A Helix 1 Peptide from UBA(1) Provides Evidence for Tertiary Interactions in the Denatured State of UBA(1)

Abstract

Studies over recent decades have shown that the denatured state ensemble (DSE) - the collection of all random-coil conformations of a denatured protein - exhibits significant conformational bias that deviates from theoretical random coil behavior. This inherent bias is thus integral to the initial stages of protein folding and can give insight into unstructured proteins, such as intrinsically disordered proteins (IDPs). In this work, we probe the denatured state of a three-helix bundle protein termed the ubiquitin-associated domain, UBA(1), derived from the DNA-excision repair protein HHR23A. UBA(1) is strongly denatured using guanidine hydrochloride (GdnHCl) and investigated using circular dichroism (CD) spectroscopy. Under moderate to strongly denaturing conditions, UBA(1) demonstrates persistent α-helical content, eventually forming significant polyproline-II (PII) helical structure at 7M GdnHCl. Isolation of the first helix of UBA(1) via insertion of a stop codon at R177 reveals substantially decreased per-residue helicity at all concentrations of denaturant when compared to full protein. This indicates that full-length UBA(1) maintains transient tertiary contacts under strongly denaturing conditions. We have also acquired high resolution NMR (15N-HSQC, HNCO, and HNCA) spectra of UBA(1) to obtain chemical shifts of the backbone atoms for each residue in denaturant. Our data provide residue-specific secondary structure information and indicate that UBA(1) forms transient native-like helices of nearly equal proportion in the DSE. These data will be compared to backbone chemical shifts of the isolated first helix of UBA(1) to further elucidate contributions of transient tertiary contacts to residual helical content in the first helix of UBA(1) in the DSE of full length UBA(1).