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Mapping Protein Conformational Landscapes Under Strongly Native Conditions With Hydrogen Exchange Mass Spectrometry Jacob Witten, Amy Marie Ruschak, Timothy Poterba, Alexis Jaramillo, Andrew D. Miranker, and Sheila S Jaswal J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b04528 • Publication Date (Web): 06 Jul 2015 Downloaded from http://pubs.acs.org on July 12, 2015
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The Journal of Physical Chemistry
Mapping Protein Conformational Landscapes under Strongly
Native
Conditions
with
Hydrogen
Exchange Mass Spectrometry Jacob Witten$, Amy Ruschak‡, Timothy Poterba$, Alexis Jaramillo$, Andrew D. Miranker*‡, Sheila S. Jaswal*‡$
AUTHOR ADDRESS $Department of Chemistry, Amherst College, PO Box 5000, Amherst, MA 01002. ‡Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 208114, New Haven, CT 06520-81114
KEYWORDS conformational landscape, chevron analysis, folding intermediate
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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.
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The Journal of Physical Chemistry
Introduction Exploring the conformational landscape accessed by proteins under native conditions is important for understanding the relationship of stability and dynamics to biological function.1 Even in conditions that favor the native state, proteins are continuously in a folding-unfolding equilibrium.2 As these excursions from the native state are transient, traditional methods to probe protein landscapes rely on perturbing the equilibrium through addition of heat, denaturant or pressure to increase the population of partially and fully unfolded conformations.3 However, extrapolation to physiological conditions is not always reliable, and destabilizing conditions may cause misfolding and aggregation.4 Given the role of protein folding in cellular processes such as turnover, membrane translocation and pathological self assembly,5-7 developing experimental tools to explore native conformational landscapes for a broader range of proteins is of growing importance.8-13 A key method to probe landscapes under native conditions is hydrogen exchange (HX), which typically involves placing protein in deuterated solvent.14 Exchange-labile amide protons that are exposed to solvent undergo rapid, base-catalyzed exchange (at pH > 4) with an intrinsic rate constant (kint). kint is dependent on the amino acid sequence, temperature and pH, and can be calculated using well established parameters.15 Amide protons that are buried or hydrogen bonded in the native state are reduced in their solvent exposure and undergo exchange with a rate constant kHX that is slower than kint. A site protected from HX in the native state can become accessible to HX through sampling of exchange competent states via local fluctuations, partial and/or global unfolding.16,17 Here we focus only on sites exchanging through global unfolding where the closed states are represented by NH and ND, and the open states by UH and UD (Fig. 1).
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NH
ku kf
kint
UH
UD
kf ku
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ND
Figure 1. Schematic of the conformational equilibrium governing global HX for a two-state protein occurring under native conditions in excess D2O. After a site undergoes HX, it no longer reports on the sampling of the conformational equilibrium (grey). For a protein under native conditions (kf > ku), the apparent rate of exchange (kHX) for sites exposed only through global unfolding is determined by the unfolding (ku), and folding (kf) rate constants governing the conformational equilibrium and kint:14,18 𝑘 𝑘
𝑘𝐻𝑋 = 𝑘 𝑢!𝑘𝑖𝑛𝑡 𝑓
𝑖𝑛𝑡
(1)
The relationship between kf and kint defines two limiting regimes in which equation (1) simplifies to enable kHX to report directly either on the kinetic barrier to unfolding or the thermodynamic stability of the native state. In EX1, kint>>kf, so kHX reports on the kinetic barrier through ku.19 In EX2, kint1.3, blue, EXX: 1.3 > ρ > -1.3, green, EX2: ρ
