# Protein dynamics under force

> **NIH NIH R35** · COLUMBIA UNIV NEW YORK MORNINGSIDE · 2020 · $392,555

## Abstract

Protein dynamics under force
My laboratory is focused on understanding protein dynamics under force, of common occurrence in biology.
Over the last 20 years of NIH funding, we have pioneered the development of protein engineering and force
spectroscopy techniques that we have used to study muscle proteins, proteins and polysaccharides of the
extracellular matrix, and since 2015, a new focus on studying proteins involved in bacterial adhesion. This
MIRA proposal will unify our current studies of protein mechanics under a single funding mechanism.
The most important discovery of force-spectroscopy over the past 20 years is that proteins do mechanical work
when they fold against an opposing mechanical force. For example, the amount of mechanical work done by a
folding titin Ig domain can be 2-3 times larger than that of the chemically powered motor. Given that titin is the
third filament of muscle, determining the role played by titin folding in the force generated by a contracting
muscle is a scientific question of fundamental significance. Many key questions need to be answered before
concluding that titin folding is a significant contributor to the mechanical work done by a contracting muscle.
This MIRA proposal will give us the flexibility to attempt to answer them. For example, a key prediction of our
model is that activation of myosin II motors by Ca++ leads to a drop in the force experienced by titin, triggering
delivery of work by folding. This proposal focuses on further developing a set of novel tools that we have
designed to directly answer such questions. The outcome of our efforts is likely to be a revolution in our
understanding of the molecular mechanisms of muscle contraction.
Since 2015, a parallel research track in my laboratory has been the study of Gram-positive pili, giant single
polypeptide proteins composed of hundreds of Ig like repeats that resemble the overall design of titin. In sharp
contrast to the inherent extensibility of titin, pili incorporate isopeptide bonds that prevent its mechanical
unfolding and resist the large forces experienced by bacteria during colonization, biofilm formation, and
infection. Since the discovery of the isopeptide bond in the shaft pilin of S. pyogenes in 2007, numerous new
structures are being reported for other Gram-positive pili. Under a mechanical load however, the structure of
the pilin proteins will rapidly change, altering its antigenic surfaces and exposing new ones. This present a
novel opportunity, well adapted to force-spectroscopy techniques, for identifying peptides that can bind to and
destabilize Gram-positive pili rendering them susceptible to proteolytic degradation. This MIRA proposal is
aimed at developing rapid assays, based on force spectroscopy, for blocking isopeptide formation in bacterial
shaft pilins, thereby using pilin mechanics to greatly expand the possibility for rationally designed antibiotics.

## Key facts

- **NIH application ID:** 9857051
- **Project number:** 5R35GM129962-02
- **Recipient organization:** COLUMBIA UNIV NEW YORK MORNINGSIDE
- **Principal Investigator:** Julio M Fernandez
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $392,555
- **Award type:** 5
- **Project period:** 2019-03-01 → 2021-02-01

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9857051

## Citation

> US National Institutes of Health, RePORTER application 9857051, Protein dynamics under force (5R35GM129962-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9857051. Licensed CC0.

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