A Molecular Flow Sensor

Overview

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Figure 1: β-Switch Region of GPIb

Ouch!! You cut your finger with a knife. Blood immediately starts coming out from the wound, and in a panic you search through your drawer for a band-aid. However, well before you can find the band-aid, and even before the "ouch" came out, just about the same time you sensed the pain, your body's self-healing mechanism was turned on. A multistep signaling cascade involving a dozen different proteins in the blood started immediately after the cut, calling for platelet cells to clog arround the wound and form a plug to stop the bleeding. How does our body sense a wound so fast? A possible answer is that the platelet cells carry on their surface a sensitive molecular flow sensor, a protein called GP1b, which senses erroneous blood flow caused by a cut in the blood vessel. It has been hypothesized that a small segment located on the alpha-subunit of GP1b, called the β-switch, transforms from a random coil to a β-hairpin in the presence of shear flow (see Figure 1). The β-switch, in its β-hairpin form, is able to bind to a protein called von Willerbrand factor, which will then anchor the platelet to the damage site. To test this hypothesis, we found a way to generate water flow in our computer simulation, mimicking real blood flow, and studied the behavior of the β-switch under flow.

Methods for Setting Up the Simulations

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Figure 2: Flow Control Technique

In most molecular dynamics simulations, proteins or other biomolecules are placed in a small box of water with periodic boundary conditions; so it is with our molecular flow sensor (see Figure 2). In order to generate constant water flow in a box like that, every water oxygen atom within a 3Å slab at the left end of the box was pushed along the x direction by a constant force f. The pushing force started the water flowing right away. However, this method alone will also cause the flow velocity to increase continuously, and before long the water molecules will be moving so fast that the protein is essentially being cooked. Our simple solution to this problem was to the couple a Langevin thermostat to every water oxygen atom. By doing that, each water molecule was subjected at any moment to a damping force proportional to its velocity. The pushing and damping forces acting on the water molecules quickly reached a balance after the start of the simulation, resulting in a nice uniform constant water flow. The velocity of the flow could by easily adjusted by changing either the applied force f or the damping coefficient of the thermostat. An extra bonus of using the thermostat for velocity control is that it also took care of the temperature at the same time, which is frequently a problem in many molecular dynamic simulations.

Results

betahairpin Figure 3: Steps towards β-hairpin formation.

To transform from a random loop to a structured β-hairpin, the β-switch needs to form six hydorgen bonds between its backbone. Not only do the backbone hydrogen bond partners have to correctly find each other, but almost all the side groups will also have to be reoriented to their correct position and pack tightly against each other for the h-bonds to form. Its almost like opening a combination lock with a long long code-----you must get every number right, or you're stuck. How can something as simple as uniform water flow induce such complicated conformational change in the β-switch? The answer, as we saw in our simulations, lies partly in the unique design of this tiny flow sensor, which allows it to perform the change in an ordered, stepwise manner, using the dragging force of the water flow. The flow-induced loop->β-hairpin transition of the b-switch was observed to happen in a stepwise manner consisting of two major steps, as schematically illustrated in Figure 3 to the right.

Step 1: Rotate

Shortly after the start of the flow, water molecules collided with the protein, dragging and extending the the protein's backbone forward. Upon extension, the backbone dihedral angles were forced to rotate in the direction that increases the length of the backbone. The β-switch is connected to the entire GP1b protein in such a way that this rotation motion enabled the potential backbone H-bond partners on the two opposite backbone segments to face each other. Moreover, side-groups rotated synchronously with the backbone. In several cases the backbone dihedral rotation caused some of the smaller side-groups to pass through the limited space between the two strands. Toward the end of this step, the backbone was fully extended and the orientation of all the side-groups were close to what they would be in the final b-hairpin state. At the same time, under the influence of the flow, the two strands of the β-hairpin started to rapidly approach each other allowing initially separated h-bond partners to come closer.

Step 2: Pack

In the second step of the transition, which immediately followed the first one, side-groups simultaneously searched for the right conformation to pack tightly with their nearest neighbors. The tight packing of side-groups is critical to the formation of stable H-bonds, since it allows the h-bond partners to stay close to each other. Under the influence of the flow, pairs of side-groups remained close to one another during the searching process. This ensures that whenever the suitable conformations were found, the side groups quickly pack with each other, and the correcponding h-bond is likely to be formed right away (a typical example is the packing involving bulky side chains of Met239, Trp230, and Gln232, which are highlighted in Figure 3, parts C and D). Once tight packing was achieved for most of the side-groups, all five H-bonds were formed, and the transition to the β-hairpin was completed. The β-hairpin is a stable conformation under flow, and breaking of individual H-bonds after the formation of all five of them was never observed.

What prevents spontaneous transtion from happening in the abscence of flow? Once we've understood the mechanism of the transition process, it seems that the unique design of the β-switch made the process relatively easy and automatic. One might worry that such an easy process will have a high rate of misfire, leading to unwanted blood clotting in healthy blood vessels. In fact, the rate of spontaneous transition in the abscence of flow is very low. The key to preventing misfire is entropy - or the number of possible conformations for a given state. As observed in our simulations under equilibrium condition, the β-switch is extremely flexible in its loop form, and can adopt a large number of different conformations. In contrast, there is essentially only one acceptable conformation in the β-hairpin state. The large difference in the number of accessible conformations indicates a significant entropic difference between the two states. Therefore, without the guidance of a unidirectional flow, the β-switch will almost always be sampling different loop conformation, and never find the right path to form β-hairpin. Its just like trying to find the band-aid in your terribly unorganized desk drawer - simply 'mission impossible'.

  • Click here for movies showing the extension of GP1b's loop to a beta-hairpin: movie1 (mpeg, 271 KB); movie2 (mpeg, 1.9 MB); movie3 (mpeg, 5.7 MB).

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