Probing Nature’s Nanomachines One Molecule at a Time

Abstract

Did you know that proteins are nanoscale machines that help us think, dance, and keep the threat of cancer at bay? Did you know that biology is a new research frontier for physicists? Here, I will discuss how biophysicists are using light-based tools to poke and examine nature’s nanomachines, one molecule at a time, uncovering the amazing acrobatic abilities that are essential for all forms of life. DNA is our genetic material that stores all the information necessary to build our cells and body. Proteins are made based on genetic information encoded in the DNA, and they are the central players that perform nearly all chemical reactions that make life possible. Because of proteins, our nerves can fire, our eyes can sense colors, and we can move our muscles. Proteins are often called nanomachines because they are unimaginably small, just a few nanometers across. How small is a nanometer? Human hair is about the thinnest object our naked eyes can see and is ∼80,000 nm thick. That is, we can fit 20,000 proteins across the width of a single hair! If your body is expanded to the size of the earth, a single cell in your body would be about as big as a good-sized city, and a single protein molecule would be about as tall as a person. Many proteins are also called nanomachines because they work as molecular motors that convert chemical energy into mechanical energy and they do so with precision and robustness that would make the best engineers cry in envy. A great example of molecular motors is a DNA packaging motor that can push thousands of basepairs of DNA into the very small volume of a virus particle, eventually reaching a pressure like that found inside a champagne bottle (1Smith D.E. Tans S.J. Bustamante C. et al.The bacteriophage straight φ29 portal motor can package DNA against a large internal force.Nature. 2001; 413: 748-752Crossref PubMed Scopus (879) Google Scholar). Another example is a protein called kinesin that carries cargoes inside the cell. Just as cars move on the highway using gasoline as the fuel, kinesins move on cellular highways called microtubules using the chemical energy released from the burning of ATP, the cellular currency of energy (2Svoboda K. Schmidt C.F. Block S.M. et al.Direct observation of kinesin stepping by optical trapping interferometry.Nature. 1993; 365: 721-727Crossref PubMed Scopus (1586) Google Scholar). These cargo-carrying motor proteins move directionally, some moving toward the cell center and others moving away from the cell center, and by selectively turning on and off a subset of motors, cells can move pigments around, allowing animals such as chameleons to change their skin color to match their surroundings. Such directional movements are also crucial for delivering cargoes to different parts of a nerve cell, which cannot rely on random, diffusional movement due to their substantial length. A better understanding of these molecular motors may help us detect and treat human diseases that are caused by defects in these proteins and may lead to the design and synthesis of artificial nanoscale machines. To study these tiny machines, we need tools that can examine biological motions at the nanoscale. In the last 20-plus years, breathtaking technological developments, often coming out of the laboratories of physicists, have allowed researchers to study nature’s nanomachines at the level of single molecules, establishing the field of single-molecule biophysics. Why are single-molecule measurements necessary and powerful? If all of the molecules under observation move in lock step, as in a marching band, averaging their signals would not obscure their movements. But this is generally not the case, and in most cases, you cannot forcibly synchronize their motion. Just imagine trying to achieve that for college students on campus! For such nonsynchronizable situations, single-molecule measurements can reveal complex dynamics that are hidden in ensemble experiments. In addition, molecules can have personalities; that is, nominally identical molecules can behave differently due to their complex composition built from thousands of atoms, and they may even be moody, changing their characters over time. Averaging over a heterogeneous population, therefore, can be misleading. Steven Chu, a Nobel laureate, famously joked that on average, one person on this planet has one ovary and one testicle. Finally, single-molecule measurements allow us to make correlations between molecular properties. Think of Americans’ views on climate change and the safety of genetically-modified-organism crops. Only when we survey individuals do we realize that conservatives tend to dismiss scientific consensus on climate change and that liberals often ignore scientific evidence supporting the safety of genetically-modified-organism crops. For these reasons, single-molecule measurement techniques that use the ability to detect and manipulate single molecules have become widely adopted by the researchers. Here, I will discuss two major fluorescence-based technologies used for single-molecule measurements. Imagine a soccer field being imaged from a faraway planet. If you attach an LED lamp to your favorite soccer player, because of the fundamental phenomenon called light diffraction, that lamp may appear to be about the size of the soccer field to that distant observer. Nevertheless, using a simple mathematical trick, we can determine the position of the lamp, or “localize” it, with great precision, say, down to the size of the player’s shoe. In principle, we can track the movements of the player and his feet with precision limited only by how many photons are being detected to form the image. For example, we can determine the size of his gait during a run, or his “step size” if you were to use the jargon of biophysicists. The same trick can be used to localize individual protein nanomachines and track their positions over time. Using this single-molecule localization-and-tracking approach, researchers have shown that myosin V, which carries a cargo along a track called the actin filament, moves in steps of 37 nm in length (3Mehta A.D. Rock R.S. Cheney R.E. et al.Myosin-V is a processive actin-based motor.Nature. 1999; 400: 590-593Crossref PubMed Scopus (675) Google Scholar). Furthermore, by labeling the “foot” of myosin V with a single dye, a 72-nm step length was observed, which is double the center-of-mass step length. The latter finding conclusively showed that myosin V walks like a human adult, with the two feet taking the leading position alternatingly, instead of crawling like an inchworm (4Yildiz A. Forkey J.N. Selvin P.R. et al.Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization.Science. 