Biology subjects

Manjunath, K.

Publications and source records attributed to Manjunath, K..

6 recordsLinked to original sources

bioRxiv2022

Inferring bound structure and residue specific contributions to binding energetics in the Intrinsically Disordered Protein, CcdA

Unlike globular proteins, mutational effects on the function of Intrinsically Disordered Proteins (IDPs) are not well-studied. Deep Mutational Scanning of a yeast surface displayed mutant library yields insights into sequence-function relationships in the CcdA IDP. The approach enables facile prediction of interface residues and local structural signatures of the bound conformation. In contrast to previous titration-based approaches which use a number of ligand concentrations, we show that use of a single rationally chosen ligand concentration can provide quantitative estimates of relative binding constants for large numbers of protein variants. This is because the extended interface of IDP ensures that energetic effects of point mutations are spread over a much smaller range than for globular proteins. Our data also provides insights into the much-debated role of helicity and disorder in partner binding of IDPs. Based on this exhaustive mutational sensitivity dataset, a model was developed to predict mutational effects on binding affinity of IDPs that form alpha-helical structures upon binding.

biophysics↗

bioRxiv2022

Structural and functional determinants inferred from deep mutational scans

Mutations that affect protein binding to a cognate partner primarily occur either at buried residues or at exposed residues directly involved in partner binding. Distinguishing between these two categories based solely on mutational phenotypes is challenging. The bacterial toxin CcdB kills cells by binding to DNA Gyrase. Cell death is prevented by binding to its cognate antitoxin CcdA, at an extended interface that partially overlaps with the GyrA binding site. Using the CcdAB toxin-antitoxin (TA) system as a model, a comprehensive site-saturation mutagenesis library of CcdB was generated in its native operonic context. The mutational sensitivity of each mutant was estimated by evaluating the relative abundance of each mutant in two strains, one resistant and the other sensitive to the toxic activity of the CcdB toxin, through deep sequencing. The ability to bind CcdA was inferred through a RelE reporter gene assay, since the CcdAB complex binds to its own promoter, repressing transcription. By analysing mutant phenotypes in the CcdB sensitive, CcdB resistant and RelE reporter strains, it was possible to assign residues to buried, CcdA interacting or GyrA interacting sites. A few mutants were individually constructed, expressed, and biophysically characterised to validate molecular mechanisms responsible for the observed phenotypes. Residues inferred to be important for antitoxin binding, are also likely to be important for rejuvenating CcdB from the CcdB-Gyrase complex. Therefore, even in the absence of structural information, when coupled to appropriate genetic screens, such high-throughput strategies can be deployed for predicting structural and functional determinants of proteins. Broader Impact StatementPartial loss-of-function mutations predominantly occur either at buried-site or exposed, active-site residues. We report a facile method to identify multiple binding sites for different interacting partners for a protein, and distinguish them from buried site and exposed non active-site residues, solely from mutational data.

biophysics↗

bioRxiv2021

Mechanistic insights into global suppressors of protein folding defects

Most amino acid substitutions in a protein either lead to partial loss of function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss of function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 {beta}-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. The suppressors both alone, and in conjunction with inactive mutants, stabilise the protein both thermodynamically and kinetically in-vitro, predominantly through acceleration of the refolding rate. When coupled to inactive mutants they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increases, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action.

biophysics↗

bioRxiv2021

Stabilizing proteins through saturation suppressor mutagenesis.

While there have been recent, transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding, however reliable screens for mutants that improve protein stability do not exist, especially for proteins that are well folded and relatively stable. We demonstrate that incorporation of a single, specific destabilizing, (parent inactive) mutation into each member of a deep mutational scanning library followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. When coupled to FACS sorting of a yeast surface display library of the bacterial toxin CcdB, followed by deep sequencing of sorted populations, multiple stabilizing mutations could be identified after a single round of sorting. Multiple libraries with different parent inactive mutations could be pooled and simultaneously screened to further enhance the accuracy of identification of stabilizing mutations. Individual stabilizing mutations could be combined to result in a multimutant with increase in thermal melting temperature of about 20 {degrees}C and enhanced tolerance to high temperature exposure. The method employs small library sizes and can be readily extended to other display and screening formats to rapidly isolate stabilized protein mutants.

biophysics↗

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↗

bioRxiv2020

A facile method of mapping neutralizing HIV-1 epitopes

Identification of specific epitopes targeted by neutralizing antibodies is essential to advance epitope-based vaccine design strategies. We report a facile methodology for rapid epitope mapping of neutralizing antibodies (NAbs) against HIV-1 Envelope (Env) at single residue resolution, using Cys labeling, viral neutralization assays and deep sequencing. This was achieved by the generation of a library of Cys mutations in Env glycoprotein on the viral surface, covalent labeling of the Cys residues using a Cys reactive label that masks epitope residues, followed by infection of the labeled mutant virions in mammalian cells in the presence of NAbs. Env gene sequencing from NAb-resistant viruses was used to accurately delineate epitopes for the NAbs VRC01, PGT128 and PGT151. These agreed well with corresponding experimentally determined structural epitopes previously inferred from NAb:Env structures. HIV-1 infection is associated with complex and polyclonal antibody responses, typically composed of multiple antibody specificities. Deconvoluting the epitope specificities in a polyclonal response is a challenging task. We therefore extended our methodology to map multiple specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as in an HIV-1 infected elite neutralizer capable of neutralizing Tier-3 pseudoviruses with high titers. The method can be readily extended to other viruses for which convenient reverse genetics or lentiviral surface display systems are available.

molecular biology↗