ahmed, s. | Search bioRxiv

bioRxiv2021

Prediction of residue-specific contributions to binding and thermal stability using yeast surface display

Accurate prediction of residue burial as well as quantitative prediction of residue-specific contributions to protein stability and activity is challenging, especially in the absence of experimental structural information. This is important for prediction and understanding of disease causing mutations, and for protein stabilization and design. Using yeast surface display of a saturation mutagenesis library of the bacterial toxin CcdB, we probe the relationship between ligand binding and expression level of displayed protein, with in vivo solubility in E.coli and in vitro thermal stability. We find that both the stability and solubility correlate well with the total amount of active protein on the yeast cell surface but not with total amount of expressed protein. We coupled FACS and deep sequencing to reconstruct the binding and expression mean fluorescent intensity of each mutant. The reconstructed mean fluorescence intensity (MFIseq) was used to differentiate between buried site, exposed non active-site and exposed active-site positions with high accuracy. The MFIseq was also used as a criterion to identify destabilized as well as stabilized mutants in the library, and to predict the melting temperatures of destabilized mutants. These predictions were experimentally validated and were more accurate than those of various computational predictors. The approach was extended to successfully identify buried and active-site residues in the receptor binding domain of the spike protein of SARS-CoV-2, suggesting it has general applicability.

biophysics↗

bioRxiv2018

Small RNA-mediated genomic silencing promotes telomere stability in the absence of telomerase

Small RNAs with homology to telomeres can map to heterochromatic segments of the C. elegans genome, although small RNAs with perfect homology to (TTAGGC)n telomere repeats are exceptionally rare. The heterochromatin mark H3K9me2 is enriched at C. elegans sub-telomeres, and we found that the HP1 protein HPL-2 that binds H3K9me2 promotes telomere stability in the absence of telomerase. Small RNA silencing can promote H3K9 methylation, and we found that telomere stability in the absence of telomerase required WAGO-1 and HRDE-1 Argonaute proteins that bind small RNAs as well as germ granule proteins MUT-7 and MUT-14 that promote small RNA biogenesis. Loss of HRDE-1, WAGO-1 or PPW-2 Argonautes or the germ granule protein MUT-16 resulted in depletion of subtelomeric small RNAs. Loss of telomerase induces biogenesis of subtelomeric small RNAs from 5 end of the telomeric lncRNA TERRA, whereas loss of both telomerase and small RNA-mediated genome silencing induces TERRA expression, telomere damage and accelerated sterility. We propose that small RNA-mediated heterochromatin formation and telomerase function redundantly to repress a form of telomeric DNA damage that is coupled to TERRA expression and subtelomeric small RNA biogenesis. Long read sequencing revealed few mutations at 5 ends of telomeres for most N2 wild type C. elegans strains. Despite abundant subtelomeric small RNAs, telomere repeats of TERRA strongly resisted RNA-dependent RNA polymerase activity, revealing an exceptional quality of telomere repeat RNA. Article summaryLoss of telomerase and either small RNA biogenesis factors or heterochromatin proteins resulted in rapid onset of telomere fusions. We identified Argonaute proteins that bind to subtelomeric small RNAs and promote telomere stability in the absence of telomerase. Telomeres are transcribed to create the non-coding RNA TERRA that is a composite of subtelomere sequence and telomere repeats. Although subtelomeric small RNAs were upregulated in the absence of telomerase, the telomere sequence of TERRA was completely resistant to small RNA biogenesis. Small RNAs and TERRA may interact to promote repair of damaged telomeres that cannot be healed by telomerase.

genetics↗