Icon for: Patrick Sims

PATRICK SIMS

University of California at Irvine
Years in Grad School: 4
Judges’
Choice

Judges’ Queries and Presenter’s Replies

  • Icon for: Qiaobing Xu

    Qiaobing Xu

    Judge
    May 20, 2013 | 11:32 p.m.

    excellent work! will the ionic strength affect the measurement? how does the enzyme constant measured by single molecule method here compare with that reported in literature (the bulk enzyme behavior)?

  • Icon for: Patrick Sims

    Patrick Sims

    Presenter
    May 21, 2013 | 06:31 p.m.

    Dear Prof. Xu,

    Thank you for the questions.

    Varying the ionic strength was one of the first experiments we tried in order to ascertain and convince ourselves that the transduction mechanism of the signal was an electrostatic effect. The hypothesis was that by varying the ionic strength or Debye length we should observe changes in the strength of the signal due to screening of charges near the nanotube. When we performed these measurements, we saw at progressively higher ionic strengths a decrease in the switching signal, and at progressively lower ionic strengths, we saw an increase in the signal. To further elucidate the electrostatic mechanism, we then created variants of lysozyme with differing charged amino acids in the vicinity of the attachment site to the nanotube in order to see how these changes would affect the signal strength. For more information on the ionic strength measurement, you can see our paper “Dissecting Single-Molecule Signal Transduction in Carbon Nanotube Circuits with Protein Engineering.” http://pubs.acs.org/doi/abs/10.1021/nl304209p

    The average behavior of binding, unbinding, and catalytic rates for a collection of single molecules using this technique compare closely with those of bulk experiments as well as with those found using other single molecule fluorescence-based techniques such as FRET. Nevertheless, the rates do vary from molecule to molecule due to static heterogeneity, i.e., slight differences present in genetically equivalent enzymes. The rates also do vary over the course of ten minute measurements due to time-dependent variations that we have attributed to dynamic disorder, i.e, slow fluctuations of the protein conformation.

  • May 21, 2013 | 04:15 p.m.

    I have a rather naive question. I understand that the charge of the protein at the binding site leads to a gating of the SWNT field effect transistor and this is the source behind the signals you measure. But it is not clear to me if we can indeed back out conformational mechanisms behind the action of the protein from the read out of the signal and if one can, how do we go about doing this. Could you explain this a little in the context of the lysozyme for example?

  • Icon for: Patrick Sims

    Patrick Sims

    Presenter
    May 21, 2013 | 06:32 p.m.

    Dear Prof. Baskaran,
    Thank you for the question.
    I will do my best to clarify. The gating is due to the movement of charges of the protein near the attachment site to the nanotube. From x-ray crystallography data and from single molecule fluorescence measurements, lysozyme has been shown to undergo a hinge-bending conformational change when performing catalysis where the hinge closes when peptidoglycan binds to the active site of the enzyme. Using x-ray crystallography data and sequence information, we selected charged amino acids in the vicinity of the attachment site that underwent large positional changes between the opened and closed state of the enzyme. By creating variants of lysozyme where these amino acids were negative, neutral, and positive, we showed that they were the key players in signal transduction. Furthermore, in the absence of peptidoglycan, this hinge-bending does not occur and the two-level switching signal is absent. From structural knowledge and from the measurements of these lysozyme variants, it suggests that the movement of these amino acids with respect to the nanotube upon hinge opening and closing cause the two-level switching signal. Without any previous knowledge from x-ray or in some cases NMR studies, it would have been difficult to back-out the conformational mechanism from just the two-level switching signal.

  • May 21, 2013 | 07:54 p.m.

    Very interesting work.
    I am not specialist in this field, and my question is probably silly. If you can see the electric signal related to enzyme activity, would be possible to do the opposite, affect the processes with electric means?

  • Icon for: Patrick Sims

    Patrick Sims

    Presenter
    May 21, 2013 | 09:00 p.m.

    Dear Prof. Noginova,

    Thank you for the question.

