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Fast hydrogen exchange affects ¹?N relaxation measurements in intrinsically disordered proteins.

ABSTRACT: Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, (1)H-(15)N HSQC, is used to measure the (15)N transverse relaxation rate (R2), the measured R2 rate is convoluted with the HX rate (kHX) and has higher apparent R2 values. Since the (15)N R2 measurement is important for analyzing protein backbone dynamics, the HX effect on the R2 measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing (15)N R 2 (CPMG) experiments on ?-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R 2 (CPMG) can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D2O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R 2 (CPMG) values be obtained by methods described herein.

SUBMITTER: Kim S

PROVIDER: S-EPMC3615062 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Fast hydrogen exchange affects ¹⁵N relaxation measurements in intrinsically disordered proteins.

Kim Seho S Wu Kuen-Phon KP Baum Jean J

Journal of biomolecular NMR 20130112 3

Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, (1)H-(15)N HSQC, is used to measure the (15)N transverse relaxation rate (R2), the measured R2 rate is convoluted with the HX rate (kHX) and has higher apparent R2 values. Since the (15)N R2 measurement is impor ...[more]

PMID: 23314729

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Project description:The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccation-induced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 - while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins - HSP70 can be co-immunoprecipitated with a polyglutamine-containing client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by Förster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability, but a bioinformatics survey does not support this, since there are no significant differences in the degree of disorder between desiccation tolerant and intolerant species. It seems clear therefore that molecular shield function is largely an intermolecular activity implemented by specialist IDPs, distinct from molecular chaperones, but with a role in proteostasis.

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Fast hydrogen exchange affects ¹?N relaxation measurements in intrinsically disordered proteins.

Publications

Fast hydrogen exchange affects ¹⁵N relaxation measurements in intrinsically disordered proteins.

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