Solid state NMR chemical shift assignment of the non-structural single-stranded DNA binding protein gVp from fd bacteriophage

Conformational Changes in Ff Phage Protein gVp upon Complexation with Its Viral Single-Stranded DNA Revealed Using Magic-Angle Spinning Solid-State NMR

“…(i) The expression and purification of gVp were described in an earlier work [45]. In brief, we used a pET-30b plasmid with kanamycin (KAN) resistance to overexpress gVp with an N-terminal His-tag.…”

“…We previously assigned the chemical shifts of the free form of gVp [45]. A comparison with the spectrum of gVp in the complex revealed distinctive chemical shift perturbations (CSPs) due to the presence of the ssDNA (Figure 8).…”

Atomic-Resolution Structure of the Protein Encoded by Gene V of fd Bacteriophage in Complex with Viral ssDNA Determined by Magic-Angle Spinning Solid-State NMR

“…34 In work previously reported by our lab, the isotropic chemical shifts of both free gVp and ssDNAbound gVp have been assigned. 35,36 Quantification and analysis of chemical shift perturbations (CSPs) led to the conclusion that gVp undergoes significant structural changes upon binding, and regions expected to undergo the most extensive structural modifications�including the DNA-binding loop, the core loop, and the C-terminus�were identified. 36 MAS ssNMR is also useful for protein structure elucidation based on acquisition of internuclear distance-dependent interactions and prediction of backbone torsion angles based on chemical shifts, 37 as well as acquisition of additional structural restraints.…”

“…Magic-angle spinning solid-state NMR (MAS ssNMR) is advantageous for probing systems of nucleotide-bound proteins, since linewidths are not dependent on the particle mass, long-range crystallinity is not required, as well as the ability to sensitively detect conformational changes, as even minor changes in the fold result in significant changes to the resonance positions . In work previously reported by our lab, the isotropic chemical shifts of both free gVp and ssDNA-bound gVp have been assigned. , Quantification and analysis of chemical shift perturbations (CSPs) led to the conclusion that gVp undergoes significant structural changes upon binding, and regions expected to undergo the most extensive structural modificationsincluding the DNA-binding loop, the core loop, and the C-terminuswere identified . MAS ssNMR is also useful for protein structure elucidation based on acquisition of internuclear distance-dependent interactions and prediction of backbone torsion angles based on chemical shifts, as well as acquisition of additional structural restraints. , Given a sufficient amount of restraints distributed along different regions of the sequence, a carefully designed calculation process can lead to a converged, viable structural model.…”