Deuteration of nonexchangeable protons on proteins affects their thermal stability, side‐chain dynamics, and hydrophobicity

Nichols, Parker J.; Falconer, Isaac B.; Griffin, Aaron; Mant, Colin T.; Hodges, Robert S.; McKnight, C. James; Vögeli, Beat; Vugmeyster, Liliya

doi:10.1002/pro.3878

Cited by 23 publications

(19 citation statements)

References 58 publications

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“…Biophysical characterization of both D-HEWL variants and commercially available unlabelled HEWL shows that both D-HEWL molecules are stable and active. The perdeuterated variants showed lower thermal stability relative to the hydrogenated protein both in D 2 O and H 2 O buffers, in line with what has been reported in several biophysical studies on protein deuteration (Berns, 1963;Hattori et al, 1965;Brockwell et al, 2001;Meilleur et al, 2004;Koruza et al, 2018;Nichols et al, 2020). Additionally, it seems that both hydrogenated and perdeuterated forms of HEWL have an increased transition temperature in D 2 O compared with H 2 O, as described in previous studies (Makhatadze et al, 1995;Efimova et al, 2007).…”

Section: Discussionsupporting

confidence: 88%

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

Ramos

Laux

Haertlein

et al. 2021

Int Union Crystallogr J

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This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.

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Section: Discussionsupporting

confidence: 88%

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

Ramos

Laux

Haertlein

et al. 2021

Int Union Crystallogr J

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“…Surprisingly, the observations suggest that in vitro refolding has a stronger impact on HEWL thermal stability than protein perdeuteration, with the respective decreases in T m being greater than 3.5 C compared with variations of smaller than 1.6 C. These changes in HEWL thermal stability are consistent throughout different buffer compositions and also in H 2 O and D 2 O solvents, where the minor deviations can be explained by the aforementioned difference between pH and pD (Glasoe & Long, 1960). Moreover, the reduction in protein thermal stability associated with protein perdeuteration is in agreement with several previous studies (Berns, 1963;Hattori et al, 1965;Brockwell et al, 2001;Meilleur et al, 2004;Koruza et al, 2018;Nichols et al, 2020;Ramos et al, 2021).…”

Section: Deuteration and In Vitro Refolding Differently Affect The Biophysical Properties Of Hewlsupporting

confidence: 91%

“…While hydrogenated and perdeuterated variants of a protein are usually close to identical in structure, several studies of the macromolecular H/D isotope effect have reported decreases in the thermal stability of perdeuterated proteins compared with their hydrogenated analogs (Berns, 1963;Hattori et al, 1965;Brockwell et al, 2001;Meilleur et al, 2004;Koruza et al, 2018;Nichols et al, 2020;Ramos et al, 2021). Observations have also been made regarding solvent H/D isotope effects which suggest that both perdeuterated and hydrogenated proteins are thermally more stable in D 2 O than in H 2 O (Hattori et al, 1965;Harrington & von Hippel, 1961;Makhatadze et al, 1995;Freyman et al, 2001;Kuhlman & Raleigh, 1998;Sasisanker et al, 2004;Efimova et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme

Ramos¹,

Laux²,

Haertlein³

et al. 2021

Acta Crystallogr D Struct Biol

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The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97–Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis.

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“…Similarly, the deuteration of non-exchangeable protons in globular proteins was found to reduce their denaturation temperature of few degrees [48,49,50],…”

Section: Swelling Behaviour Of Pnipam Microgelsmentioning

confidence: 97%

The role of polymer structure on water confinement in poly(N-isopropylacrylamide) dispersions

Buratti¹,

Tavagnacco²,

Zanatta³

et al. 2022

Preprint

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Poly(N -isopropylacrylamide) (PNIPAM) is a synthetic polymer that is widely studied for its thermoresponsive character. However, recent works also reported evidence of a low temperature (protein-like) dynamical transition around 225 K in concentrated PNIPAM suspensions, independently of the polymer architecture, i.e., both for linear chains and for microgels. In this work, we investigate water-polymer interactions by extensive differential scanning calorimetry (DSC) measurements of both systems, in order to understand the effect of the different topological structures on the solution behaviour, in particular regarding crystallization and melting processes. In addition, we compare protiated and deuterated microgels, in both water and deuterated water. The DSC results are complemented by dynamic light scattering experiments, which confirm that the selective isotopic substitution differently affects the solution behaviour. Our findings highlight the important role played by the polymer architecture on the solution behaviour: indeed, microgels turn out to be more efficient confining agents, able to avoid water crystallization in a wider concentration range with respect to linear chains.

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Deuteration of nonexchangeable protons on proteins affects their thermal stability, side‐chain dynamics, and hydrophobicity

Cited by 23 publications

References 58 publications

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme

The role of polymer structure on water confinement in poly(N-isopropylacrylamide) dispersions

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