Cardiac muscle contraction is regulated by Ca2+ binding to the N-domain of troponin C (cNTnC). Following Ca2+ association, the relocation of troponin I from actin to troponin C triggers contraction. In a diseased heart, there is a desensitization of the myocardium for Ca2+, and one treatment strategy is to use pharmaceuticals that stabilize the open conformation of cNTnC, and thus enhance its interaction with troponin I (cTnI147-163). Another option would be to engineer variants of troponin C that resemble the drug-induced open state of cNTnC. One possible mutant, L48Q, has been shown to increase thin filament Ca2+-sensitivity. L48 is involved in forming crucial hydrophobic interactions with F20 and A23 in both the apo and Ca2+-bound forms of cNTnC. The replacement of leucine with glutamine decreases the hydrophobicity in this region, and therefore may destabilize the closed state of cNTnC. We used nuclear magnetic resonance (NMR) to investigate how the L48Q mutation might increase thin filament Ca2+-sensitivity. We found that the affinity of L48Q-cNTnC for cTnI147-163 was enhanced by ∼3 fold, with a KD ∼ 50 ∈μM (wt-cNTnC; KD ∼ 150 ∈μM). We have developed a computational method to predict the tertiary structural changes in cNTnC by comparing the 1H,15N - HSQC spectra with control spectra from open and closed forms of cNTnC. The chemical shift patterns of residues in the defunct Ca2+-binding site I of L48Q-cNTnC resemble the cTnI147-163-bound form of wt-cNTnC, indicative of a more open state. We conclude that the L48Q mutation disrupts the hydrophobic packing of cNTnC such that it stabilizes a more open state of cNTnC, and it is this structural perturbation that is responsible for the enhanced affinity of L48Q-cNTnC for cTnI147-163.