A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets

International audienceHere, we enhanced the popular yeast display method by multiple rounds of DNA and protein engineering. We introduced surface exposure-tailored reporters, eUnaG2 and DnbALFA, creating a new platform of C and N terminal fusion vectors. The optimization of eUnaG2 resulted in five times brighter fluorescence and 10°C increased thermostability than UnaG. The optimized DnbALFA has 10-fold the level of expression of the starting protein. Following this, different plasmids were developed to create a complex platform allowing a broad range of protein expression organizations and labeling strategies. Our platform showed up to five times better separation between nonexpressing and expressing cells compared with traditional pCTcon2 and c-myc labeling, allowing for fewer rounds of selection and achieving higher binding affinities. Testing 16 different proteins, the enhanced system showed consistently stronger expression signals over c-myc labeling. In addition to gains in simplicity, speed, and cost-effectiveness, new applications were introduced to monitor protein surface exposure and protein retention in the secretion pathway that enabled successful protein engineering of hard-to-express proteins. As an example, we show how we optimized the WD40 domain of the ATG16L1 protein for yeast surface and soluble bacterial expression, starting from a nonexpressing protein. As a second example, we show how using the here-presented enhanced yeast display method we rapidly selected high-affinity binders toward two protein targets, demonstrating the simplicity of generating new protein−protein interactions. While the methodological changes are incremental, it results in a qualitative enhancement in the applicability of yeast display for many applications

Simulation of energy-resolved mass spectrometry distributions from surface-induced dissociation

Understanding the relationship between protein structure and experimental data is crucial for utilizing experiments to solve biochemical problems and optimizing the use of sparse experimental data for structural interpretation. Tandem mass spectrometry (MS/MS) can be used with a variety of methods to collect structural data for proteins. One example is surface-induced dissociation (SID), which is used to break apart protein complexes (via a surface collision) into intact subcomplexes and can be performed at multiple laboratory frame SID collision energies. These energy-resolved tandem MS/MS experiments have shown that the profile of the breakages depends on the acceleration energy of the collision. It is possible to extract an appearance energy (AE) from energy-resolved mass spectrometry (ERMS) data, which shows the relative intensity of each type of subcomplex as a function of SID acceleration energy. We previously determined that these AE values for specific interfaces correlated with structural features related to interface strength. In this study, we further examined the structural relationships by developing a method to predict the full ERMS plot from structure, rather than extracting a single value. First, we noted that for proteins with multiple interface types, we could reproduce the correct shapes of breakdown curves, further confirming previous structural hypotheses. Next, we demonstrated that interface size and energy density (measured using Rosetta) correlated with data derived from the ERMS plot (R^2 = 0.71). Furthermore, based on this trend, we used native crystal structures to predict ERMS. The majority of predictions resulted in good agreement, and the average root-mean-square error (RMSE) was 0.20 for the 20 complexes in our dataset. We also show that if additional information on cleavage as a function of collision energy could be obtained, the accuracy of predictions improved further. Finally, we demonstrated that ERMS prediction results were better for the native than for inaccurate models in 17/20 cases. An application to run this simulation has been developed in Rosetta, which is freely available for use

Protein quaternary structures in solution are a mixture of multiple forms

Over half the proteins in the E. coli cytoplasm form homo or hetero-oligomeric structures. Experimentally determined structures are often considered in determining a protein's oligomeric state, but static structures miss the dynamic equilibrium between different quaternary forms. The problem is exacerbated in homo-oligomers, where the oligomeric states are challenging to characterize. Here, we re-evaluated the oligomeric state of 17 different bacterial proteins across a broad range of protein concentrations and solutions by native mass spectrometry (MS), mass photometry (MP), size exclusion chromatography (SEC), and small-angle X-ray scattering (SAXS), finding that most exhibit several oligomeric states. Surprisingly, some proteins did not show mass-action driven equilibrium between the oligomeric states. For approximately half the proteins, the predicted oligomeric forms described in publicly available databases underestimated the complexity of protein quaternary structures in solution. Conversely, AlphaFold multimer provided an accurate description of the potential multimeric states for most proteins, suggesting that it could help resolve uncertainties on the solution state of many proteins

SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution.

International audienceHumans can be infected by SARS-CoV-2 either through inhalation of airborne viral particles or by touching contaminated surfaces. Structural and functional studies have shown that a single RBD of the SARS-CoV-2 homotrimer spike glycoprotein interacts with ACE2, which serves as its receptor 1,2. Binding of spike (S) protein to ACE2 and subsequent cleavage by the host protease transmembrane serine protease 2 (TMPRSS2) results in cell and virus membrane fusion and cell entry 1. Blocking of the ACE2 receptor by specific antibodies prevents viral entry 1,3-5. In vitro binding measurements have shown that SARS-CoV-2 S protein binds ACE2 with an affinity of around 10 nM, which is about tenfold tighter than the binding of the SARS-CoV S protein 2,4,6. It has been suggested that this is, at least partially, responsible for the higher infectivity of SARS-CoV-2 7. Recently, three major SARS-CoV2 variants of concern have emerged and mutations in the RBD of the spike proteins of these variants have further strengthened this hypothesis. Deep-mutational scanning of the RBD domain showed that the N501Y mutation in the Alpha variant to enhances binding to ACE2 7. The Beta variant has three altered residues in the ACE2-binding site (K417N, E484K and N501Y), and has spread extremely rapidly, becoming the dominant lineage in the Eastern Cape and Western Cape Provinces within weeks 8. The Gamma variant, with independent K417T, E484K and N501Y mutations, similar to the B.1.351 variant is spreading rapidly from the Amazon region 9. Another S mutation associated wit