Single Molecule Force Spectroscopy Reveals Critical Roles of Hydrophobic Core Packing in Determining the Mechanical Stability of Protein GB1

Abstract

Understanding molecular determinants of protein mechanical stability is important not only for elucidating how elastomeric proteins are designed and functioning in biological systems but also for designing protein building blocks with defined nanomechanical properties for constructing novel biomaterials. GB1 is a small α/β protein and exhibits significant mechanical stability. It is thought that the shear topology of GB1 plays an important role in determining its mechanical stability. Here, we combine single molecule atomic force microscopy and protein engineering techniques to investigate the effect of side chain reduction and hydrophobic core packing on the mechanical stability of GB1. We engineered seven point mutants and carried out mechanical φ-value analysis of the mechanical unfolding of GB1. We found that three mutations, which are across the surfaces of two subdomains that are to be sheared by the applied stretching force, in the hydrophobic core (F30L, Y45L, and F52L) result in significant decrease in mechanical unfolding force of GB1. The mechanical unfolding force of these mutants drop by 50-90 pN compared with wild-type GB1, which unfolds at around 180 pN at a pulling speed of 400 nm/s. These results indicate that hydrophobic core packing plays an important role in determining the mechanical stability of GB1 and suggest that optimizing hydrophobic interactions across the surfaces that are to be sheared will likely be an efficient method to enhance the mechanical stability of GB1 and GB1 homologues.

Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

“…Single-molecule protein unfolding experiments using (I27) 5 were completed at four different pulling speeds (160, 400, 1000, and 2000 nm s −1 ). Each experimental forceextension trace contains between one and five unfolding events.…”

“…(I27) 5 polyprotein constructs were expressed and purified according to methods described previously [58]. All domains in the protein constructs were assumed to be folded under the conditions used in the AFM experiments, based on characterization of the stability of (I27) 5 described elsewhere [58].…”

“…Single-molecule force spectroscopy is a commonly used tool for the mechanical characterization of polymers and biological molecules, such as proteins and DNA [1][2][3][4][5][6][7][8][9][10], and ligand-protein interactions [11][12][13][14][15][16][17][18][19]. These experiments can be performed using a variety of experimental setups, including optical tweezers [3,20], the atomic force microscope (AFM) [1,4], magnetic tweezers [21,22], and the biomembrane force probe [23].…”

“…2. Four schematics depicting possible reasons why five unfolding events for (I27) 5 polyprotein constructs are not always seen, despite every polyprotein construct containing five domains of I27. In the first schematic, five unfolding events should be seen; in the second, due to two domains being attached nonspecifically to the surface, only three unfolding events will be seen; in the third schematic, only three domains are between the cantilever tip and the surface due to the position at which the polyprotein construct has been picked up; and in the fourth schematic, the polyprotein construct detaches from the cantilever tip before all of the I27 domains have unfolded.…”

“…So, while the MC simulation that is used to model the data assumes an equal distribution of unfolding events, each experiment will have a unique distribution that is unlikely to be equal. Here, we complete single-molecule experiments on the polyprotein (I27) 5 and measure the probability distribution of unfolding events ranging from two to five domains. Next, we consider the effect of the number of unfolding events observed for a polyprotein on the measured unfolding force for each individual protein domain in the polyprotein chain at a range of pulling speeds.…”