The State of Biophysics - Biophysical Journal

1006

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FIGURE 1 How to record the dancing moves of a rap musician. Labeling the right hand with a green dye and the left hand with a red dye makes it possible to follow the distance changes between the two hands as a function of time. When the two hands are very close to each other (horseback posture), fluorescence excitation energy is trans- ferred from the green dye to the red dye, in a pro- cess called FRET, so that the red signal is stronger than the green. When the two hands are far away from each other (cowboy), there is little energy transfer and the green signal is stronger. Once the dance finishes and the red dye is released from the musician, only green signal is observed. In the case of protein nanomachines, FRET occurs over a length scale of a few nanometers. Because of close proximity, the two dyes would not appear as two separate objects in single-molecule imaging. Instead, one sees a single object that changes color over time. This illustration was created by Dr. Jin- gyi Fei at the University of Chicago. To see this figure in color, go online.

small forces, down to 10 12 N of force, and measure the response mechanically at the nanometer level. For example, optical tweezers have been used to measure the step size of many molecular motors ( 2 ). Recently, the precision of opti- cal tweezers has improved to the angstrom scale, almost the size of a water molecule ( 7 ). In optical-tweezers measurements, it is as if you are clos- ing your eyes and using your hands to manipulate and mea- sure the response of an object, whereas in fluorescence measurements, you have your hands tied in back and make passive observations with your eyes. By combining the two, we can hope to sample the best of both worlds. For example, we can use optical tweezers to measure the activities of single proteins such as helicases, which unwind DNA into single strands using the energy of ATP molecules and at the same time measure the conformational changes of the protein using FRET. Using such a hybrid instrument, it was shown that a helicase called UvrD unwinds DNA when it takes the ‘‘closed’’ conformation and rezips DNA when it takes the ‘‘open’’ conformation ( 8 ). When a related helicase was forced to maintain the closed form, it became a superhelicase that can unwind thousands of basepairs without falling off, even against a very strong opposing force ( 9 ). This superhelicase may be useful for various biotechnological amplifications, such as rapid pathogenic DNA detection and sequencing in developing countries or during surgery in hospitals.

one-dimensional (1D) sliding. This is akin to joining a book club to sample a few dozen of its members in search of a po- tential mate. The filament around broken DNA can bind to a random DNA sequence but slides along the DNA back and forth over hundreds of basepairs. This local search can be much more effective when combined with a three-dimen- sional search. If the local search is unsuccessful, the fila- ment can dissociate and bind another region of DNA, which would be equivalent to joining a different club, such as a knitting club. Using FRET, one can probe for such 1D sliding activity. A green dye on the filament and a red dye on the target DNA would result in fluctuations in FRET, anticorrelated changes in the intensities of green and red signals, providing evi- dence for 1D sliding ( 6 ). But can the filament really find the target sequence through sliding? To address this ques- tion, we can put two near-matching sequences on the target DNA. The filament would spend some time on one near- matching sequence and after it realizes that the match is not perfect, it will leave and slide, and will land on the other near-matching sequence, and this can continue back and forth. This would be like dating two twin brothers, each of whom is not a perfect match. Overall, single-molecule FRET measurements suggest that such 1D sliding may accelerate the finding of the matching sequence by as much as 250 times, possibly aiding DNA repair greatly. Marriage of single-molecule FRET and optical tweezers Another single-molecule technique, called optical tweezers, or what I call ‘‘chopsticks made of light,’’ can apply very

Future outlook

Single-molecule measurements have revealed the amazingly complex but elegant abilities of nature’s nanomachines to

Biophysical Journal 110(5) 1004–1007