Beyond defining the structure and stability of folded states of proteins, primary amino acid sequences determine all of the features of their conformational landscapes. Characterizing how sequence modulates the population of protein excited states or folding pathways requires atomic level detailed structural and energetic information. Such insight is essential for improving protein design strategies, as well as for interpreting protein evolution. Here, high pressure NMR and molecular dynamics simulations were combined to probe the conformational landscape of a small model protein, the tryptophan cage variant, TcSb. Pressure effects on protein conformation are based on volume differences between states, providing a subtle continuous variable for perturbing conformations. 2D proton TOCSY spectra of TcSb were acquired as a function of pressure at different temperature, pH, and urea concentration. In contrast to urea and pH which lead to unfolding of TcSb, pressure resulted in modulation of the structures that are populated within the folded state basin. The results of molecular dynamics simulations on TcSb displayed remarkable agreement with the NMR data. Principal component analysis identified two structural subensembles in the folded state basin, one of which was strongly destabilized by pressure. The pressure-dependent structural perturbations observed by NMR coincided precisely with the changes in secondary structure associated with the shifting populations in the folded state basin observed in the simulations. These results highlight the deep structural insight afforded by pressure perturbation in conjunction with high resolution experimental and advanced computational tools.