Biomedical subjects

Mark C Leake

Publications and source records attributed to Mark C Leake.

13 recordsLinked to original sources

PubMed2026

A protein-dependent riboswitch activates ribosomal frameshifting in cardioviruses.

Programmed -1 ribosomal frameshifting (PRF) is a translational control mechanism used by RNA viruses to regulate the relative abundance of proteins encoded in different reading frames. Cardioviruses exhibit the highest known PRF efficiency, with ∼85% of ribosomes shifting into the -1 frame. This unusual event requires an interaction between the viral 2A protein and a stimulatory element in the RNA genome, but the basis for protein dependence is unclear. To address this, here we investigate the structure and dynamics of the PRF signal in Theiler's murine encephalitis virus (TMEV). By combining X-ray crystallography, small-angle X-ray scattering (SAXS), and single-molecule fluorescence resonance energy transfer (smFRET), we show that 2A binding switches the RNA from a stem-loop conformation into a pseudoknot, and we demonstrate that pseudoknot formation is essential for efficient PRF in vitro and in cells. Together, these findings illustrate how the cardiovirus PRF element behaves as a protein-dependent riboswitch, defining the molecular mechanism by which frameshifting is conditionally activated.

Frameshifting, Ribosomal↗

PubMed2006

Stoichiometry and turnover in single, functioning membrane protein complexes.

Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media. Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall. Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli. We monitored motor function by rotation of a tethered cell body, and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP-MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains approximately 22 copies of GFP-MotB, consistent with approximately 11 stators each containing two MotB molecules. We also observed a membrane pool of approximately 200 GFP-MotB molecules diffusing at approximately 0.008 microm2 s(-1). Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP-MotB between the membrane pool and motor with a rate constant of the order of 0.04 s(-1): the dwell time of a given stator in the motor is only approximately 0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine.

Bacterial Proteins↗

PubMed2006

The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11.

Torque is generated in the rotary motor at the base of the bacterial flagellum by ion translocating stator units anchored to the peptidoglycan cell wall. Stator units are composed of the proteins MotA and MotB in proton-driven motors, and they are composed of PomA and PomB in sodium-driven motors. Strains of Escherichia coli lacking functional stator proteins produce flagella that do not rotate, and induced expression of the missing proteins leads to restoration of motor rotation in discrete speed increments, a process known as "resurrection." Early work suggested a maximum of eight units. More recent indications that WT motors may contain more than eight units, based on recovery of disrupted motors, are inconclusive. Here we demonstrate conclusively that the maximum number of units in a motor is at least 11. Using back-focal-plane interferometry of 1-mum polystyrene beads attached to flagella, we observed at least 11 distinct speed increments during resurrection with three different combinations of stator proteins in E. coli. The average torques generated by a single unit and a fully induced motor were lower than previous estimates. Speed increments at high numbers of units are smaller than those at low numbers, indicating that not all units in a fully induced motor are equivalent.

Bacterial Proteins↗

PubMed2006

Mechanical properties of cardiac titin's N2B-region by single-molecule atomic force spectroscopy.

Titin is a giant protein responsible for passive-tension generation in muscle sarcomeres. Here, we used single-molecule AFM force spectroscopy to investigate the mechanical characteristics of a recombinant construct from the human cardiac-specific N2B-region, which harbors a 572-residue unique sequence flanked by two immunoglobulin (Ig) domains on either side. Force-extension curves of the N2B-construct revealed mean unfolding forces for the Ig-domains similar to those of a recombinant fragment from the distal Ig-region in titin (I91-98). The mean contour length of the N2B-unique sequence was 120 nm, but there was a bimodal distribution centered at approximately 95 nm (major peak) and 180 nm (minor peak). These values are lower than expected if the N2B-unique sequence were a permanently unfolded entropic spring, but are consistent with the approximately 100 nm maximum extension of that segment measured in isolated stretched cardiomyofibrils. A contour-length below 200 nm would be reasonable, however, if the N2B-unique sequence were stabilized by a disulphide bridge, as suggested by several disulphide connectivity prediction algorithms. Since the N2B-unique sequence can be phosphorylated by protein kinase A (PKA), which lowers titin-based stiffness, we studied whether addition of PKA (+ATP) affects the mechanical properties of the N2B-construct, but found no changes. The softening effect of PKA on N2B-titin may require specific conditions/factors present inside the cardiomyocytes.

Amino Acid Sequence↗

PubMed2006

The molecular elasticity of the insect flight muscle proteins projectin and kettin.

