Biology subjects

Wysocki, V. H.

Publications and source records attributed to Wysocki, V. H..

23 records · Page 2Linked to original sources

bioRxiv2022

Structure of a Rad52 homolog from bacteriophage in complex with a novel duplex intermediate of DNA annealing

Human Rad52 protein binds to ssDNA and promotes the annealing of complementary strands. This activity is central to multiple DNA repair pathways and Rad52 is a target for cancer therapeutics. Previous crystal structures of the DNA binding domain of Rad52 revealed an 11-mer ring that binds to ssDNA in an extended conformation with the bases exposed for homology recognition. While this complex is likely involved in the early stages of annealing, there is no structure of Rad52 with two strands of DNA bound simultaneously, and its mechanism of annealing is poorly understood. To approach this problem, we have turned to the RecT/Red{beta} family of annealing proteins from bacteriophage, which are distant homologs of Rad52 that form stable complexes with a duplex intermediate of annealing. We have used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 [A] structure of a RecT homolog from a prophage of Listeria innocua (LiRecT) in complex with two complementary 83-mer oligonucleotides that were added to the protein sequentially. The structure reveals a left-handed helical filament of the protein bound to a novel conformation of DNA duplex that is highly extended and under-wound. The duplex is bound at a stoichiometry of 5 bp/monomer to a deep, narrow, positively-charged groove that runs along the outer surface of the filament. Data from native mass spectrometry confirm that the filament complex seen by cryo-EM also exists in solution. Collectively, these data provide new insights into the mechanism of annealing by LiRecT and by homologous proteins including human Rad52.

biochemistry↗

bioRxiv2021

Protein shape sampled by ion mobility mass spectrometry consistently improves protein structure prediction

Among a wide variety of mass spectrometry (MS) methodologies available for structural characterizations of proteins, ion mobility (IM) provides structural information about protein shape and size in the form of an orientationally averaged collision cross-section (CCS). While IM data have been predominantly employed for the structural assessment of protein complexes, CCS data from IM experiments have not yet been used to predict tertiary structure from sequence. Here, we are showing that IM data can significantly improve protein structure determination using the modeling suite Rosetta. The Rosetta Projection Approximation using Rough Circular Shapes (PARCS) algorithm was developed that allows for fast and accurate prediction of CCS from structure. Following successful rigorous testing for accuracy, speed, and convergence of PARCS, an integrative modelling approach was developed in Rosetta to use CCS data from IM experiments. Using this method, we predicted protein structures from sequence for a benchmark set of 23 proteins. When using IM data, the predicted structure improved or remained unchanged for all 23 proteins, compared to the predicted models in the absence of CCS data. For 15/23 proteins, the RMSD (root-mean-square deviation) of the predicted model was less than 5.50 [A], compared to only 10/23 without IM data. We also developed a confidence metric that successfully identified near-native models in the absence of a native structure. These results demonstrate the ability of IM data in de novo structure determination.

biophysics↗

bioRxiv2020

De novo design of transmembrane beta-barrels

The ability of naturally occurring transmembrane {beta}-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a "hypothesis, design and test" approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the {beta}-turns and {beta}-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history. One sentence summarySuccess in de novo design of transmembrane {beta}-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

synthetic biology↗

bioRxiv2020

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via -helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by -helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

synthetic biology↗

bioRxiv2020

HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal

The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. While the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native mass spectrometry to investigate RNA binding of wild-type Gag and Gag lacking the p6 domain (Gag{Delta}p6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. Gag{Delta}p6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically nucleates Gag-Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.

biochemistry↗