a clever way of doing this with
a “multilayer grafting” approach in which successive injections of streptavidin
and nanodiscs more effectively fill the capillary. The combination of a more
hydrophilic filling and multilayer grafting increased the column capacity for nanodiscs
by three-fold.
The researchers tested their approach
on the adenosine-A2A receptor (AA2AR), which has frequently been
used as a model GPCR. Two previously reported weak ligands, both with
affinities around 0.2 mM, could be detected, and competition with an
orthosteric binder revealed that they were binding specifically.
This is a nice, how-to guide for
performing WAC on membrane proteins, and the paper includes detailed equations for
calculating affinities from differences in retention times. I look forward to
seeing the technique used in de novo screens.
No comments:
Post a Comment