Data from: Nanomechanics of wild-type and mutant dimers of the tip-link protein protocadherin 15
Data files
Oct 26, 2023 version files 87.78 GB
Abstract
Mechanical force controls the opening and closing of mechanosensitive ion channels atop the hair bundles of the inner ear. The filamentous tip link connecting transduction channels to the tallest neighboring stereocilium modulates the force transmitted to the channels and thus changes their probability of opening. Each tip link comprises four molecules: a dimer of protocadherin 15 and a dimer of cadherin 23, all of which are stabilized by Ca2+ binding. Using a high-speed optical trap to examine dimeric PCDH15, we find that the protein's configuration is sensitive to Ca2+ and that the molecule exhibits limited unfolding at a physiological Ca2+ concentration. PCDH15 can therefore modulate its stiffness without undergoing large unfolding events in physiological Ca2+ conditions. The experimentally determined stiffness of PCDH15 accords with published values for the stiffness of the gating spring, the mechanical element that controls the opening of mechanotransduction channels. When PCDH15 has a point mutation, V507D, associated with non-syndromic hearing loss, unfolding events occur more frequently under tension and refolding events occur less often than in the wild-type protein. Our results suggest that the maintenance of appropriate tension in the gating spring is critical to the appropriate transmission of force to transduction channels, and hence to hearing.
README: Nanomechanics of wild-type and mutant dimers of the tip-link protein protocadherin 15
Camila M. Villasante1, Xinyue Deng1, Joel E. Cohen2,3,4, and A. J. Hudspeth1,5
1Laboratory of Sensory Neuroscience, The Rockefeller University, New York, NY 10065 USA; 2Laboratory of Populations, The Rockefeller University, New York, NY 10065 USA; 3Earth Institute and Department of Statistics, Columbia University, New York, NY 10027 USA; 4Department of Statistics, University of Chicago, Chicago, IL 60637 USA; 5Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065 USA
Dryad DOI: https://doi.org/10.5061/dryad.stqjq2c8s
Associated with bioRxiv pre-print: https://doi.org/10.1101/2023.10.17.562769
Description of the data and file structure
All datasets underlying the work in Nanomechanics of wild-type and mutant dimers of the tip-link protein protocadherin 15 are included in this repository. There are three constructs: wild-type PCDH15, V507D PCDH15, and the anchors only. There are three buffer conditions: saturating (3 mM) Ca2+, physiological (20 uM) Ca2+, and no Ca2+ (1 mM EDTA).
Each dataset is an .hdf file containing a dictionary. The keys of the dictionary are: 'ywavecut', 'forcewavecut', 'ywavesmooth', and 'forcewavesmooth', and each corresponds to a Numpy array.
'ywavecut': end-to-end distance data (nm) that has been calibrated and the zero position adjusted to be three standard deviations below the mean position of the construct at the low inter-ramp resting force. The time range includes all cycles from a given dataset. The temporal resolution of the data is 10 μs.
'forcewavecut': force data (pN) that has been calibrated. The time range is the same as ywavecut. The temporal resolution of the data is 10 μs.
'ywavesmooth': end-to-end distance data (nm) that is the same as 'ywavecut' except it has been smoothed with a Savitzky-Golay filter with a window of 101 points, which reduced the temporal resolution of the data from 10 μs to 1 ms.
'forcewavesmooth': force data (pN) that is the same as 'forcewavecut' except it has been smoothed with a Savitzky-Golay filter with a window of 101 points, which reduced the temporal resolution of the data from 10 μs to 1 ms.
Code/Software
Scripts used to analyze this data are located in https://github.com/cvillasante/VDCH-2023.
Methods
These are force-extension datasets collected using a custom-built optical trap system. Custom written LabVIEW (2013) software was used to acquire the raw data. It has been calibrated and the zero position adjusted to correspond to three standard deviations below the mean position of the construct at the inter-ramp resting force of 1 pN.