MotAB-like machinery drives the movement of MreB filaments during bacterial gliding motility.

MreB is a bacterial actin that is important for cell shape and cell wall biosynthesis in many bacterial species. MreB also plays crucial roles in Myxococcus xanthus gliding motility, but the underlying mechanism remains unknown. Here we tracked the dynamics of single MreB particles in M. xanthus using single-particle tracking photoactivated localization microscopy. We found that a subpopulation of MreB particles moves rapidly along helical trajectories, similar to the movements of the MotAB-like gliding motors. The rapid MreB motion was stalled in the mutants that carried truncated gliding motors. Remarkably, M. xanthus MreB moves one to two orders of magnitude faster than its homologs that move along with the cell wall synthesis machinery in Bacillus subtilis and Escherichia coli, and this rapid movement was not affected by the inhibitors of cell wall biosynthesis. Our results show that in M. xanthus, MreB provides a scaffold for the gliding motors while the gliding machinery drives the movement of MreB filaments, analogous to the interdependent movements of myosin motors and actin in eukaryotic cells.

Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots.

Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ∼10-fold more efficiently than existing SNAP ligands. Single-molecule analysis of these QDs shows 99% of time spent in the fluorescent on-state, ∼4-fold higher quantum efficiency than standard CdSe/ZnS QDs, and 350 million photons detected before photobleaching. Bright signals of these QDs enable us to track the stepping movement of a kinesin motor in vitro, and the improved labeling efficiency enables tracking of single kinesins in live cells.

Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin.

Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, but not by DNA methylation, histone deacetylation, or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres accumulate DDR signals and become more accessible to telomere-associated proteins. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery.

The polarity of myxobacterial gliding is regulated by direct interactions between the gliding motors and the Ras homolog MglA.

Gliding motility in Myxococcus xanthus is powered by flagella stator homologs that move in helical trajectories using proton motive force. The Frz chemosensory pathway regulates the cell polarity axis through MglA, a Ras family GTPase; however, little is known about how MglA establishes the polarity of gliding, because the gliding motors move simultaneously in opposite directions. Here we examined the localization and dynamics of MglA and gliding motors in high spatial and time resolution. We determined that MglA localizes not only at the cell poles, but also along the cell bodies, forming a decreasing concentration gradient toward the lagging cell pole. MglA directly interacts with the motor protein AglR, and the spatial distribution of AglR reversals is positively correlated with the MglA gradient. Thus, the motors moving toward lagging cell poles are less likely to reverse, generating stronger forward propulsion. MglB, the GTPase-activating protein of MglA, regulates motor reversal by maintaining the MglA gradient. Our results suggest a mechanism whereby bacteria use Ras family proteins to modulate cellular polarity.

G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding.

Human telomeres terminate with a single-stranded 3' G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K(+), POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA's access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na(+), in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.

Flagella stator homologs function as motors for myxobacterial gliding motility by moving in helical trajectories.

Many bacterial species use gliding motility in natural habitats because external flagella function poorly on hard surfaces. However, the mechanism(s) of gliding remain elusive because surface motility structures are not apparent. Here, we characterized the dynamics of the Myxococcus xanthus gliding motor protein AglR, a homolog of the Escherichia coli flagella stator protein MotA. We observed that AglR decorated a helical structure, and the AglR helices rotated when cells were suspended in liquid or when cells moved on agar surfaces. With photoactivatable localization microscopy, we found that single molecules of AglR, unlike MotA/MotB, can move laterally within the membrane in helical trajectories. AglR slowed down transiently at gliding surfaces, accumulating in clusters. Our work shows that the untethered gliding motors of M. xanthus, by moving within the membrane, can transform helical motion into linear driving forces that push against the surface.

Characterizing the dynamics of functionally relevant complexes of formate dehydrogenase.

The potential for femtosecond to picosecond time-scale motions to influence the rate of the intrinsic chemical step in enzyme-catalyzed reactions is a source of significant controversy. Among the central challenges in resolving this controversy is the difficulty of experimentally characterizing thermally activated motions at this time scale in functionally relevant enzyme complexes. We report a series of measurements to address this problem using two-dimensional infrared spectroscopy to characterize the time scales of active-site motions in complexes of formate dehydrogenase with the transition-state-analog inhibitor azide (N(3)(-)). We observe that the frequency-frequency time correlation functions (FFCF) for the ternary complexes with NAD(+) and NADH decay completely with slow time constants of 3.2 ps and 4.6 ps, respectively. This result suggests that in the vicinity of the transition state, the active-site enzyme structure samples a narrow and relatively rigid conformational distribution indicating that the transition-state structure is well organized for the reaction. In contrast, for the binary complex, we observe a significant static contribution to the FFCF similar to what is seen in other enzymes, indicating the presence of the slow motions that occur on time scales longer than our measurement window.

Examination of enzymatic H-tunneling through kinetics and dynamics.

