A number of crystal structures of membrane proteins have been reported, providing structural bases for physiologically important processes. However, the snapshots from the crystal structures do not necessarily provide sufficient information on understanding the functional mechanism of the membrane proteins. Solution NMR is a complementary method of X-ray crystallography, which reveals structural change and dynamics related to the functions. Here, we review what in membrane proteins can be analyzed by solution NMR, and its applications to functional analyses of membrane proteins.
Two examples of our most recent investigations using high-speed atomic force microscopy were provided. (1) The calcium pump reaction cycle was captured as up-and-down motions, corresponding to the conformational changes of the pump during ATP-mediated ion transport. The motion pictures obtained suggest that the pump would not exactly follow the Post-Alberts schema in the physiological condition. (2) The binding of two agonists (glycine and aspartate) to the hetero-tetrameric GluN1/GluN2A receptor triggered a structural change in the extracellular domain of the receptor without desensitization. This change was abolished in the presence of a selective antagonist, D-(−)-2-amino-5-phosphonopentanoic acid.
Channelrhodopsin (ChR) is a light-gated cation channel derived from algae. Since the inward flow of cations triggers the neuron firing, neurons expressing ChRs can be optically controlled even within freely moving mammals. Although ChR has been broadly applied to neuroscience research, little is known about its molecular mechanisms. We determined the crystal structure of chimeric ChR at 2.3 Å resolution and revealed its molecular architecture. The integration of structural and electrophysiological analyses provided insight into the molecular basis for the channel function of ChR, and paved the way for the principled design of ChR variants with novel properties.
cDNA display is a robust and versatile in vitro protein selection tool developed on the basis of mRNA display. In this tool, a protein is covalently linked with its coding cDNA via a specially-designed puromycin-linker to improve the stability of the molecule. As a result of the stabilization, cDNA display succeeded in extending the variety of libraries (e.g., modified peptide library) and target molecules (e.g., cell surface molecules). Thus, this technology will enable to design functional proteins and peptides which could not be evolved by the previous in vitro protein selection tools.
A number of structural and kinetic studies of DNA polymerases have proposed the catalytic mechanism of the nucleotidyl-transfer reaction. However, the actual process has never been visualized. Here we show the nucleotidyl-transfer reaction process catalyzed by human DNA polymerase η using time-resolved protein crystallography. In sequence, the nucleophile 3′-OH is deprotonated, the deoxyribose at the primer end converts from C2′-endo to C3′-endo, and the nucleophile and the α-phosphate of dATP approach each other to form the new bond. A third Mg2+ ion, which arrives with the new bond and stabilizes the intermediate state, may be an unappreciated feature of the two-metal-ion mechanism.
Utilizing quick-freeze deep-etch-replica electron-microscopy, we observed that actin-attached myosin during in vitro sliding was oppositely-bent, considering the direction of the putative pre-power stroke configuration. Since SH1–SH2-cross-linked myosin was a good candidate with a similar appearance, we devised a new procedure to define the relative angle of the catalytic-domain and the lever-arm from the averaged images, and built its 3-D model. The projection angle of the lever-arm in that model was compatible with actin-sliding cross-bridge structure. Introducing this conformer as the structural analogue of the transient intermediate, we propose a revised scheme of the crossbridge-cycle compatible with various experimental results.