Limitations of Time-Resolved Fluorescence Suggested by Molecular Simulations: Assessing the Dynamics of T cell Receptor Binding Loops

Abstract

Time-resolved fluorescence anisotropy (TRFA) has a rich history in evaluating protein dynamics. Yet as often employed, TRFA assumes that the motional properties of a covalently tethered fluorescent probe accurately portray the motional properties of the protein backbone at the probe attachment site. In an extensive survey using TRFA to study the dynamics of the binding loops of a αβ T cell receptor, we observed multiple discrepancies between the TRFA data and previously published results that led us to question this assumption. We thus simulated several of the experimentally probed systems using a protocol that permitted accurate determination of probe and protein time correlation functions. We found excellent agreement in the decays of the experimental and simulated correlation functions. However, the motional properties of the probe were poorly correlated with those of the backbone of both the labeled and unlabeled protein. Our results warrant caution in the interpretation of TRFA data and suggest further studies to ascertain the extent to which probe dynamics reflect those of the protein backbone. Meanwhile, the agreement between experiment and computation validates the use of molecular dynamics simulations as an accurate tool for exploring the molecular motion of T cell receptors and their binding loops.

Differential utilization of binding loop flexibility in T cell receptor ligand selection and cross-reactivity

“…S1A). However, as we demonstrated previously 25 , incorporation of a fluorescent label can alter the motional properties of TCR loops, such that measurements on labeled proteins may not reflect the behavior of unlabeled proteins. We thus performed two separate 100 ns molecular dynamics simulations of the DMF5 TCR incorporating the cysteine and attached fluorescent label at positions 27α or 100β.…”

“…For the A6 TCR, we previously performed a comprehensive experimental validation of our molecular simulation methodology using fluorescence anisotropy 25 . To ask if the simulation with the DMF5 protein performed similarly well, we fluorescently labeled positions in the DMF5 CDR1α loop and the CDR3β loop.…”

“…However, while structural, thermodynamic, and kinetic data can give insight into conformational changes and their relationship to motion 3 , there have been few investigations into the motional properties of TCR CDR loops and their relation to receptor binding properties. Although some progress has been made using fluorescence and NMR 18 20 25 34 , due to the technical challenges presented by recombinant TCRs and TCR-pMHC complexes (e.g., production yields, protein stability, binding affinities, etc. ), insight in the near future is likely to emerge from computational studies 35 .…”

“…Protein labeling and fluorescence anisotropy was performed as described previously 25 33 . Briefly, approximately 25–50 μM of purified wild type and cysteine mutant DMF5 TCR was combined with a ten-fold excess of fluorescein-5-maleimide and 20uM TCEP-HCl.…”

Peptide and Peptide-Dependent Motions in MHC Proteins: Immunological Implications and Biophysical Underpinnings

“…In one notable study, fluorescence polarization was used along with MD simulations to show that allelic variations in HLA-B27 led to differences in peptide motions ( 35 ). A limitation of fluorescence is a reliance on bulky fluorescent labels, which can perturb structure and dynamics ( 63 ) [though in at least one case, intrinsic tryptophan fluorescence has been used to study MHC-I behavior ( 62 )]. Infrared spectroscopy is another approach that may prove to be of use in studying the motions of peptides bound to MHC proteins, as it can report on fast motions using approaches analogous to NMR, but with reporters that are less perturbative—and sometimes non-perturbative in the case of C-D probes—to the native structure and dynamics of interest ( 64 , 65 ).…”