Using Atomic Force Microscopy to Aid in Study of Human Protein

Multi-department research team makes discovery that could influence investigation of treatment options for Duchenne Muscular Dystrophy

In a collaborative undertaking, scientists from Prof. Murti Salapaka’s research group, and multiple other University departments are the first to observe and report the mechanical characteristics of utrophin using atomic force microscopy (AFM). Data gathered in the study indicate differences in the mechanical properties of utrophin as compared to dystrophin that could influence its potential use in the treatment of Duchenne muscular dystrophy (DMD). The findings are reported in Nature’s Scientific Reports under the title, “Distinct Mechanical Properties in Homologous Spectrin-like Repeats of Utrophin.

Muscular dystrophy is a group of diseases that causes weakness and loss of muscle mass. Progressive in nature, DMD is the most common type of muscular dystrophy and tends to affect boys more than girls. Like other forms of muscular dystrophy, DMD is a genetic disorder caused by the absence of dystrophin, a protein that helps keep muscles intact, supporting muscle fiber strength, and maintaining stability. Dystrophin acts as a shock absorber within muscles and its absence causes muscle damage and progressive weakness, typically beginning in early childhood. DMD has devastating consequences for patients and their families. Most patients are wheelchair bound in childhood and because lack of dystrophin can affect muscles in the heart, diaphragm and other respiratory organs, it can impact heart health and lung function.

The published paper documents the first single molecule mechanical characterization of the spring-like utrophin. Utrophin is a homologue and is currently being investigated as a therapeutic replacement for dystrophin. (Utrophin, which stands for ubiquitous dystrophin, occurs in the human fetus during muscle development, but is later replaced by dystrophin at the sarcolemma after birth.)

The team studied the mechanical properties of expressed and purified full-length utrophin and relevant fragments using an atomic force microscope (AFM), at the level of a single molecule.

Sivaraman Rajaganapathy (Salapaka group) explains the workings of the AFM: “An AFM primarily consists of a sharp tip (25 nm) at the end of a flexible cantilever, whose deflection can be measured with nano-meter precision. This allows the application and measurement of very small forces (10-100s of pico Newtons) applied on single protein molecules.”

The current experiment involved stretching individual protein molecules between the tip of the AFM cantilever and a substrate, to extract the force versus extension characteristics of the molecules.

Jackie L. McCourt (Ervasti group) summarizes the significance of their work: “Homologous protein domains within utrophin displayed markedly different mechanical properties despite having almost identical thermodynamic properties. Another key finding was the unfolding forces measured for utrophin were significantly higher than forces previously reported for similar proteins, and this has implications for the physiological role of utrophin in muscle.”

The findings of the collaborative undertaking are significant, and potentially indicative of the road ahead. The cross-departmental team plans to continue the collaboration and move on to studying the mechanical characteristics of dystrophin, optimize protocols for precise AFM measurements, and develop a model for the unique mechanical properties observed in the single molecule force measurements.  

The research is a joint undertaking, with work being carried out by scientists from ECE’s Nanodynamics Lab (led by Prof. Murti Salapaka), BMBB’s Ervasti Lab (led by Dr. James Ervasti), and the Department of Rehabilitation Medicine.