| 1. |
- Albet-Torres, Nuria, et al.
(författare)
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Molecular motors on lipid bilayers and silicon dioxide: different driving forces for adsorption
- 2010
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Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 6:14, s. 3211-3219
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Tidskriftsartikel (refereegranskat)abstract
- Understanding how different types of interactions govern adsorption of the myosin motor fragment heavy meromyosin (HMM) onto different substrates is important in functional studies of actomyosin and for the development of motor powered lab-on-a-chip applications. In this study, we have combined in vitro motility assays and quartz crystal microbalance with dissipation (QCM-D) monitoring to investigate the underlying adsorption mechanisms of HMM onto supported lipid bilayers in comparison with pure and silanized SiO2. The QCM-D results, combined with data showing actin transportation by HMM adsorbed onto positively charged supported lipid bilayers, suggest reversible HMM surface adsorption via the negatively charged coiled-coil tail region. In contrast, the QCM-D data for HMM adsorption onto negatively charged lipids support a model according to which HMM adsorbs onto negatively charged surfaces largely via the positively charged actin binding regions. Adsorption studies at low (30-65 mM) and high (185-245 mM) ionic strengths onto piranha cleaned SiO2 surfaces (contact angle
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| 2. |
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| 3. |
- Balaz, Martina, et al.
(författare)
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Effects of surface adsorption on catalytic activity of heavy meromyosin studied using a fluorescent ATP analogue
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:24, s. 7233-7251
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Tidskriftsartikel (refereegranskat)abstract
- Biochemical studies in solution and with myosin motor fragments adsorbed to surfaces (in vitro motility assays) are invaluable for elucidation of actomyosin function. However, there is limited understanding of how surface adsorption affects motor properties, e.g., catalytic activity. Here we address this issue by comparing the catalytic activity of heavy meromyosin (HMM) in solution and adsorbed to standard motility assay surfaces [derivatized with trimethylchlorosilane (TMCS)]. For these studies we first characterized the interaction of HMM and actomyosin with the fluorescent ATP analogue adenosine 5'-triphosphate Alexa Fluor 647 2'- (or 3'-) O-(N-(2-aminoethyl)urethane) hexa(triethylammonium) salt (Alexa-ATP). The data suggest that Alexa-ATP is hydrolyzed by HMM in solution at a slightly higher rate than ATP but with a generally similar mechanism. Furthermore, Alexa-ATP is effective as a fuel for HMM-propelled actin filament sliding. The catalytic activity of HMM on TMCS surfaces was studied using (1) Alexa-ATP in total internal reflection fluorescence (TIRF) spectroscopy experiments and (2) Alexa-ATP and ATP in HPLC-aided ATPase measurements. The results support the hypothesis of different HMM configurations on the surface. However, a dominant proportion of the myosin heads were catalytically active, and their average steady-state hydrolysis rate was slightly higher (with Alexa-ATP) or markedly higher (with ATP) on the surface than in solution. The results are discussed in relation to the use of TMCS surfaces and Alexa-ATP for in vitro motility assays and single molecule studies. Furthermore, we propose a novel TIRF microscopy method to accurately determine the surface density of catalytically active myosin motors.
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| 5. |
- Balaz, Martina, et al.
(författare)
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Protein-surface Interactions and Functional Geometry of Surface-adsorbed Myosin Motor Fragments
- 2009
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 96:3 Suppl. 1, s. 495A-495A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Biophysical studies with myosin motor fragments (heavy meromyosin; HMM and subfragment 1; S1) adsorbed to artificial surfaces, are important for elucidation of actomyosin function. In spite of the widespread use of such in vitro motility assays and single molecule studies, little is known about the adsorption geometry and effects of protein-surface interactions on the motor properties. Here, we investigate these factors with focus on HMM using quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) spectroscopy based ATPase assays. In the latter, we monitored the turnover of Alexa-fluor647-ATP (Alexa-ATP) by surface adsorbed HMM. Studies were performed with HMM/S1 adsorbed to model hydrophilic (SiO2) or hydrophobic (trimethyl-chlorosilane [TMCS] - derivatized) surfaces. The results suggest that adsorption of HMM is weakened on SiO2 (but not on TMCS) at high (245 mM) compared to low (65 mM) ionic strengths. The changes in ionic strength were also associated with structural changes in the protein layer according to QCM-D studies. Moreover, the TIRF based ATPase assay suggested a larger fraction of HMM molecules with low catalytic activity on SiO2. These and other TIRF and QCM-D results, suggest that HMM preferentially adsorbs to negatively charged hydrophilic surfaces via the actin-binding region. In contrast, the majority of the HMM molecules seem to adsorb via their C-terminal tail on moderately hydrophobic surfaces. In the latter case the catalytic sites appear to be close to, but not immobilized on the surface. The results with HMM were compared to, and found consistent with, QCM-D and TIRF-data obtained with S1 motor fragments.
