Mapping Protein Conformational Landscapes under Strongly Native Conditions with Hydrogen Exchange Mass Spectrometry

Abstract

The thermodynamic stability and kinetic barriers separating protein conformations under native conditions are critical for proper protein function and understanding dysfunction in diseases of protein conformation. Traditional methods to probe protein unfolding and folding employ denaturants and highly non-native conditions, which may destabilize intermediate species or cause irreversible aggregation especially at the high protein concentrations typically required. Hydrogen exchange (HX) is ideal for detecting conformational behavior under native conditions without the need for denaturants, but detection by NMR is limited to small highly soluble proteins. Mass spectrometry (MS) can, in principle, greatly extend the applicability of native-state HX to larger proteins and lower concentrations. However, quantitative analysis of HXMS profiles is currently limited by experimental and theoretical challenges. Here we address both limitations, by proposing an approach based on using standards to eliminate the systematic experimental artifacts in HXMS profiles, and developing the theoretical framework to describe HX behavior across all regimes based on the Linderstrøm-Lang formalism. We demonstrate proof of principle by a practical application to native-state HX of a globular protein. The framework and the practical tools developed advance the ability of HXMS to extract thermodynamic and kinetic conformational parameters of proteins under native conditions.

Stepwise Assembly of β-Sheet Structure during the Folding of an SH3 Domain Revealed by a Pulsed Hydrogen Exchange Mass Spectrometry Study

“…MS data were analyzed by discrete fitting as well as by global fitting (Supporting Information), assuming that the mass distributions of all the species populated during refolding could be described as Gaussian distributions. Analysis of pulsed HX-MS data by fitting mass profiles to the sum of Gaussian distributions, each arising from HX into a distinct protein conformation, is an effective way to delineate the mechanism of exchange and structural transitions . The mass profiles obtained from the HX studies for the intact protein and for the different segments of the protein were analyzed as the sum of two, three, or four Gaussian distributions using eq : where a represents the area under each Gaussian distribution and b and c represent the values of the peak m / z position and the width, respectively, of each of n Gaussian distributions.…”

“…Analysis of pulsed HX-MS data by fitting mass profiles to the sum of Gaussian distributions, each arising from HX into a distinct protein conformation, is an effective way to delineate the mechanism of exchange and structural transitions. 55 The mass profiles obtained from the HX studies for the intact protein and for the different segments of the protein were analyzed as the sum of two, three, or four Gaussian distributions using eq 1:…”

Lessons from a quarter century of being human in protein science

“…In fact, we demonstrated that a simple analytical model for the exchange of a 2-state protein undergoing HXMS due to global unfolding came very close to replicating the apparent 3-state experimental HXMS behavior for the amyloidogenic protein β2M under nearnative equilibrium conditions. 27 My computational undergraduates (mostly those who are majoring in Mathematics or Computer Science in addition to Chemistry or Biochemistry and Biophysics) have built a numerical simulations approach to fit complex HXMS time course data and clearly distinguish whether the spectra arise from a 2-state (no intermediate) or 3-state (possesses a folding intermediate) folder. My wet-lab students have been using model proteins to develop the experimental NHXMS workflow.…”

CcdB at pH 4 Forms a Partially Unfolded State with a Dry Core

“…Analysis was carried out by plot-ting observed mean mass as a function of time of exchange, assuming that exchange is occurring under the EX2 regime, although peak width analysis suggests that the slowest phase of exchange at pH 7 could be under the EXX regime (Fig. S8 b) (67,68) because it shows significant increase in peak width as a function of time. The exchange at pH 4 does not show such increase in peak width, suggesting an EX2 exchange mechanism.…”