| 1. |
- Albet-Torres, Nuria, et al.
(författare)
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Long-Term Storage of Surface-Adsorbed Protein Machines
- 2011
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 27:11, s. 7108-7112
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Tidskriftsartikel (refereegranskat)abstract
- The effective and simple long-term storage of complex functional proteins is critical in achieving commercially viable biosensors. This issue is particularly challenging in recently proposed types of nanobiosensors, where molecular-motor-driven transportation substitutes microfluidics and forms the basis for novel detection schemes. Importantly, therefore, we here describe that delicate heavy meromyosin (HMM)-based nanodevices (HMM motor fragments adsorbed to silanized surfaces and actin bound to HMM) fully maintain their function when stored at -20 degrees C for more than a month. The mechanisms for the excellent preservation of acto-HMM motor function upon repeated freeze thaw cycles are discussed. The results are important to the future commercial implementation of motor-based nanodevices and are of more general value to the long-term storage of any protein-based bionanodevice.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- Korten, Slobodanka, et al.
(författare)
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Sample solution constraints on motor-driven diagnostic nanodevices
- 2013
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 13:5, s. 866-876
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Tidskriftsartikel (refereegranskat)abstract
- The last decade has seen appreciable advancements in efforts towards increased portability of lab-on-a-chip devices by substituting microfluidics with molecular motor-based transportation. As of now, first proof-of-principle devices have analyzed protein mixtures of low complexity, such as target protein molecules in buffer solutions optimized for molecular motor performance. However, in a diagnostic workup, lab-on-a-chip devices need to be compatible with complex biological samples. While it has been shown that such samples do not interfere with crucial steps in molecular diagnostics (for example antibody-antigen recognition), their effect on molecular motors is unknown. This critical and long overlooked issue is addressed here. In particular, we studied the effects of blood, cell lysates and solutions containing genomic DNA extracts on actomyosin and kinesin-microtubule-based transport, the two biomolecular motor systems that are most promising for lab-on-a-chip applications. We found that motor function is well preserved at defined dilutions of most of the investigated biological samples and demonstrated a molecular motor-driven label-free blood type test. Our results support the feasibility of molecular-motor driven nanodevices for diagnostic point-of-care applications and also demonstrate important constraints imposed by sample composition and device design that apply both to kinesin-microtubule and actomyosin driven applications.
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| 5. |
- Kumar, Saroj, et al.
(författare)
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Magnetic capture from blood rescues molecular motor function in diagnostic nanodevices
- 2013
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Ingår i: Journal of Nanobiotechnology. - : BioMed Central (BMC). - 1477-3155. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Background: Introduction of effective point-of-care devices for use in medical diagnostics is part of strategies to combat accelerating health-care costs. Molecular motor driven nanodevices have unique potentials in this regard due to unprecedented level of miniaturization and independence of external pumps. However motor function has been found to be inhibited by body fluids.Results: We report here that a unique procedure, combining separation steps that rely on antibody-antigen interactions, magnetic forces applied to magnetic nanoparticles (MPs) and the specificity of the actomyosin bond, can circumvent the deleterious effects of body fluids (e.g. blood serum). The procedure encompasses the following steps: (i) capture of analyte molecules from serum by MP-antibody conjugates, (ii) pelleting of MP-antibody-analyte complexes, using a magnetic field, followed by exchange of serum for optimized biological buffer, (iii) mixing of MP-antibody-analyte complexes with actin filaments conjugated with same polyclonal antibodies as the magnetic nanoparticles. This causes complex formation: MP-antibody-analyte-antibody-actin, and magnetic separation is used to enrich the complexes. Finally (iv) the complexes are introduced into a nanodevice for specific binding via actin filaments to surface adsorbed molecular motors (heavy meromyosin). The number of actin filaments bound to the motors in the latter step was significantly increased above the control value if protein analyte (50-60 nM) was present in serum (in step i) suggesting appreciable formation and enrichment of the MP-antibody-analyte-antibody-actin complexes. Furthermore, addition of ATP demonstrated maintained heavy meromyosin driven propulsion of actin filaments showing that the serum induced inhibition was alleviated. Detailed analysis of the procedure i-iv, using fluorescence microscopy and spectroscopy identified main targets for future optimization.Conclusion: The results demonstrate a promising approach for capturing analytes from serum for subsequent motor driven separation/detection. Indeed, the observed increase in actin filament number, in itself, signals the presence of analyte at clinically relevant nM concentration without the need for further motor driven concentration. Our analysis suggests that exchange of polyclonal for monoclonal antibodies would be a critical improvement, opening for a first clinically useful molecular motor driven lab-on-a-chip device.