In recent years, kinetic measurements of isotope effects of enzyme-catalyzed reactions and their temperature dependence led to the development of theoretical models that were used to rationalize the findings. These models suggested that motions at the femto- to picosecond (fs to ps) time scale modulate the environment of the catalyzed reaction. Due to the fast nature of motions that directly affect the cleavage of a covalent bond, it is challenging to correlate the enzyme kinetics and dynamics related to that step. We report a study of formate dehydrogenase (FDH) that compares the temperature dependence of intrinsic kinetic isotope effects (KIEs) to measurements of the environmental dynamics at the fs-ps time scale (Bandaria et al. J. Am. Chem. Soc. 2008, 130, 22-23). The findings from this comparison of experimental kinetics and dynamics are consistent with models of environmentally coupled H-tunneling models, also known as Marcus-like models. Apparently, at tunneling ready conformations, the donor-acceptor distance, orientation, and fluctuations seems to be well tuned for H-transfer and are not affected by thermal fluctuations slower than 10 ps. This phenomenon has been suggested in the past to be quite general in enzymatic reactions. Here, the kinetics and the dynamics measurements on a single chemical step and on fs-ps time scale, respectively, provide new insight and support for the relevant theoretical models. Furthermore, this methodology could be applied to other systems and be used to examine mutants for which the organization of the donor and acceptor is not ideal, or enzymes with different rigidity and different temperature optimum.

Exploring the molecular origins of protein dynamics in the active site of human carbonic anhydrase II.

We present three-pulse vibrational echo measurements of azide ion bound to the active site Zn of human carbonic anhydrase II (HCA II) and of two separate active-site mutants Thr199 --> Ala (T199A) and Leu198 --> Phe (L198F). Because structural motions of the protein active site influence the frequency of bound ligands, the differences in the time scales of the frequency-frequency correlation functions (FFCFs) obtained from global fits to each set of data allow us to make inferences about the time scales of the active site dynamics of HCA II. Surprisingly, the deletion of a potential electrostatic interaction in results in very little change in the FFCF, but the insertion of the bulky phenylalanine ring in causes much faster dynamics. We conclude that the fast, sub-picosecond time scale in the correlation function is attributable to hydrogen bond dynamics, and the slow, apparently static contribution is due to the conformational flexibility of Zn-bound azide in the active site.

Efforts toward developing probes of protein dynamics: vibrational dephasing and relaxation of carbon-deuterium stretching modes in deuterated leucine.

The spectral position of C-D stretching absorptions in the so-called "transparent window" of protein absorption (1800-2300 cm(-1)) makes them well suited as probes of protein dynamics with high temporal and structural resolution. We have previously incorporated single deuterated amino acids into proteins to site-selectively follow protein folding and ligand binding by steady-state FT IR spectroscopy. Ultimately, our goal is to use C-D bonds as probes in time-resolved IR spectroscopy to study dynamics and intramolecular vibrational energy redistribution (IVR) in proteins. As a step toward this goal, we now present the first time-resolved experiments characterizing the population and dephasing dynamics of selectively excited C-D bonds in a deuterated amino acid. Three differently deuterated, Boc-protected leucines were selected to systematically alter the number of additional C-D bonds that may mediate IVR out of the initially populated bright C-D stretching mode. Three-pulse photon echo experiments show that the steady-state C-D absorption linewidths are broadened by both homogeneous and inhomogeneous effects, and transient grating experiments reveal that IVR occurs on a subpicosecond time scale and is nonstatistical. The results have important implications for the interpretation of steady-state C-D spectra and demonstrate the potential utility of C-D bonds as probes of dynamics and IVR within a protein.

Fast enzyme dynamics at the active site of formate dehydrogenase.

The role of femtosecond-picosecond structural dynamics of proteins in enzyme-catalyzed reactions is a hotly debated topic. We report infrared photon echo measurement of the formate dehydrogenase-NAD+-azide ternary complex. In contrast to earlier studies of protein dynamics, the data show complete spectral diffusion on the femtosecond-picosecond time scale with no static heterogeneity. This result indicates that this transition-state analogue complex completely samples the distribution of structures that determine the distribution of azide vibrational frequencies within a few picoseconds and that there are no slower motions that perturb the H-bond network at the active site.

Vibrational relaxation of C-D stretching vibrations in CDCl3, CDBr3, and CDI3.

We present time-resolved transient grating measurements of the vibrational relaxation rates of the C-D stretching vibrations of deuterated haloforms in benzene and acetone. We compare our results with previous measurements of excited C-H stretches in the same solvents to obtain insight into the solvent effect on the vibrational relaxation. In deuterated molecules, there are more low-order-coupled states and the states are closer in energy to the C-D stretch than in the unlabeled isotopologs. Therefore, the relaxation is faster for the deuterated molecules. The relaxation also shows a significant solvent dependence. Bromoform and iodoform form charge-transfer complexes with both benzene and acetone which enhance the relaxation rate. For chloroform, hydrogen bonding to acetone is expected to be a more favorable interaction. Surprisingly, however, the vibrational relaxation of CDCl(3) is slower in acetone than in benzene.