Inner ear hair cells mediate our senses of hearing and balance by transforming mechanical stimuli from sound and head movements into electrochemical signals that can be processed by the brain. At the core of hair cell function are tip links, fine protein filaments essential for normal hearing and that are involved in inherited deafness. Two enormous cadherin proteins have been shown to form the mature tip link (cadherin-23 and protocadherin-15), which is thought to be a straight filament with tightly, bound calcium ions that give it rigidity and strength. Transient, immature protocadherin-15 tip link variants have been suggested to be important during development and tip link regeneration after noise-induced damage. There might be additional physiologically relevant variants formed by multiple isoforms of cadherin-23 and protocadherin-15. The overall goal of this project is to determine the biophysical and biochemical behavior of tip links when exposed to different calcium concentrations, when made by different protein variants, and when they carry deafness-related mutations. In aim 1, force spectroscopy and various biophysical and biochemical methods will be used to determine how changing calcium concentration modifies tip link properties and how non-canonical calcium binding sites of protocadherin-15 alter its elasticity. In aim 2, x-ray crystallography and computational modeling will be used to obtain and characterize models of tip link variants involving all extracellular isoforms of cadherin-23 and protocadherin-15. In aim 3, various methods will be used to determine the biochemical and biophysical consequences of missense mutations and deletions associated with inherited deafness, both to explain the severity of observed phenotypes and to predict the effects of yet to be found mutations and deletions in tip link proteins. Results of this project will provide a clear molecula view of tip link properties and function under various physiologically relevant circumstances, and may inform treatment strategies and molecular therapies for inherited deafness.