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| 6. |
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| 7. |
- Graczyk, Mariusz, et al.
(författare)
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Fabrication of bottle-shaped nanochannels in fused silica using a self-closing effect
- 2012
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Ingår i: Microelectronic Engineering. - : Elsevier BV. - 1873-5568 .- 0167-9317. ; 97, s. 173-176
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Tidskriftsartikel (refereegranskat)abstract
- The spatial control of molecular motor function, using nanostructured surfaces, is of great interest for the development of commercial devices for diagnostics and high-throughput drug screening with molecular motors as targets. In the present study we have fabricated 100-300 nm wide nanochannels, completely subsurfaced on fused silica chips, with the aim to interface them with a microfluidic system. Such a system will allow for changes in the chemical environment surrounding molecular motors, with minimal influence on their directional motion. This will be achieved by changing the chemical environment in a perpendicular direction to the motor motion and allowing the chemical substances to diffuse in and out of the nanochannels via a small slit (5-10 nm) on the top of the nanochannels. To create this slit, and to control its width, we here demonstrate the use of a self-closing effect based on the volume increase (2.27 times) during oxidation of silicon. The details of the fabrication steps (EBL, RIE and oxidation) are discussed. (C) 2012 Elsevier B.V. All rights reserved.
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| 8. |
- Graczyk, Mariusz, et al.
(författare)
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Optimization of a self-closing effect to produce nanochannels with top slits in fused silica
- 2012
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Ingår i: Journal of Vacuum Science and Technology B. - : American Vacuum Society. - 1520-8567. ; 30:6
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Tidskriftsartikel (refereegranskat)abstract
- The authors report on the fabrication of subsurfaced 100-600 nm wide nanochannels in fused silica with top slit openings in the size range of 5-10 nm. Such nanochannels can be used in combination with a nanofluidics system to guide molecular motors and quickly switch the chemical environment inside the nanochannels through diffusion via the top slits. To realize nanochannel top slits in this size range, the authors here demonstrate the use of a self-closing effect based on the volume expansion of a thin Si layer during oxidation. A high contrast electron beam lithography exposure step in conjunction with dry etching of SiO2 by reactive ion etching (RIE) and Si by inductively coupled plasma-RIE followed by wet etching of a fused silica substrate is used to create the initial slit before oxidation. The details of nanochannel fabrication steps are described and discussed. (C) 2012 American Vacuum Society. [http://dx.doi.org/10.1116/1.4766317]
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| 9. |
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| 10. |
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| 11. |
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| 12. |
- Månsson, Alf, et al.