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| 6. |
- 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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| 7. |
- 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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| 8. |
- Sundberg, Mark, et al.
(författare)
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Actin filament guidance on a chip: Toward high-throughput assays and lab-on-a-chip applications
- 2006
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 22:17, s. 7286-7295
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Tidskriftsartikel (refereegranskat)abstract
- Biological molecular motors that are constrained so that function is effectively limited to predefined nanosized tracks may be used as molecular shuttles in nanotechnological applications. For these applications and in high-throughput functional assays (e. g., drug screening), it is important that the motors propel their cytoskeletal filaments unidirectionally along the tracks with a minimal number of escape events. We here analyze the requirements for achieving this for actin filaments that are propelled by myosin II motor fragments (heavy meromyosin; HMM). First, we tested the guidance of HMM-propelled actin filaments along chemically defined borders. Here, trimethylchlorosilane (TMCS)-derivatized areas with high-quality HMM function were surrounded by SiO2 domains where HMM did not bind actin. Guidance along the TMCS-SiO2 border was almost 100% for filament approach angles between 0 and 20 degrees but only about 10% at approach angles near 90 degrees. A model (Clemmens, J.; Hess, H.; Lipscomb, R.; Hanein, Y.; Bohringer, K. F.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Langmuir 2003, 19, 10967-10974) accounted for essential aspects of the data and also correctly predicted a more efficient guidance of actin filaments than previously shown for kinesin- propelled microtubules. Despite the efficient guidance at low approach angles, nanosized (< 700 nm wide) TMCS tracks surrounded by SiO2 were not effective in guiding actin filaments. Neither was there complete guidance along nanosized tracks that were surrounded by topographical barriers (walls and roof partially covering the track) unless there was also chemically based selectivity between the tracks and surroundings. In the latter case, with dually defined tracks, there was close to 100% guidance. A combined experimental and theoretical analysis, using tracks of the latter type, suggested that a track width of less than about 200-300 nm is sufficient at a high HMM surface density to achieve unidirectional sliding of actin filaments. In accord with these results, we demonstrate the long- term trapping of actin filaments on a closed-loop track (width < 250 nm). The results are discussed in relation to lab-on-a-chip applications and nanotechnology-assisted assays of actomyosin function.
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| 9. |
- Torres, Nuria Albet, et al.
(författare)
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Drug effect unveils inter-head cooperativity and strain-dependent ADP release in fast skeletal actomyosin
- 2009
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:34, s. 22926-22937
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Tidskriftsartikel (refereegranskat)abstract
- Amrinone is a bipyridine compound with characteristic effects on the force-velocity relationship of fast skeletal muscle, including a reduction in the maximum shortening velocity and increased maximum isometric force. Here we performed experiments to elucidate the molecular mechanisms for these effects, with the additional aim to gain insight into the molecular mechanisms underlying the force-velocity relationship. In vitro motility assays established that amrinone reduces the sliding velocity of heavy meromyosin-propelled actin filaments by 30% at different ionic strengths of the assay solution. Stopped-flow studies of myofibrils, heavy meromyosin and myosin subfragment 1, showed that the effects on sliding speed were not because of a reduced rate of ATP-induced actomyosin dissociation because the rate of this process was increased by amrinone. Moreover, optical tweezers studies could not detect any amrinone-induced changes in the working stroke length. In contrast, the ADP affinity of acto-heavy meromyosin was increased about 2-fold by 1 mM amrinone. Similar effects were not observed for acto-subfragment 1. Together with the other findings, this suggests that the amrinone-induced reduction in sliding velocity is attributed to inhibition of a strain-dependent ADP release step. Modeling results show that such an effect may account for the amrinone-induced changes of the force-velocity relationship. The data emphasize the importance of the rate of a strain-dependent ADP release step in influencing the maximum sliding velocity in fast skeletal muscle. The data also lead us to discuss the possible importance of cooperative interactions between the two myosin heads in muscle contraction.
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| 10. |
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