Proteins tethered to solid supports are of increasing interest in bioanalytical chemistry and protein science in general. However, the extent to which tethering modifies the energy landscape and dynamics of the protein is most often unknown because there are few biophysical methods that can determine secondary and tertiary structures and explore conformational equilibria and dynamics of a tethered protein with site-specific resolution. Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) offers a unique opportunity for this purpose. Here, we employ a general strategy using unnatural amino acids that enables efficient and site-specific tethering of a spin-labeled protein to a Sepharose solid support. Remarkably, EPR spectra of spin-labeled T4 lysozyme (T4L) reveal that a single site-specific attachment suppresses rotational motion of the protein sufficiently to allow interpretation of the spectral line shape in terms of protein internal dynamics. Importantly, line shape analysis and distance mapping using double electron-electron resonance reveal that internal dynamics, the tertiary fold, conformational equilibria, and ligand binding of the tethered proteins were similar to those in solution, in contrast to random attachment via native lysine residues. The results of this study set the stage for the development of an EPR-based flow system that will house soluble and membrane proteins immobilized site-specifically, thereby enabling facile screening of structural and dynamical effects of binding partners.