Electron Transfer Flavoproteins (ETFs) act as electron carriers and have been found in every kingdom of life. While the ETFs, characterized in literature, are expressed as heterodimers, we’ve discovered a monomeric ETF (mETF), wherein the N-terminus is homologous to EtfB, the C-terminus EtfA, and contains a linker region to connect the two. Consistent with the origin from the thermoacidophilic archaeon Sulfolobus accidocaldarius, initial tests reveal this monomeric ETF to be very stable in a variety of conditions. The new mETF, therefore, provides the opportunity to use NMR to characterize possible domain-scale dynamics, that are believed to gate electron transfer.
Uniformly 15N-labelled, protonated mETF (67 kDa) is sedimented into a 0.7 mm diameter rotor that allows spinning rates of up to 111 kHz in the magnet. The mETF’s notably sharp lines permit comparison of isolated peaks seen in CP-based H-N correlation and analogous INEPT-based experiments to distinguish rigid and dynamic regions of the protein. In particular, resonances of six, well-dispersed tryptophans are tracked as a function of temperature to assess attenuation of rigidity with increasing temperature in different regions of the protein.
A second aim is to observe chemical shifts of the nitrogen (N) atoms of the two flavin isoalloxazine rings, while limiting signal contributions of the 67 kDa protein. To do so, unlabeled protein is refolded around selectively-labeled 15N FAD—a strenuous protocol that serves as a testament to the stability of the protein of interest. Dynamic Nuclear Polarization (DNP) is used to enhance the signals, attributable exclusively to the flavin Ns. While weeks are required to obtain good flavin 15N signals via conventional NMR, DNP has rendered the same experiments possible in hours.
Further investigations will probe the noncovalent interactions, namely protonation states and hydrogen bonding with nearby amino acids, which dictate unique chemistries at both flavin cofactors.