    This is an excellent point. In fact, we are actively exploring this question and trying to use time-varying electric fields to perturb enzymes and drive their mechanical motions. We have some preliminary, unpublished results, but we are still figuring out all the details.

  • Icon for: Qi-Huo Wei

    Qi-Huo Wei

    Judge
    May 21, 2013 | 10:03 p.m.

    Beautiful work. Do the CNT FET sensors vary in sensitivity from device to device (depending on SD distance, CNT property), and does it matter to your results?

  • Icon for: Patrick Sims

    Patrick Sims

    Presenter
    May 22, 2013 | 03:52 p.m.

    Dear Prof. Wei,

    Thank you for the question.

    The CNT FET sensors do vary from device-to-device due to properties inherent to the nanotube and due to experimental variability in the fabrication of these types of devices. Because of this variability, each device can have a different amount current flowing at a set source-drain voltage bias. Nevertheless, we have found that the sensitivity of the device is determined by the transconductance of the device, i.e, how the current varies with gate-voltage or an applied external electric field. This allows us to map the current levels of the switching signals to the corresponding value of gate-voltage that would give that current for each device. While the currents differ from device-to-device, this change in gate voltage tends to depend only on the enzyme attached to the nanotube device. This provided us with a metric that we used to assess and compare the response of a collection of devices to differently charged lysozyme variants.

  • May 21, 2013 | 11:29 p.m.

    I have a naive question. How can you control the number of enzyme bound to the SWNT?

  • Icon for: Patrick Sims

    Patrick Sims

    Presenter
    May 22, 2013 | 03:55 p.m.

    Dear Prof. Kong,

    Thank you for the question.

    Controlling the number of enzymes bound to the SWNT required a considerable amount of empirical testing and calibration. On average, we get an enzyme attachment for every micron of exposed nanotube. The exact procedure slightly varies for different types of enzymes, but the basic framework of the protocols is conserved. We first soak the device in a solution of a bifunctional linker molecule pyrene maleimide in ethanol for about 30 minutes. We then use a series of washes that range from 10 to 30 minutes using a surfactant such as Tween-20 to remove excess pyrene maleimide from the surface. Next, we soak the device in a solution containing the enzyme we want to attach for about an hour. Then, we perform another series of washes containing a surfactant to remove non-selectively adsorbed protein from the surface. We then image the devices using atomic force microscopy in liquid to confirm protein attachment to the nanotube. To get the desired attachment density, we empirically tailored the concentration of the enzyme in solution as well as the wash steps. For all the details involved in the protein conjugation for lysozyme, you can check out the supporting information of this paper “Single-Molecule Lysozyme Dynamics Monitored by an Electronic Circuit”
    http://www.sciencemag.org/content/335/6066/319/...

  • Further posting is closed as the competition has ended.

Presentation Discussion
  • Small default profile

    Dr. Arthur

    Guest
    May 21, 2013 | 07:37 p.m.

    Interesting application of carbon tubes, this technique may have the power to determine structural dynamics of proteins in a manner that is new to science. Go for it.

  • May 23, 2013 | 12:51 p.m.

    Great job with the video, Patrick!

  • Icon for: Patrick Sims

    Patrick Sims

    Presenter
    May 23, 2013 | 09:49 p.m.

    Thanks!

  • Icon for: Geoffrey Harlow

    Geoffrey Harlow

    Trainee
    May 23, 2013 | 07:21 p.m.

    This was an excellent video, I especially liked the Pac Man analogy and animation. But what is the metaphorical cherry that causes the lysozyme to speed up? I like your enthusiasm, this was cool.

  • Icon for: Patrick Sims

    Patrick Sims

    Presenter
    May 23, 2013 | 10:06 p.m.

    Thank you and thanks for the question.

    The metaphorical cherry, at least for lysozyme, are cross-links of the substrate. For substrates where these cross-links are present (e.g. pieces of bacterial cell wall), lysozyme switches to a rapid, non productive state in order transverse over a cross-link. This allows lysozyme to zig-zag across a bacterial cell wall as it breaks it apart.

  • Further posting is closed as the competition has ended.