Transporters are suggested to transport the substrates across the membrane using the alternating access mechanism in which the proteins have three functionally distinct conformational states, outward-facing, occluded and inward-facing. The structures from a secondary active transporter Mhp1 were recently determined in three states of the alternating access mechanism. Comparison of the structures shows that the conformational changes between these states are achieved by the rigid body movement of the four helices relative to the rest of the protein. The molecular basis of the alternating access mechanism is likely to be common among the LeuT superfamily.
Time-resolved resonance Raman spectroscopy is able to provide site-selective information on protein dynamics. In this review, we will describe our apparatus of picosecond time-resolved ultraviolet resonance Raman spectroscopy and recent application to protein dynamics of bacteriorhodopsin.
Cells actively probe mechanical properties of their enviroment by exerting internally generated forces. The response they encounter profoundly affects their behavior. Here we measure in a simple geometry the force a cell exerts suspended by two optical traps. Our assay quantifies both the overall force and the fraction of that force transmitted to the enviroment. We found that the force transmission is highly dependent on the ratio between internal and extrernal compliance. Since the transmitted force is detected at the mechano-sensor located at cell membrane, our results suggest cells measure the mechanical properties of their environments using their own stiffness as a standard.
The transcription factor, cAMP response element binding protein (CREB), is involved in formation of long-term memory in the brain of many species. There are at least two different isoforms of CREB: the activator isoform (CREB1) and the repressor isoform (CREB2). By use of quantitative real-time PCR, we succeeded in determining CREB1 and CREB2 mRNA copy numbers in the single neuron that plays a key role in the associative learning of the pond snail Lymnaea stagnalis. Further, we characterized the spliced variants of CREB1 mRNA and analyzed the relation between the learning behavior of snails and the expression pattern of these CREB1 mRNA variants in the brain of the learned snails. We here discuss the function and the quantitative changes in CREB isoforms and variants of the snail brain in long-term memory.
Proteins are dynamic in nature and work at the single-molecule level. Reflecting this fact, single-molecule fluorescence microscopy has been widely exploited to understand how proteins operate. However, what we can observe thereby is the dynamic behaviour of individual fluorescent spots (each being emitted from a fluorophore attached to a selected locus of the molecule), not of the protein molecules themselves. The structure of proteins has been studied by electron microscopy, X-ray crystallography and NMR, but the obtained structures are essentially static. This long-standing problem prevailing throughout biological research has been recently overcome by the development of high-speed atomic force microscopy that enables simultaneous recording of the structure and dynamics of functioning biomolecules with high spatiotemporal resolution.