Dataset Information

Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore.

ABSTRACT: "Fluorescence-Activating and absorption-Shifting Tag" (FAST) is a well-studied fluorogen-activating protein with high brightness and low size, able to activate a wide range of fluorogens. This makes FAST a promising target for both protein and fluorogen optimization. Here, we describe the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen. Using the spatial structure of the FAST/N871b complex, NMR relaxation analysis, and computer simulations, we identify the mobile regions in the complex and suggest mutations that could stabilize both the protein and the ligand. Two of our mutants appear brighter than the wild-type FAST, and these mutants provide up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo. Analysis of the mutants by NMR reveals that brighter mutants demonstrate the highest stability and lowest length of intermolecular H-bonds. Computer simulations provide the structural basis for such stabilization.

SUBMITTER: Goncharuk MV

PROVIDER: S-EPMC9287381 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore.

Goncharuk Marina V MV Baleeva Nadezhda S NS Nolde Dmitry E DE Gavrikov Alexey S AS Mishin Alexey V AV Mishin Alexander S AS Sosorev Andrey Y AY Arseniev Alexander S AS Goncharuk Sergey A SA Borshchevskiy Valentin I VI Efremov Roman G RG Mineev Konstantin S KS Baranov Mikhail S MS

Communications biology 20220715 1

"Fluorescence-Activating and absorption-Shifting Tag" (FAST) is a well-studied fluorogen-activating protein with high brightness and low size, able to activate a wide range of fluorogens. This makes FAST a promising target for both protein and fluorogen optimization. Here, we describe the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen. Using the spatial structure of the FAST/N871b complex, NMR relaxation analysis, and computer simulations, we iden ...[more]

PMID: 35840781

Similar Datasets

Project description:Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157-165 cancer-testis epitope. Fifty-four percent of the designed sequence replacements exhibited improved pMHC binding as compared to the native TCR, with up to 150-fold increase in affinity, while preserving specificity. Genetically engineered CD8(+) T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity, K D?=??1?-?5??M. Beyond the affinity threshold at K D?<?1??M we observed an attenuation in cellular function, in line with the "half-life" model of T cell activation. Our computer-aided protein-engineering approach requires the 3D-structure of the TCR-pMHC complex of interest, which can be obtained from X-ray crystallography. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes when experimental data is not available. Since the accuracy of the models depends on the prediction of the TCR orientation over pMHC, we have complemented the approach with a simplified rigid method to predict this orientation and successfully assessed it using all non-redundant TCR-pMHC crystal structures available. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of stage IV melanoma.

Dataset Information

Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore.

Publications

Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets