Scientists envision many functions transferred to the molecular machines instead of man-made devices in the future. In order to create molecular machine mechanical stability of the protein complex has to be well understood.
Considering small size of the proteins (what is the range of sizes of the tertiary structure of proteins? (1 балл).

Q1 ::: Answer :::
The size of the protein ranges from ~ 2-5 nm (globular protein) to several micrometers (for example, elastin in sarcomere).

Atomic force microscope seems to be good tool for studies of the mechanical properties of the proteins, specifically unfolding. The size of the proteins implies that unfolding force will be quite small as well (what is the range of unfolding forces for different proteins?) (2 балла).

Q2 ::: Answer :::
Unfolding force for the protein depends on many factors with pulling speed being the main one. In general the range for unfolding forces accessible by AFM is ~40pN – 500 pN (for more details see A. Borgia, P.M. Williams, J. Clarke, “Single-molecules studies of protein folding”, Ann. Rev. of Biochem., 2008, 77, 101-125.). Depending on the rate and force unfolding pathways of the protein can be very different.

The standard experiment for measuring protein unfolding is approaching surface with adsorbed proteins with microscope tip, until tip touches the surface, then pull the tip off the surface (please draw schematically force curve as function of tip displacement in experiment described above, consider two cases: molecule/no molecule between tip and the surface. Please describe physical processes happening at each step. (4 балла).

Q3 ::: Answer :::
The process of tip approach is presented on the figure 1. Far away from the surface only long range Van-der-Waals (VdW) forces are acting between tip and the surface. As tip approaches the surface the magnitude of long-range VdW increases (see region B on the image, brown curve). When tip is close enough to the surface, it snaps into contact with the surface (brown curve). While in contact short range VdW forces are acting between tip and the surface (brown curve). Let’s assume no molecule situation: when tip is retracted from the surface (blue curve), the contact force is responsible for the increase in the force on the region D. As soon as pulling force exceeds contact force the force on the tip zeros and the tip will continue to follow (brown curve). So the retraction curve will look like blue curve in the range from 400 nm to 700 nm, than it will continue as brown curve. Let’s assume that tip picks up molecule: the tip will still follow blue curve in the distance ranges between 400 – 700 nm. Then protein starts to stretch (700 – 750 nm), protein unfolds at ~ 750nm. Number of domains in the protein determines the maximum number of unfolding cycles. Sometimes protein detaches from either tip or surface.

As an experimentalist in this project you have to work with small displacements and small forces, what internalstandard you would use to calibrate your system (please list several types of proteins you would use. (2 балла).

Q4 ::: Answer :::
There are several requirements for the proteins to be standards for protein unfolding: reproducible unfolding pattern, known parameters of domain lengths and unfolding forces for selected protein, the presence of multiple domains in protein structure. Well known structure of protein helps to correlate unfolding events with the change in protein structure. Usually titin I27 is used as standard. Sometimes it is beneficial to have protein of similar structure as the sample of interest and use that one as standard. For example for filamentous protein spectin might be a good standard, because its unfolding curve is well known.

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