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The HIV-1 capsid protein (CA) assembles into a conical shell during viral maturation, encasing and protecting the viral RNA genome. The C-terminal domain (CTD) of the two-domain capsid protein dimerizes, and this dimer connects individual chains in the mature capsid lattice. Previous NMR studies have shown that different dimer arrangements can be formed by isolated capsid protein chains and in assembled capsid lattices; however, the dynamics and functional relevance of these alternate dimers are unknown. To explore the conformational landscape of the CA-CTD dimer, we carried out atomistic molecular dynamics simulations using the weighted ensemble path sampling strategy, generating an ensemble of conformations. Focusing on the two dimer forms previously observed via solution NMR, we refined the conformational ensemble to highlight two metastable states using a Markov state model. Experimentally, we measured the interconversion rates between the two alternate dimers using¹⁹F NMR, and these rates showed good agreement with the interconversion rates derived from the simulations. After identifying the key interactions that distinguish the dimer states, the alternate dimer was further experimentally verified through disulfide crosslinking. Our results demonstrate the advantages of pairing weighted ensemble path sampling with¹⁹F NMR to gain atomistic insights into the hidden dimer state of the HIV-1 capsid protein. 

Obtaining atomic-level information on components in the cell is a major focus in structural biology. Elucidating specific structural and dynamic features of proteins and their interactions in the cellular context is crucial for understanding cellular processes. We introduce¹⁹F dynamic nuclear polarization (DNP) combined with fast magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy as a powerful technique to study proteins in mammalian cells. We demonstrate our approach on the severe acute respiratory syndrome coronavirus 2 5F-Trp-N^NTDprotein, electroporated into human cells. DNP signal enhancements of 30- to 40-fold were observed, translating into over 1000-fold experimental time savings. High signal-to-noise ratio spectra were acquired on nanomole quantities of a protein in cells in minutes. 2D¹⁹F-¹⁹F dipolar correlation spectra with remarkable sensitivity and resolution were obtained, exhibiting¹⁹F-¹⁹F cross peaks associated with fluorine atoms as far as ~10 angstroms apart. This work paves the way for¹⁹F DNP-enhanced MAS NMR applications in cells for probing protein structure, dynamics, and ligand interactions. 

Comparative study of the performance of different spin labels for¹⁹F electron-nuclear double resonance (ENDOR) for short-range (1.0–1.5 nm) distance measurement in proteins.