(författare)
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In vitro assays of molecular motors - impact of motor-surface interactions
- 2008
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Ingår i: Frontiers in Bioscience. - 1093-9946 .- 1093-4715. ; 13:May 1, s. 5732-5754
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Forskningsöversikt (övrigt vetenskapligt/konstnärligt)abstract
- In many types of biophysical studies of both single molecules and ensembles of molecular motors the motors are adsorbed to artificial surfaces. Some of the most important assay systems of this type (in vitro motility assays and related single molecule techniques) will be briefly described together with an account of breakthroughs in the understanding of actomyosin function that have resulted from their use. A poorly characterized, but potentially important, entity in these studies is the mechanism of motor adsorption to surfaces and the effects of motor surface interactions on experimental results. A better understanding of these phenomena is also important for the development of commercially viable nanotechnological applications powered by molecular motors. Here, we will consider several aspects of motor surface interactions with a particular focus on heavy meromyosin (HMM) from skeletal muscle. These aspects will be related to heavy meromyosin structure and relevant parts of the vast literature on protein-surface interactions for non-motor proteins. An overview of methods for studying motor-surface interactions will also be given. The information is used as a basis for further development of a model for HMM-surface interactions and is discussed in relation to experiments where nanopatterning has been employed for in vitro reconstruction of actomyosin order. The challenges and potentials of this approach in biophysical studies, compared to the use of self-assembly of biological components into supramolecular protein aggregates (e. g. myosin filaments) will be considered. Finally, this review will consider the implications for further developments of motor-powered lab-on-a-chip devices.
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| 13. |
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| 17. |
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| 18. |
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| 19. |
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| 20. |
- Niman, Cassandra, et al.
(författare)
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Fluidic switching in nanochannels for the control of Inchworm: a synthetic biomolecular motor with a power stroke.
- 2014
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Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 6:24, s. 15008-15019
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Tidskriftsartikel (refereegranskat)abstract
- Synthetic molecular motors typically take nanometer-scale steps through rectification of thermal motion. Here we propose Inchworm, a DNA-based motor that employs a pronounced power stroke to take micrometer-scale steps on a time scale of seconds, and we design, fabricate, and analyze the nanofluidic device needed to operate the motor. Inchworm is a kbp-long, double-stranded DNA confined inside a nanochannel in a stretched configuration. Motor stepping is achieved through externally controlled changes in salt concentration (changing the DNA's extension), coordinated with ligand-gated binding of the DNA's ends to the functionalized nanochannel surface. Brownian dynamics simulations predict that Inchworm's stall force is determined by its entropic spring constant and is ∼0.1 pN. Operation of the motor requires periodic cycling of four different buffers surrounding the DNA inside a nanochannel, while keeping constant the hydrodynamic load force on the DNA. We present a two-layer fluidic device incorporating 100 nm-radius nanochannels that are connected through a few-nm-wide slit to a microfluidic system used for in situ buffer exchanges, either diffusionally (zero flow) or with controlled hydrodynamic flow. Combining experiment with finite-element modeling, we demonstrate the device's key performance features and experimentally establish achievable Inchworm stepping times of the order of seconds or faster.
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| 21. |
- Persson, Malin, 1983-, et al.
(författare)
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Heavy Meromyosin Molecules Extending More Than 50 nm above Adsorbing Electronegative Surfaces
- 2010
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 26:12, s. 9927-9936
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Tidskriftsartikel (refereegranskat)abstract
- In the in vitro motility assay, actin filaments are propelled by surface-adsorbed myosin motors, or rather, myosin motor fragments such as heavy meromyosin (HMM). Recently, efforts have been made to develop actomyosin powered nanodevices on the basis of this assay but such developments are hampered by limited understanding of the HMM adsorption geometry. Therefore, we here investigate the HMM adsorption geometries on trimethylchlorosilane-[TMCS-] derivatized hydrophobic surfaces and on hydrophilic negatively charged surfaces (SiO2). The TMCS surface is of great relevance in fundamental studies of actomyosin and both surface substrates are important for the development of motor powered nanodevices. Whereas both the TMCS and SiO2 surfaces were nearly saturated with HMM (incubation at 120 mu g mL(-1)) there was little actin binding on SiO2 in the absence of ATP and no filament sliding in the presence of ATP. This contrasts with excellent actin-binding and motility on TMCS. Quartz crystal microbalance with dissipation (QCM-D) studies demonstrate a HMM layer with substantial protein mass up to 40 nm above the TMCS surface, considerably more than observed for myosin subfragment 1 (SI; 6 nm). Together with the excellent actin transportation on TMCS, this strongly suggests that HMM adsorbs to TMCS mainly via its most C-terminal tail part. Consistent with this idea, fluorescence interference contrast (FLIC) microscopy showed that actin filaments are held by HMM 38 +/- 2 nm above the TMCS-surface with the catalytic site, on averge, 20-30 nm above the surface. Viewed in a context with FLIC, QCM-D and TIRF results, the lack of actin motility and the limited actin binding on SiO2 shows that HMM adsorbs largely via the actin-binding region on this surface with the C-terminal coiled-coil tails extending > 50 nm into solution. The results and new insights from this study are of value, not only for the development of motor powered nanodevices but also for the interpretation of fundamental biophysical studies of actomyosin function and for the understanding of surface protein interactions in general.