2003; 300: 2061-2065Crossref PubMed Scopus (1553) Google Scholar). These types of studies also showed that proteins can slide along the length of DNA and determined the rapidity of their motion and whether the motion was unidirectional or not. As I will discuss below, a protein’s motion on DNA in search of a target can be very important in keeping the threat of cancer at bay. In addition, if we label a single protein with one dye and one dye only, we can deduce how many proteins are working together at a given time by determining the brightness of the protein assembly; that is, if it is four times as bright as a single dye, we can conclude that four copies of the same protein are needed for a certain function (5Ulbrich M.H. Isacoff E.Y. Subunit counting in membrane-bound proteins.Nat. Methods. 2007; 4: 319-321Crossref PubMed Scopus (514) Google Scholar). If multiple dyes of different colors are used, we can also determine how many different kinds of proteins are functioning in the same biological process. This type of “stoichiometric” information is often difficult or tedious to obtain using other approaches. However, single-molecule localization alone cannot follow the internal motion of the molecule. We can see how quickly a soccer player can change direction and how fast he runs with a ball, but this alone does not tell us what makes Maradona a great player instead of a merely average player. We need to increase the number of degrees of freedom that are being observed. One powerful technique that can follow the conformational changes of a molecule is fluorescence resonance energy transfer (FRET). “Conformation” is jargon used by biologists to denote the shape of a molecule. Just as a soccer player needs to change his bodily shape or posture to run and handle a ball, proteins need to change their conformations repeatedly to carry out their duties. In FRET, dyes of two different colors, say green and red, are attached to two sites of a protein. Normally, when we excite the green dye with a laser, we would see only green photons coming out. But when the two are very close to each other, within a few nanometers, the two dyes communicate with each other and the excitation energy is transferred from the green dye to the red dye such that we now see red photons come out. The relative ratio of the two colors is then used as a measure of their distance from each other, and if we know where the dyes are attached on the protein, we can deduce conformational changes of the molecule. Fig. 1 shows a cartoon of a rap musician dancing, undergoing conformational changes between different postures that are detected as anticorrelated changes in the intensities of green and red signals. Let me illustrate the use of FRET to study DNA repair. Inside every cell of our body there are two meters of DNA. Because there are ∼1014 human cells in our body, with the estimated renewal of 100 times for an average cell during our lifetime, our body would need to make about one light year length of DNA. DNA is under constant threat of damage. For example, sunlight and smoking can cause DNA damage that can accumulate and eventually lead to cancer. If DNA repair did not exist when we need to make so much DNA, we would die of cancer at a far younger age than is usually the case. One major mechanism of DNA repair is called homologous recombination. When a segment of DNA is broken, the cell uses another copy of the same DNA as a template to repair the breakage. To aid this process, a protein filament is formed around the broken DNA, and this filament then searches for a matching DNA sequence in a sea of millions of basepairs of DNA. This is no easy task and is often compared to finding a needle in a haystack. How does the cell accomplish this feat rapidly yet accurately? One possibility is to perform a three-dimensional search. Let the filament land on a random location of the target DNA, and if there is no sequence match, then dissociate and repeat until a match is found. It is equivalent to dating random people on the street until a soul mate is found, which would be exceedingly time consuming if you live in a big city. Another possibility is to perform one-dimensional (1D) sliding. This is akin to joining a book club to sample a few dozen of its members in search of a potential 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-dimensional search. If the local search is unsuccessful, the filament 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 evidence for 1D sliding (6Ragunathan K. Liu C. Ha T. RecA filament sliding on DNA facilitates homology search.eLife. 2012; 1: e00067Crossref Scopus (76) Google Scholar). But can the filament really find the target sequence through sliding? To address this question, 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. Another single-molecule technique, called optical tweezers, or what I call “chopsticks made of light,” can apply very 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 (2Svoboda K. Schmidt C.F. Block S.M. et al.Direct observation of kinesin stepping by optical trapping interferometry.Nature. 1993; 365: 721-727Crossref PubMed Scopus (1586) Google Scholar). Recently, the precision of optical tweezers has improved to the angstrom scale, almost the size of a water molecule (7Abbondanzieri E.A. Greenleaf W.J. Block S.M. et al.Direct observation of base-pair stepping by RNA polymerase.Nature. 2005; 438: 460-465Crossref PubMed Scopus (675) Google Scholar). In optical-tweezers measurements, it is as if you are closing your eyes and using your hands to manipulate and measure 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 (8Comstock M.J. Whitley K.D. Chemla Y.R. et al.Protein structure. Direct observation of structure-function relationship in a nucleic acid-processing enzyme.Science. 2015; 348: 352-354Crossref PubMed Scopus (133) Google Scholar). 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 (9Arslan S. Khafizov R. Ha T. et al.Protein structure. Engineering of a superhelicase through conformational control.Science. 2015; 348: 344-347Crossref PubMed Scopus (65) Google Scholar). 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. Single-molecule measurements have revealed the amazingly complex but elegant abilities of nature’s nanomachines to perform life’s central functions. Yet these molecules by themselves are not alive, and they function only when they work in the cellular milieu. How living cells come alive through the concerted actions of individual molecules is a major challenge for future research. Researchers are making progress on multiple fronts. On one front we might call “extreme in vitro,” we can measure multiple properties from single-molecular assemblies. FRET can be extended up to four colors, allowing us to measure six distances simultaneously. This can also be combined with multiaxis optical tweezers to study, for example, DNA replication, which is an amazingly rapid and accurate process that requires more than a dozen different proteins, with single basepair resolution, while at the same time probing how many proteins of which kind are present at each step of the reaction. On another front, we can push the technology to the cellular level, and even to the tissue level, ultimately hoping to view single-molecule activities in living cells in full glorious detail. Finally, we might find a third way, where protein complexes can be captured from freshly sacrificed cells or animals, or patient tissues, for detailed single-molecule analysis.