Projectin and kettin are titin-like proteins mainly responsible for the high passive stiffness of insect indirect flight muscles, which is needed to generate oscillatory work during flight. Here we report the mechanical properties of kettin and projectin by single-molecule force spectroscopy. Force-extension and force-clamp curves obtained from Lethocerus projectin and Drosophila recombinant projectin or kettin fragments revealed that fibronectin type III domains in projectin are mechanically weaker (unfolding force, F(u) approximately 50-150 pN) than Ig-domains (F(u) approximately 150-250 pN). Among Ig domains in Sls/kettin, the domains near the N terminus are less stable than those near the C terminus. Projectin domains refolded very fast [85% at 15 s(-1) (25 degrees C)] and even under high forces (15-30 pN). Temperature affected the unfolding forces with a Q(10) of 1.3, whereas the refolding speed had a Q(10) of 2-3, probably reflecting the cooperative nature of the folding mechanism. High bending rigidities of projectin and kettin indicated that straightening the proteins requires low forces. Our results suggest that titin-like proteins in indirect flight muscles could function according to a folding-based-spring mechanism.

Animals↗

PubMed2005

Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells.

The energy-transducing cytoplasmic membrane of bacteria contains pumps and antiports maintaining the membrane potential and ion gradients. We have developed a method for rapid, single-cell measurement of the internal sodium concentration ([Na(+)](in)) in Escherichia coli using the sodium ion fluorescence indicator, Sodium Green. The bacterial flagellar motor is a molecular machine that couples the transmembrane flow of ions, either protons (H(+)) or sodium ions (Na(+)), to flagellar rotation. We used an E. coli strain containing a chimeric flagellar motor with H(+)- and Na(+)-driven components that functions as a sodium motor. Changing external sodium concentration ([Na(+)](ex)) in the range 1-85 mM resulted in changes in [Na(+)](in) between 5-14 mM, indicating a partial homeostasis of internal sodium concentration. There were significant intercell variations in the relationship between [Na(+)](in) and [Na(+)](ex), and the internal sodium concentration in cells not expressing chimeric flagellar motors was 2-3 times lower, indicating that the sodium flux through these motors is a significant fraction of the total sodium flux into the cell.

Bacterial Outer Membrane Proteins↗

PubMed2005

Direct observation of steps in rotation of the bacterial flagellar motor.

The bacterial flagellar motor is a rotary molecular machine that rotates the helical filaments that propel many species of swimming bacteria. The rotor is a set of rings up to 45 nm in diameter in the cytoplasmic membrane; the stator contains about ten torque-generating units anchored to the cell wall at the perimeter of the rotor. The free-energy source for the motor is an inward-directed electrochemical gradient of ions across the cytoplasmic membrane, the protonmotive force or sodium-motive force for H+-driven and Na+-driven motors, respectively. Here we demonstrate a stepping motion of a Na+-driven chimaeric flagellar motor in Escherichia coli at low sodium-motive force and with controlled expression of a small number of torque-generating units. We observe 26 steps per revolution, which is consistent with the periodicity of the ring of FliG protein, the proposed site of torque generation on the rotor. Backwards steps despite the absence of the flagellar switching protein CheY indicate a small change in free energy per step, similar to that of a single ion transit.

Adenosine Triphosphate↗

PubMed2004

Multiple sources of passive stress relaxation in muscle fibres.

The forces developed during stretch of nonactivated muscle consist of velocity-sensitive (viscous/viscoelastic) and velocity-insensitive (elastic) components. At the myofibrillar level, the elastic-force component has been described in terms of the entropic-spring properties of the giant protein titin, but entropic elasticity cannot account for viscoelastic properties, such as stress relaxation. Here we examine the contribution of titin to passive stress relaxation of isolated rat-cardiac myofibrils depleted of actin by gelsolin treatment. Monte Carlo simulations show that, up to approximately 5 s after a stretch, the time course of stress relaxation can be described assuming unfolding of 1-2 immunoglobulin domains per titin molecule. For extended periods of stress relaxation, the simulations failed to correctly describe the myofibril data, suggesting that in situ, titin-Ig domains may be more stable than predicted in earlier single-molecule atomic-force-microscopy studies. The reasons behind this finding remain unknown; simply assuming a reduced unfolding probability of domains--an effect found here by AFM force spectroscopy on titin-Ig domains in the presence of a chaperone, alpha-B-crystallin--did not help correctly simulate the time course of stress relaxation. We conclude that myofibrillar stress relaxation likely has multiple sources. Evidence is provided that in intact myofibrils, an initial, rapid phase of stress relaxation results from viscous resistance due to the presence of actin filaments.

Actins↗

PubMed2004

Developmentally regulated switching of titin size alters myofibrillar stiffness in the perinatal heart.

