Construction of Polyproteins for use in Atomic Force Microscopy Studies

Client Mayo Clinic, Rochester, MN, Dr. Julio Fernandez, Principal Investigator

Engineering a polyprotein

The atomic force microscope (AFM) is a remarkably simple instrument. Originally used for measuring surface contours of individual molecules, it has provided important information about the structure of proteins. In the force-measuring mode, AFM is capable of measuring forces down to piconewtons and can resolve force changes caused by the displacement of its probe by a fraction of a nanometer¹. Single molecule measurements are routinely done (see Fisher et al. for a review²). Since measurements can be made on many individual molecules in a short period of time, a statistical evaluation of the measurements can be performed. Perhaps the most powerful aspect of AFM is that the measurements can be done in an aqueous environment, allowing for the study of biological material under conditions that resemble those in vivo. The folding and unfolding of the protein can be studied in the presence of substrate, product, and co-factors. The temperature can be controlled as can the buffer conditions, i.e., ionic strength, pH, solvents, etc.

The AFM functions like a miniature phonograph. A sharp tip mounted on a cantilever interacts with the sample, causing minute deflections that can be calibrated as a force. The design of our AFM is shown in Figure 1. An example of how we have applied AFM to the analysis of a protein is described in detail below. In a typical experiment, the protein sample is placed on a gold-coated cover slip that is attached to a piezoelectric positioner. We engineered recombinant proteins so that one end (the C-terminus) is anchored to the gold substrate. Random segments of the protein (titin) are then picked up by adsorption to the AFM tip and stretched for up to several hundred nanometers (Dz_p, Fig. 1B). When the protein is stretched, it pulls on the cantilever causing small cantilever deflections (Dz_c) that can be converted into units of force using the cantilever spring constant, k_c (Fig. 1C). As the protein is stretched the force increases in a non-linear (non-Hookean) fashion; this is the energy required to decrease the degrees of freedom of the polypeptide (an entropic-spring behavior that is a common property of polymers). If the protein is further stretched the probability for the unfolding of individual domains becomes higher. When a domain unfolds, the AFM tip travels back to its resting position and the force goes back to zero (Fig. 1B and 1C).

Figure 1.
A) Schematics of our custom built atomic force microscope. The AFM was constructed using a Digital Instruments (DI, Santa Barbara, CA, USA) AFM detector head (AFM-689) mounted on top of a Physik Instrumente (PI; Waldbronn, Germany) single axis piezoelectric positioner. The PI positioner has a capacitative sensor resulting in a Z axis resolution of 0.1 nm (P-732.ZC). The data acquisition (Force) and the voltage control of the movement of the piezoelectric positioner (Dz_p) are done by means of a PC-mounted data acquisition board (AT-MIO-16X; National Instruments) and controlled by custom made LabView (NI) software. The spring constant, k_c, of each individual AFM tip (Si₃N₄ tip NPS, DI) is calibrated in solution, before each experiment. K_c varies between 30-120 mN/m. The force is measured by the deflection of the cantilever and the extension can be calculated from the piezo travel (Dz_p).

B) Stretching and unfolding Ig-type domains with AFM. 1) shows an unstretched polypeptide with 4 Ig domains adsorbed to an AFM tip; 2) the stretching of the protein requires force and this is monitored as a deflection of the cantilever; 3) the unfolding of a domain increases the protein length, relaxing the cantilever back to its resting position.

C) Idealized force-extension curve for the stretching of a multi-domain polypeptide and the subsequent unfolding of a single domain. The numbers correspond to the stages marked in B.

Construction of a polyprotein is necessary because the mechanical properties of a single module could not be studied using current AFM techniques. A single module is only 89 residues and will extend only for a short distance. The length of a single domain falls into a region where we always observe a large amount of non-specific interactions between the AFM tip and the substrate (<30nm). In contrast, tandem repeats of many modules extend well beyond the region of nonspecific interactions and generate periodical patterns that amplify the features of the individual modules and allow for a high signal-to-noise ratio. One of the aims of our research has been to reduce the interference from non-specific interactions.

Recombinant DNA techniques were used to construct direct tandem repeats of a single Ig domain from I band titin (I27). Two methods were employed to synthesize and express recombinant multimers of I27, with similar results. One of them was a multiple step cloning technique modified from a previously described method³ that makes use of four restriction sequences (BamHI, BglII, SmaI and KpnI ) to build even multiples of I27. This approach adds two new amino acids (Arg and Ser) to the I27 sequence such that the repeated monomer becomes [I27-RS]_n. Using this method we have constructed a protein composed of eight tandem repeats (I27^RS₈ , Fig. 2).

Figure 2.Construction of poly-I27 proteins.
A) Agarose gel stained with ethidium bromide showing the size of the I27-RS multiples (right lane).

B) Coomassie blue staining of the purified I27^RS₈ protein (~90 kDa) separated by SDS-PAGE.

C, D) Summary of the sequence of the I27^RS₈ and I27^LG₁₁ constructs.

The second method was based on single step cloning of high order multimers into a custom-made expression vector^4,5. This method relies on a single type of restriction site, AvaI, to build linear multimers of the I27 monomer. The AvaI sequences add two new amino acids (Leu and Gly) to the I27 sequence such that the repeated monomer becomes [I27-LG]_n. Using this method we constructed a protein composed of eleven tandem repeats (I27^LG₁₁, Fig. 2D). Expanding on this theme, we have since constructed polyproteins for the titin domains I28 through I34 including an alternating I27-I28 construct. Each was expressed in E. coli, purified by Ni-affinity chromatography, and subjected to AFM analysis.