Analysis of folded structure and folding thermodynamics in heterogeneous-backbone proteomimetics

Recent years have seen a growing number of examples of designed oligomeric molecules with artificial backbone connectivity that are capable of adopting complex folded tertiary structures analogous to those seen in natural proteins. A range of experimental techniques from structural biology and biophysics have been brought to bear in the study of these proteomimetic agents. Here, we discuss some considerations encountered in the characterization of high-resolution folded structure as well as folding thermodynamics of protein-like artificial backbones. We provide an overview of the use of X-ray crystallography and NMR spectroscopy in such systems and review example applications of these methods in the primary literature. Further, we provide detailed protocols for two experiments that have proved useful in our prior and ongoing efforts to compare folding thermodynamics between natural protein domains and heterogeneous-backbone counterparts.