Acid‐induced denaturation of Escherichia coli ribonuclease HI analyzed by CD and NMR spectroscopies

Abstract

Acid-induced denaturation of the ribonuclease HI protein from Escherichia coli was analyzed by CD and NMR spectroscopies. The CD measurement revealed that the acid denaturation at 10 degrees C proceeds from the native state (N-state) to a molten globule-like state (A-state), through an apparently more unfolded state (U(A)-state). In (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, cross peaks from the N-state and those from the other two states are distinctively observed, while the U(A)-state and A-state are not distinguished from each other. Cross peaks from the U(A)/A-states showed a small pH dependence, which suggests a similarity in the backbone structure between the two states. The direct hydrogen-deuterium (H-D) exchange measurement at pH with the largest population of U(A)-state revealed that at least alpha-helix I is highly protected in the structure of the U(A)-state. A pH-jump H-D exchange analysis showed that the protection of alpha-helix I is highest also in the A-state. The profile of hydrogen-bond protection indicated that the structure of the A-state is closely related to that of the kinetic folding intermediate.

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin

“…With the exception of Tyr 114 , these protection factors are low and do not exceed a value of ϳ300 (Table 1). These protection factors are comparable with those observed for the molten globules of apomyoglobin (36), apoleghemoglobin (41), and RNase HI (42). In contrast, backbone amides of native apoflavodoxin have protection factors up to 2 ϫ 10 6 .…”

Three dimensional structure directs T-cell epitope dominance associated with allergy

“…Protease- and MHCII-binding sites form hydrogen bonds and other non-covalent contacts with allergen backbone and sidechain groups that, in the native allergen, stabilize three-dimensional structure. Low pH in the antigen-processing compartment is expected to destabilize the structure of allergens, but many proteins remain in a native or native-like conformation at low pH's [ 8 , 9 ]. The tendency for proteases to cleave initially in domain linkers and in flexible loops is well known, and the energetic penalty for unfolding 10–12 residues of polypeptide was found sufficient to explain the site selectivity of a serine protease acting on a native protein substrate [ 10 ].…”

DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science

“…The molten globule (MG) state, which has a substantial amount of secondary structure but lacks the specific tertiary side-chain packing characteristics of native proteins, is an equilibrium intermediate state under mildly denaturing conditions for numerous globular proteins, many with more than ~100 residues [22][23][24]. In the 1990s, the structural characterizations of the MG state by H/D-exchange 2D NMR were carried out for a number of globular proteins, including apomyoglobin [25], cytochrome c [26,27], α-lactalbumin [28][29][30][31][32], Ca 2+ -binding milk lysozyme [33,34], and other proteins [35][36][37][38][39][40][41]. The P values of slowly exchanging NH protons in the MG state range from 10 2 to 10 3 , which is more than three orders of magnitude smaller than the values in the native (N) state.…”