The thioamide is a compelling isostere of the peptide backbone. The altered chemical and physical properties provide the opportunity to stabilize, probe, and thereby study peptide and protein systems via a single atom substitution. The thioamide is also a naturally occurring modification. With only two characterized proteins with thioamide post-translational modifications, the biological function of the thioamide remains unknown. To determine both the role of the thioamide in nature, as well as to promote its use as a biophysical probe, a better understanding of the fundamental impact of thioamide incorporation on protein structure is required. Thioamides have different hydrogen-bonding properties than oxoamides and because beta-sheet proteins are composed of intricate networks of hydrogen bonds, they provide a biologically relevant system in which to study the structural impact of thioamides in proteins. In this work, I describe improved methods to produce thioamide-containing beta-sheet proteins and investigate the structural impact with nuclear magnetic resonance (NMR) spectroscopy and other biophysical techniques. Improved methods include increasing the scale of semi-synthesis by 10-fold and demonstrating that thioamides are compatible with Knorr pyrazole thioester formation for native chemical ligation (NCL). To understand the structural impact in proteins, I first incorporated thioamides into a minimal beta-sheet system, the beta-hairpin, and analyzed the structural impact with NMR. To generate and study thioamide-containing proteins more rapidly, a host-guest strategy is outlined where I use the SpyCatcher003/ SpyTag003 complex to study the impact of thioamide location on the rate of complex ligation. Thioamide incorporation tailored the rate of ligation and even resulted in a faster ligation than wild type. In both the beta-hairpin and the SpyCatcher systems, we find that the effect of the thioamide was dependent on the microenvironment of the residue, so that simple rules are not sufficient. The data gathered here represent early steps toward a structure-based predictive model on the effect of thioamide substitution on protein or peptide structure.