Assessing site-specific enhancements imparted by hyperpolarized water in folded and unfolded proteins by 2D HMQC NMR

Or Szekely; Gregory Lars Olsen; Mihajlo Novakovic; Rina Rosenzweig; Lucio Frydman

doi:10.1021/jacs.0c00807

Assessing site-specific enhancements imparted by hyperpolarized water in folded and unfolded proteins by 2D HMQC NMR

Or Szekely, Gregory Lars Olsen, Mihajlo Novakovic, Rina Rosenzweig, Lucio Frydman^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

20 Citations (Scopus)

Abstract

Hyperpolarized water can be a valuable aid in protein NMR, leading to amide group H-1 polarizations that are orders of magnitude larger than their thermal counterparts. Suitable procedures can exploit this to deliver 2D H-1-N-15 correlations with good resolution and enhanced sensitivity. These enhancements depend on the exchange rates between the amides and the water, thereby yielding diagnostic information about solvent accessibility. This study applied this "HyperW" method to four proteins exhibiting a gamut of exchange behaviors: PhoA((330-471)), an unfolded 122-residue fragment; barstar, a fully folded ribonuclease inhibitor; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, a protein domain whose folded and unfolded forms interchange rapidly and with temperature-dependent population ratios. For PhoA((330-471)) HyperW sensitivity enhancements were >= 300X, as expected for an unfolded protein sequence. Though fully folded, barstar also exhibited substantial enhancements; these, however, were not uniform and, according to CLEANEX experiments, reflected the solvent-exposed residues. R17 showed the expected superposition of >= 100-fold enhancements for its unfolded form, coexisting with more modest enhancements for their folded counterparts. Unexpected, however, was the behavior of drkN SH3, for which HyperW enhanced the unfolded but, surprisingly, enhanced even more certain folded protein sites. These preferential enhancements were repeatedly and reproducibly observed. A number of explanations-including three-site exchange magnetization transfers between water and the unfolded and folded states; cross-correlated relaxation processes from hyperpolarized "structural" waters and labile side-chain protons; and the possibility that faster solvent exchange rates characterize certain folded sites over their unfolded counterparts-are considered to account for them.

Original language	English
Pages (from-to)	9267-9284
Number of pages	18
Journal	Journal of the American Chemical Society
Volume	142
Issue number	20
Early online date	27 Apr 2020
DOIs	https://doi.org/10.1021/jacs.0c00807
Publication status	Published - 20 May 2020

Funding

This work would not have been possible without the technical assistance of the late Koby Zibzener. We are also grateful to Dr. S.F. Cousin for assistance in the cross-relaxation calculations, to Dr. Shira Albeck (ISPC, Weizmann Institute of Science) for the barstar protein, and to Ms. Ivana Petrovic for expression of 13C-labeled drkN-SH3. This work was supported by the Kimmel Institute for Magnetic Resonance (Weizmann Institute), the EU Horizon 2020 program (Marie Sklodowska-Curie Grant 642773), Israel Science Foundation Grant 965/18, and the Perlman Family Foundation. RR was also supported by the Israel Science Foundation Grant 1889/18 and the Minerva Foundation Research Grant.

All Science Journal Classification (ASJC) codes

Catalysis
General Chemistry
Biochemistry
Colloid and Surface Chemistry

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10.1021/jacs.0c00807Licence: CC BY

Cite this

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abstract = "Hyperpolarized water can be a valuable aid in protein NMR, leading to amide group H-1 polarizations that are orders of magnitude larger than their thermal counterparts. Suitable procedures can exploit this to deliver 2D H-1-N-15 correlations with good resolution and enhanced sensitivity. These enhancements depend on the exchange rates between the amides and the water, thereby yielding diagnostic information about solvent accessibility. This study applied this {"}HyperW{"} method to four proteins exhibiting a gamut of exchange behaviors: PhoA((330-471)), an unfolded 122-residue fragment; barstar, a fully folded ribonuclease inhibitor; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, a protein domain whose folded and unfolded forms interchange rapidly and with temperature-dependent population ratios. For PhoA((330-471)) HyperW sensitivity enhancements were >= 300X, as expected for an unfolded protein sequence. Though fully folded, barstar also exhibited substantial enhancements; these, however, were not uniform and, according to CLEANEX experiments, reflected the solvent-exposed residues. R17 showed the expected superposition of >= 100-fold enhancements for its unfolded form, coexisting with more modest enhancements for their folded counterparts. Unexpected, however, was the behavior of drkN SH3, for which HyperW enhanced the unfolded but, surprisingly, enhanced even more certain folded protein sites. These preferential enhancements were repeatedly and reproducibly observed. A number of explanations-including three-site exchange magnetization transfers between water and the unfolded and folded states; cross-correlated relaxation processes from hyperpolarized {"}structural{"} waters and labile side-chain protons; and the possibility that faster solvent exchange rates characterize certain folded sites over their unfolded counterparts-are considered to account for them.",

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AB - Hyperpolarized water can be a valuable aid in protein NMR, leading to amide group H-1 polarizations that are orders of magnitude larger than their thermal counterparts. Suitable procedures can exploit this to deliver 2D H-1-N-15 correlations with good resolution and enhanced sensitivity. These enhancements depend on the exchange rates between the amides and the water, thereby yielding diagnostic information about solvent accessibility. This study applied this "HyperW" method to four proteins exhibiting a gamut of exchange behaviors: PhoA((330-471)), an unfolded 122-residue fragment; barstar, a fully folded ribonuclease inhibitor; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, a protein domain whose folded and unfolded forms interchange rapidly and with temperature-dependent population ratios. For PhoA((330-471)) HyperW sensitivity enhancements were >= 300X, as expected for an unfolded protein sequence. Though fully folded, barstar also exhibited substantial enhancements; these, however, were not uniform and, according to CLEANEX experiments, reflected the solvent-exposed residues. R17 showed the expected superposition of >= 100-fold enhancements for its unfolded form, coexisting with more modest enhancements for their folded counterparts. Unexpected, however, was the behavior of drkN SH3, for which HyperW enhanced the unfolded but, surprisingly, enhanced even more certain folded protein sites. These preferential enhancements were repeatedly and reproducibly observed. A number of explanations-including three-site exchange magnetization transfers between water and the unfolded and folded states; cross-correlated relaxation processes from hyperpolarized "structural" waters and labile side-chain protons; and the possibility that faster solvent exchange rates characterize certain folded sites over their unfolded counterparts-are considered to account for them.

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Assessing site-specific enhancements imparted by hyperpolarized water in folded and unfolded proteins by 2D HMQC NMR

Abstract

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