Protein complex prediction using Rosetta, AlphaFold, and mass spectrometry covalent labeling.

Covalent labeling (CL) in combination with mass spectrometry can be used as an analytical tool to study and determine structural properties of protein-protein complexes. However, data from these experiments is sparse and does not unambiguously elucidate protein structure. Thus, computational algorithms are needed to deduce structure from the CL data. In this work, we present a hybrid method that combines models of protein complex subunits generated with AlphaFold with differential CL data via a CL-guided protein-protein docking in Rosetta. In a benchmark set, the RMSD (root-mean-square deviation) of the best-scoring models was below 3.6 Å for 5/5 complexes with inclusion of CL data, whereas the same quality was only achieved for 1/5 complexes without CL data. This study suggests that our integrated approach can successfully use data obtained from CL experiments to distinguish between nativelike and non-nativelike models.

Solid phase synthesis and spectroscopic characterization of the active and inactive forms of bacteriophage S21 pinholin protein.

The mechanism for the lysis pathway of double-stranded DNA bacteriophages involves a small hole-forming class of membrane proteins, the holins. This study focuses on a poorly characterized class of holins, the pinholin, of which the S21 protein of phage ϕ21 is the prototype. Here we report the first in vitro synthesis of the wildtype form of the S21 pinholin, S2168, and negative-dominant mutant form, S21IRS, both prepared using solid phase peptide synthesis and studied using biophysical techniques. Both forms of the pinholin were labeled with a nitroxide spin label and successfully incorporated into both bicelles and multilamellar vesicles which are membrane mimetic systems. Circular dichroism revealed the two forms were both >80% alpha helical, in agreement with the predictions based on the literature. The molar ellipticity ratio [θ]222/[θ]208 for both forms of the pinholin was 1.4, suggesting a coiled-coil tertiary structure in the bilayer consistent with the proposed oligomerization step in models for the mechanism of hole formation. 31P solid-state NMR spectroscopic data on pinholin indicate a strong interaction of both forms of the pinholin with the membrane headgroups. The 31P NMR data has an axially symmetric line shape which is consistent with lamellar phase proteoliposomes lipid mimetics.