Amide Hydrogen Exchange (HX)

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Contents 1 Introduction 2 H/D exchange experiments 2.1 Freeze-drying protein samples 2.2 NMR Experiments suitable to follow H/D exchange 3 Factors affecting rate of amide proton exchange 3.1 Hydrogen bonding and secondary structure 3.2 Water accessibility 3.3 pH 3.4 Temperature 3.5 Co-solutes 4 References

Introduction

Amide hydrogen exchange is a type of chemical exchange process. Exchange half-times of the amide hydrogen atoms can range from minutes ( or less ? ) to years [1]. Because of large variations in the timescale of the exchange reaction, monitoring the exchange reaction by NMR almost always requires recording of multiple data sets and even different types of experiments (what types?). The exchange rate is slower when the amide is buried in the core of the protein or when it is involved in a H bond, it's faster when amide groups exposed to solvent. In the proteins mutations could strongly influence the exposure of amide groups to solvent, local dynamics, and therefore strongly affect the exchange rates.

H/D exchange experiments

In H/D exchange experiments amide hydrogen exchange is observed by disappearance or appearance of signals in ¹H¹⁵N-HSQC or TOCSY (without water suppression, for samples in D₂O) [2] spectra after rapid change of solvent from H₂O to D₂O or vice-versa.

H/D exchange can be initiated using different techniques: quick dissolution of lyophilized sample [1, 2] in D₂O or H₂O (depending on whether the original sample was deuterated or protonated), dilution of sample into D₂O or H₂O, solvent exchange using desalting columns.

Freeze-drying protein samples

When freeze-drying protein samples, one should keep in mind that co-solutes can affect solubility and folding state or protein upon reconstitution.

Thiocyanate under those conditions promotes aggregation of proteins into unsoluble phase, addition of sucrose to the contrary - counteracts aggregation[3] (Protein unfolding in solids can be followed by infrared spectroscopy by monitoring amide strech frequencies [3]) Preferential binding [4] is the currently accepted mechanism of denaturant (such as thiocyanate and urea) action, wherein denaturant molecules spontaneously "envelope" the exposed protein surface. As the proten conformation samples unfolded states, more dentaturant binds to the newly exposed surface, and so on. Stabilizing effect of sucrose and other cosmotropic agents is explained by the opposite effect - their exclusion [4] from the protein molecule exposed surface - those (kosmotropic) molecules prefer to be solvated rather then bound to the protein.

NMR Experiments suitable to follow H/D exchange

Peak intensities of faster exchanging protons are frequently followed by ¹H-¹⁵N HSQC spectra. HSQC spectra do not produce a reliable internal standard of peak intensity since in principle, all observable amide protons are subject to exchange with water. Therefore most often sample is kept in the spectrometer without changing tuning of the probe, while measuring multiple spectra.

It is impractical to keep sample in the spectrometer to follow slow exchange. Slower exchanging protons can be monitored by TOCSY experiments without water suppression in D₂O [2]. In that case peak intensities of non-exhangeable protons can be used to calibrate intensity scales of spectra recorded at different spectrometer conditions and separated by significant time intervals. Also, if sample is stored outside spectrometer - it must be kept in a thermostat.

Factors affecting rate of amide proton exchange

Hydrogen bonding and secondary structure

Hydrogen bonding is not necessarily causing slow amide hydrogen exchange. In bovine pancreatic trypsin inhibitor (BPTI), for example, there are three buried amide protons (I55,L56 and N59) exhibiting slow exchange that are not hydrogen bonded to the protein acceptor [5]. Also, there are some strongly hydrogen bound protons that are in fast exchange because they are exposed on the surface.

See also hydrogen bonding in proteins.

Water accessibility

pH

Article [5] gives a reference to another article discussing pH dependence of amide hydrogen exchange.

Temperature

Co-solutes

Tadeo et. al [1] have quite extensively analyzed effect of salts on the rates of amide proton exchange. Salts can significantly (within 1-3 orders of mangitude at ~1M salt concentration) affect the rate of amide proton exchange [1]: cosmotropic salts (sodium sulphates and chloride) decrease rate of exchange 1.2-100 -fold (at 1M concentration), while chaotropic salts (sodium thiocyanate) increase rate of amide exchange 10 -fold on average (at 1M concentration). Effect of ion's nature on the amide hydrogen exchange seems to be in agreement with the Hofmeister series of ions [1, 6], also ΔG_ex is lineraly proportional to salt concentration for moderately charged proteins [1].

The corollary of rate modulation effect by the co-solutes is that adding an appropriate co-solute can allow accessing rates that might be too fast or too slow to measure directly. Secondly - one must be very careful about controlling buffer conditions used in amide proton exchange experiments.

References

1. Tadeo, X and Castano, D and Millet, O. Anion modulation of the 1H/2H exchange rates in backbone amide protons monitored by NMR spectroscopy. Protein Science , 2007. BibTeX [salt]

2. Michel, LV and Ye, T and Bowman, SEJ and Levin, BD and Hahn, MA and Russell, BS and Elliott, SJ and Bren, KL. Heme Attachment Motif Mobility Tunes Cytochrome c Redox Potential. Biochemistry 46(42):11753--11760, 2007. BibTeX [heme]

3. Error fetching PMID 8889176: [lyoph]

4. Timasheff, SN. Water as ligand: Preferential binding and exclusion of denaturants in protein unfolding. Biochemistry 31(41):9857--9864, 1992. BibTeX [timasheff]

5. Baxter, NJ and Williamson, MP. Temperature dependence of 1 H chemical shifts in proteins. Journal of Biomolecular NMR 9(4):359--369, 1997. BibTeX [baxter97]

6. Hofmeister, F. Zur Lehre von der Wirkung der Salze. Naunyn-Schmiedeberg's Archives of Pharmacology 25(1):1--30, 1888. BibTeX [hofmeister]

7. Craven, CJ and Derix, NM and Hendriks, J and Boelens, R and Hellingwerf, KJ and Kaptein, R. Probing the Nature of the Blue-Shifted Intermediate of Photoactive Yellow Protein in Solution by NMR: Hydrogen-Deuterium Exchange Data and pH Studies. Biochemistry 39(47):14392--14399, 2000. BibTeX [two]

8. Kay, LE. Protein dynamics from NMR. Biochem Cell Biol 76:145--152, 1998. BibTeX [dynamic]

9. Yamasaki, K and Akasako-Furukawa, A and Kanaya, S. Structural stability and internal motions of Escherichia coli ribonuclease HI: 15N relaxation and hydrogen-deuterium exchange analyses. Journal of Molecular Biology 277(3):707--722, 1998. BibTeX [dynbyex]