Periodic Reporting for period 2 - ArtMotor (Artificial Motor Proteins: toward a designed, autonomous protein motor built from non-motor parts)
Reporting period: 2023-03-01 to 2024-08-31
Molecular motors and machines are essential for all cellular processes that together enable life. Much structural and biophysical information on their function has been acquired and has enabled the development of models for motor function. However, it remains difficult to discern detailed mechanisms, for example about the relative role of different force generation mechanisms, or how information is communicated across a protein to achieve the necessary coordination.
The purpose of ArtMotor is to design and build functional, synthetic protein motors capable of moving and transducing energy, based on existing, non-motor protein modules of known molecular function. By combining approaches from computational protein design, structural and molecular biology, and single-molecule detection, we aim to (a) construct relatively simple protein motors that will require external control, and (b) construct, step by step, an autonomous protein motor capable of moving along a track.
The overarching objectives of ArtMotor are to generate new insights into mechanisms of energy transduction in proteins, and to help inspire complex protein designs that may lead to advances in fields from enzyme design to nano-engineering.
The purpose of ArtMotor is to design and build functional, synthetic protein motors capable of moving and transducing energy, based on existing, non-motor protein modules of known molecular function. By combining approaches from computational protein design, structural and molecular biology, and single-molecule detection, we aim to (a) construct relatively simple protein motors that will require external control, and (b) construct, step by step, an autonomous protein motor capable of moving along a track.
The overarching objectives of ArtMotor are to generate new insights into mechanisms of energy transduction in proteins, and to help inspire complex protein designs that may lead to advances in fields from enzyme design to nano-engineering.
Our major drive has been towards the realization of a simple protein motor that requires external control, a so-called clocked walker that we call the tumbleweed (TW). The aim is to observe and measure walking of TW on a DNA track in response to added ligands. To this end, we have refined our TW design to make it better fit for walking. We have improved our DNA track design so as to balance the response of the three distinct feet to their respective ligands. Finally, we are close to eliminating an unwanted side reaction that may stall TW. Two measures of walking along a DNA track are coming together: bulk SPR measurements of TW binding to DNA as we change the ligands; and single molecule measurements indicating that TW takes steps in response to ligands.
In parallel, we have made advances on two autonomous, bipedal walkers, one based on the TrpR repressor (also used in TW) and the other based on the LacI repressor. With TrpR, we can express the required single chain constructs to make a simple foot. We are altering the individual DNA-binding helices and the DNA track so as to achieve directional binding (asymmetry). With the LacI design, we have created monomeric forms and we are testing heterodimeric forms so as to achieve symmetry in track binding. For LacI, we are at the stage of designing enzymatic sites to control binding. The designs are at an advanced stage, in silico. We will be screening for expression and enzymatic activity shortly.
In parallel, we have made advances on two autonomous, bipedal walkers, one based on the TrpR repressor (also used in TW) and the other based on the LacI repressor. With TrpR, we can express the required single chain constructs to make a simple foot. We are altering the individual DNA-binding helices and the DNA track so as to achieve directional binding (asymmetry). With the LacI design, we have created monomeric forms and we are testing heterodimeric forms so as to achieve symmetry in track binding. For LacI, we are at the stage of designing enzymatic sites to control binding. The designs are at an advanced stage, in silico. We will be screening for expression and enzymatic activity shortly.
In work under review for publication (preprint available at https://arxiv.org/abs/2109.10293) we report the synthesis and characterization of the Lawnmower, an autonomous, protein-based artificial molecular motor comprised of a spherical hub decorated with proteases. To our knowledge, this is the first artificial molecular motor completely based on proteins. Its “burnt-bridge” motion is directed by cleavage of a peptide lawn, promoting motion towards unvisited substrate. We find that Lawnmowers exhibit directional motion with average speeds of up to 80 nm/s, comparable to biological motors. By selectively patterning the peptide lawn on microfabricated tracks, we furthermore show that the Lawnmower is capable of track-guided motion. We believe that this is the first time an artificial motor is guided by a nanofabricated track.