Before birth, the compliance of the heart is limited predominantly by extracardiac constraint. Reduction of this constraint at birth requires that myocardial compliance be determined mainly by the heart's own constituents. Because titin is a principal contributor to ventricular passive tension (PT), we studied the expression and mechanics of cardiac-titin isoforms during perinatal rat heart development. Gel electrophoresis and immunoblotting revealed a single, 3.7-MDa, N2BA isoform present 6 days before birth and an additional, also previously unknown, N2BA isoform of 3.5 to 3.6 MDa expressed in the near-term fetus. These large isoforms rapidly disappear after birth and are replaced by a small N2B isoform (3.0 MDa) predominating in 1-week-old and adult rats. In addition, neonatal pig hearts showed large N2BA-titin isoforms distinct from those present in the adult porcine myocardium. By quantitative reverse transcriptase-polymerase chain reaction, developmentally expressed titin-mRNA species were detected in rat heart. Titin-based PT was much lower (approximately 15 times) in fetal than adult rat cardiomyocytes, and measured PT levels were readily predictable with a model of worm-like chain titin elasticity. Immunofluorescence microscopy showed the extensibility of the differentially spliced molecular spring regions of fetal/neonatal titin isoforms in isolated rat cardiomyofibrils. Whereas the titin-isoform shift by 700 kDa ensures high passive stiffness of the postnatal cardiac myofibrils, the expression of specific fetal/neonatal cardiac-titin isoforms may also have important functions for contractile properties, myofibril assembly or turnover, and myocardial signaling during perinatal heart development.

Alternative Splicing↗

PubMed2004

The elasticity of single titin molecules using a two-bead optical tweezers assay.

Titin is responsible for the passive elasticity of the muscle sarcomere. The mechanical properties of skeletal and cardiac muscle titin were characterized in single molecules using a novel dual optical tweezers assay. Antibody pairs were attached to beads and used to select the whole molecule, I-band, A-band, a tandem-immunoglobulin (Ig) segment, and the PEVK region. A construct from the PEVK region expressing >25% of the full-length skeletal muscle isoform was chemically conjugated to beads and similarly characterized. By elucidating the elasticity of the different regions, we showed directly for the first time, to our knowledge, that two entropic components act in series in the skeletal muscle titin I-band (confirming previous speculations), one associated with tandem-immunoglobulin domains and the other with the PEVK region, with persistence lengths of 2.9 nm and 0.76 nm, respectively (150 mM ionic strength, 22 degrees C). Novel findings were: the persistence length of the PEVK component rose (0.4-2.7 nm) with an increase in ionic strength (15-300 mM) and fell (3.0-0.3 nm) with a temperature increase (10-60 degrees C); stress-relaxation in 10-12-nm steps was observed in the PEVK construct and hysteresis in the native PEVK region. The region may not be a pure random coil, as previously thought, but contains structured elements, possibly with hydrophobic interactions.

Animals↗

PubMed2003

Association of the chaperone alphaB-crystallin with titin in heart muscle.

alphaB-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. alphaB-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309-319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of alphaB-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. alphaB-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, alphaB-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between alphaB-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by alphaB-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with alphaB-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.

Animals↗

PubMed2003

Damped elastic recoil of the titin spring in myofibrils of human myocardium.

The giant protein titin functions as a molecular spring in muscle and is responsible for most of the passive tension of myocardium. Because the titin spring is extended during diastolic stretch, it will recoil elastically during systole and potentially may influence the overall shortening behavior of cardiac muscle. Here, titin elastic recoil was quantified in single human heart myofibrils by using a high-speed charge-coupled device-line camera and a nanonewtonrange force sensor. Application of a slack-test protocol revealed that the passive shortening velocity (Vp) of nonactivated cardiomyofibrils depends on: (i) initial sarcomere length, (ii) release-step amplitude, and (iii) temperature. Selective digestion of titin, with low doses of trypsin, decelerated myofibrillar passive recoil and eventually stopped it. Selective extraction of actin filaments with a Ca2+-independent gelsolin fragment greatly reduced the dependency of Vp on release-step size and temperature. These results are explained by the presence of viscous forces opposing myofibrillar passive recoil that are caused mainly by weak actin-titin interactions. Thus, Vp is determined by two distinct factors: titin elastic recoil and internal viscous drag forces. The recoil could be modeled as that of a damped entropic spring consisting of independent worm-like chains. The functional importance of myofibrillar elastic recoil was addressed by comparing instantaneous Vp to unloaded shortening velocity, which was measured in demembranated, fully Ca2+-activated, human cardiac fibers. Titin-driven passive recoil was much faster than active unloaded shortening velocity in early phases of isotonic contraction. Damped myofibrillar elastic recoil could help accelerate active contraction speed of human myocardium during early systolic shortening.

Actins↗

PubMed2003

The elasticity of single kettin molecules using a two-bead laser-tweezers assay.

Kettin is a high molecular mass protein of insect muscle associated with thin filaments and alpha-actinin in the Z-disc. It is thought to form a link between thin and thick filaments towards its C-terminus, contributing significantly to passive sarcomere stiffness. Here the elastic properties were characterised by mechanical stretches on an antibody-delimited region of the single molecule using two independent optical traps capable of exerting forces up to 150 pN. Step-like events were observed in the force-extension relationships consistent with the unfolding of Ig domains at moderate force and refolding of these domains at significantly higher forces than have been observed for related modular proteins.

Animals↗