Periodic Reporting for period 2 - ProForce (Mechano-Regulation of Proteins at Low Forces: Paving the Way for Therapeutic Interventions)
Reporting period: 2023-09-01 to 2025-02-28
Mechanical forces play critical roles in the regulation of biological functions. For example, the environment pushing and pulling on cells can change their development, affect their movement, and help seal wounds after injury. Incorrect responses to mechanical inputs can lead to human disease, including cancer and blood clots. In biological systems, proteins sense forces by undergoing changes in their 3D conformation when external forces act. These changes, in turn can trigger other signaling events.
Despite its importance, the mechanical regulation at the fundamental level of single-proteins remains poorly understood, in part due to a lack of suitable techniques. To understand mechanical regulation in proteins, we need to probe very small forces, down to 1 pN, i.e. 1/1,000,000,000,000 of the weight of a chocolate bar.
The project ProForce wants to understand the mechanical regulation at the level of individual proteins in the previously inaccessible regime of very low forces to, ultimately, develop ways to directly interfere with and correct incorrect responses to forces. By probing proteins involved in several human diseases, we want to better understand the molecular basis of disease and to develop approaches to treat them. For this purpose, use so-called magnetic tweezers, which are a powerful tool to manipulate individual molecules by attaching them with one end to a surface and with the other end to a magnetic bead. Using a microscope connected to a camera, not too different from the ones in smart phones, we can track many molecules at the same time, which helps to generate measurement statistics. We complement magnetic tweezers with other experimental approaches, including fluorescence, X-rays, and other imaging techniques. The aim of ProForce is to understand mechano-regulation at the single-protein level and to establish force response as a potential drug target.
Despite its importance, the mechanical regulation at the fundamental level of single-proteins remains poorly understood, in part due to a lack of suitable techniques. To understand mechanical regulation in proteins, we need to probe very small forces, down to 1 pN, i.e. 1/1,000,000,000,000 of the weight of a chocolate bar.
The project ProForce wants to understand the mechanical regulation at the level of individual proteins in the previously inaccessible regime of very low forces to, ultimately, develop ways to directly interfere with and correct incorrect responses to forces. By probing proteins involved in several human diseases, we want to better understand the molecular basis of disease and to develop approaches to treat them. For this purpose, use so-called magnetic tweezers, which are a powerful tool to manipulate individual molecules by attaching them with one end to a surface and with the other end to a magnetic bead. Using a microscope connected to a camera, not too different from the ones in smart phones, we can track many molecules at the same time, which helps to generate measurement statistics. We complement magnetic tweezers with other experimental approaches, including fluorescence, X-rays, and other imaging techniques. The aim of ProForce is to understand mechano-regulation at the single-protein level and to establish force response as a potential drug target.
So far, we have tackled several problems and achieved exciting results. In a first line of work, we have improved the magnetic tweezers methodology, by developing a new approach to determining the forces applied in the instrument, essentially providing a force gauge at the scale of individual molecules. The new approach, based on a mathematical quantity known as Hadamard variance, is particular insensitive to drift, which is always a problem in very precise measurements and occurs e.g. through slight changes in temperature. In related work, we have carried out measurements and developed a mathematical model that enables us to much better understand the flucuations in the instrument, i.e. tiny variations in the length of molecules causes by collision with water moelcules around them, in particular if DNA molecules are tethered in the magnetic tweezers.
In a second line work work, we have developed a new method to detect very small changes in the 3D structure of proteins. The method is based on attaching tiny gold particles that are approximately 1 nm in size (100,000 smaller than the diameter of a human hair) and determining their relative position using X-rays. Our approach can detect changes on the order of 0.1 nm, which is less than the size of a typical atom.
Finally, we have used magnetic tweezers to determine how the Coronavirus attaches to cells. For this purpose, we have developed an approach that has the tip of the Spike protein and the key protein from the human receptor ACE2 connected by a peptide linker. We then use magnetic tweezers to directly measure the strength of the virus-cell interaction. We find that SARS-CoV-2 (which causes COVID-19) can withstand higher forces compared to SARS-CoV-1 (which was responsible for the 2002/03 pandemic), which helps explain the different infection patterns of the two viruses. We then look at different mutated viruses, so-called variants of concern, and find differences in force stability that help rationalize the epidemiology of the different variants.
In a second line work work, we have developed a new method to detect very small changes in the 3D structure of proteins. The method is based on attaching tiny gold particles that are approximately 1 nm in size (100,000 smaller than the diameter of a human hair) and determining their relative position using X-rays. Our approach can detect changes on the order of 0.1 nm, which is less than the size of a typical atom.
Finally, we have used magnetic tweezers to determine how the Coronavirus attaches to cells. For this purpose, we have developed an approach that has the tip of the Spike protein and the key protein from the human receptor ACE2 connected by a peptide linker. We then use magnetic tweezers to directly measure the strength of the virus-cell interaction. We find that SARS-CoV-2 (which causes COVID-19) can withstand higher forces compared to SARS-CoV-1 (which was responsible for the 2002/03 pandemic), which helps explain the different infection patterns of the two viruses. We then look at different mutated viruses, so-called variants of concern, and find differences in force stability that help rationalize the epidemiology of the different variants.
We will continue to improve our methods to detect protein conformational changes and to apply and resolve forces in the magnetic tweezers. At the same time, we are currently pushing the applications of our approaches to additional proteins that are relevant for human health and disease.