Cooperativity of a Protein Folding Reaction Probed at Multiple Chain Positions by Real-Time 2D NMR Spectroscopy

Abstract

The refolding reaction of S54G/P55N ribonuclease T1 is a two-step process, where fast formation of a partly folded intermediate is followed by the slow reaction to the native state, limited by a trans --> cis isomerization of Pro39. The hydrodynamic radius of this kinetic folding intermediate was determined by real-time diffusion NMR spectroscopy. Its folding to the native state was monitored by a series of 128 very fast 2D (15)N-HMQC spectra, to observe the kinetics of 66 individual backbone amide probes. We find that the intermediate is as compact as the native protein with many native chemical shifts. All 66 analyzed amide probes follow the rate-limiting prolyl isomerization, which indicates that this cooperative refolding reaction is fully synchronized. The stability of the folding intermediate was determined from the protection factors of 45 amide protons derived from a competition between refolding and H/D exchange. The intermediate has already gained 40% of the Gibbs free energy of refolding with many protected amides in not-yet-native regions.

A remote prolyl isomerization controls domain assembly via a hydrogen bonding network

“…Two sets of signals (31,35) were analyzed: The signals of set 1 originate from amide NH that are already in a native environment in the intermediate I. They show identical chemical shifts and identical intensities in I and N, and thus they vanish when the kinetic spectrum is subtracted from the reference spectrum.…”

“…amide HX from the folding intermediate follows an EX2 mechanism, the protection factor P of individual NH can be calculated with Eq. 1 (31). The rate of folding from the intermediate to the native state (k f ϭ 1.6 ϫ 10 Ϫ4 /s) is taken from our previous work (22), and the intrinsic exchange rates (k int ) were taken from reference (37).…”

“…Both spectra were measured for 8 h, the first (kinetic) spectrum during the folding reaction, and the second (reference) spectrum directly after the completion of refolding. Then the kinetic spectrum was subtracted from the reference spectrum to obtain the difference spectrum (31). The entire experiment was reproducibly repeated once.…”

“…It represents the final rate-limiting step of folding of G3P as well as the transition between the partially folded, functionally active state and the fully folded but functionally inactive state. Therefore, G3P belongs to the increasing number of examples reported recently, where the isomeric state of a proline modulates activity, ligand binding, and physiological regulation (4,8,(26)(27)(28)(29).Here we used real-time 2D NMR spectroscopy (30) and amide hydrogen exchange experiments (31,32) to analyze the structure and the stability of the folding intermediate of G3P at the level of individual residues. In the final domain docking reaction a chain of interconnecting hydrogen bonds gets stabilized.…”

“…Here we used real-time 2D NMR spectroscopy (30) and amide hydrogen exchange experiments (31,32) to analyze the structure and the stability of the folding intermediate of G3P at the level of individual residues. In the final domain docking reaction a chain of interconnecting hydrogen bonds gets stabilized.…”