posted on 2023-11-08, 19:03authored byMariah
R. Pierce, Jingjing Ji, Sadie X. Novak, Michelle A. Sieburg, Shivangi Nangia, Shikha Nangia, James L. Hougland
Membrane proteins
are difficult to isolate and purify due to their
dependence on the surrounding lipid membrane for structural stability.
Detergents are often used to solubilize these proteins, with this
approach requiring a careful balance between protein solubilization
and denaturation. Determining which detergent is most appropriate
for a given protein has largely been done empirically through screening,
which requires large amounts of membrane protein and associated resources.
Here, we describe an alternative to conventional detergent screening
using a computational modeling approach to identify the most likely
candidate detergents for solubilizing a protein of interest. We demonstrate
our approach using ghrelin O-acyltransferase (GOAT),
a member of the membrane-bound O-acyltransferase
family of integral membrane enzymes that has not been solubilized
or purified in active form. A computationally derived GOAT structural
model provides the only structural information required for this approach.
Using computational analysis of detergent ability to penetrate phospholipid
bilayers and stabilize the GOAT structure, a panel of common detergents
were rank-ordered for their proposed ability to solubilize GOAT. The
simulations were performed at all-atom resolution for a combined simulation
time of 24 μs. Independently, we biologically screened these
detergents for their solubilization of fluorescently tagged GOAT constructs.
We found computational prediction of protein structural stabilization
was the better predictor of detergent solubilization ability, but
neither approach was effective for predicting detergents that would
support GOAT enzymatic function. The current rapid expansion of membrane
protein computational models lacking experimental structural information
and our computational detergent screening approach can greatly improve
the efficiency of membrane protein detergent solubilization, supporting
downstream functional and structural studies.