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| 22. |
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| 23. |
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| 24. |
- Sundberg, Mark, et al.
(författare)
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Selective spatial localization of actomyosin motor function by chemical surface patterning
- 2006
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 22:17, s. 7302-7312
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Tidskriftsartikel (refereegranskat)abstract
- We have previously described the efficient guidance and unidirectional sliding of actin filaments along nanosized tracks with adsorbed heavy meromyosin (HMM; myosin II motor fragment). In those experiments, the tracks were functionalized with trimethylchlorosilane (TMCS) by chemical vapor deposition (CVD) and surrounded by hydrophilic areas. Here we first show, using in vitro motility assays on nonpatterned and micropatterned surfaces, that the quality of HMM function on CVD-TMCS is equivalent to that on standard nitrocellulose substrates. We further examine the influences of physical properties of different surfaces (glass, SiO2, and TMCS) and chemical properties of the buffer solution on motility. With the presence of methylcellulose in the assay solution, there was HMM-induced actin filament sliding on both glass/SiO2 and on TMCS, but the velocity was higher on TMCS. This difference in velocity increased with decreasing contact angles of the glass and SiO2 surfaces in the range of 20-67 degrees (advancing contact angles for water droplets). The corresponding contact angle of CVD-TMCS was 81 degrees. In the absence of methylcellulose, there was high-quality motility on TMCS but no motility on glass/SiO2. This observation was independent of the contact angle of the glass/SiO2 surfaces and of HMM incubation concentrations (30-150 mu g mL(-1)) and ionic strengths of the assay solution (20-50 mM). Complete motility selectivity between TMCS and SiO2 was observed for both nonpatterned and for micro- and nanopatterned surfaces. Spectrophotometric analysis of HMM depletion during incubation, K/EDTA ATPase measurements, and total internal reflection fluorescence spectroscopy of HMM binding showed only minor differences in HMM surface densities between TMCS and SiO2/glass. Thus, the motility contrast between the two surface chemistries seems to be attributable to different modes of HMM binding with the hindrance of actin binding on SiO2/glass.
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| 25. |
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| 26. |
- Vikhorev, Petr, et al.
(författare)
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Diffusion dynamics of motor driven transport: gradient production and self-organization of surfaces.
- 2008
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 24:23, s. 13509-13517
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between cytoskeletal filaments (e.g., actin filaments) and molecular motors (e.g., myosin) is the basis for many aspects of cell motility and organization of the cell interior. In the in vitro motility assay (IVMA), cytoskeletal filaments are observed while being propelled by molecular motors adsorbed to artificial surfaces (e.g., in studies of motor function). Here we integrate ideas that cytoskeletal filaments may be used as nanoscale templates in nanopatterning with a novel approach for the production of surface gradients of biomolecules and nanoscale topographical features. The production of such gradients is challenging but of increasing interest (e.g., in cell biology). First, we show that myosin-induced actin filament sliding in the IVMA can be approximately described as persistent random motion with a diffusion coefficient D) given by a relationship analogous to the Einstein equation (D = kT/gamma). In this relationship, the thermal energy (kT) and the drag coefficient (gamma) are substituted by a parameter related to the free-energy transduction by actomyosin and the actomyosin dissociation rate constant, respectively. We then demonstrate how the persistent random motion of actin filaments can be exploited in conceptually novel methods for the production of actin filament density gradients of predictable shapes. Because of regularly spaced binding sites (e.g., lysines and cysteines) the actin filaments act as suitable nanoscale scaffolds for other biomolecules (tested for fibronectin) or nanoparticles. This forms the basis for secondary chemical and topographical gradients with implications for cell biological studies and biosensing.
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