Interfacial Water at Protein Surfaces: Wide-Line NMR and DSC Characterization of Hydration in Ubiquitin Solutions

Tompa, K.; Bánki, P.; Bokor, M.; Kamasa, P.; Lasanda, G.; Tompa, Péter

doi:10.1016/j.bpj.2008.11.038

Cited by 44 publications

(60 citation statements)

References 39 publications

(64 reference statements)

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“…The 1 H linewidth of less then 1 kHz that we measured for the hydration shell of ubiquitin and AFP III corresponds well to previously reported hydration water linewidths of up to 0.85 kHz (23,25). The chemical shift we measured for the hydration water is 5ppm, which, given the broad linewidth, is not distinct from the value for bulk liquid water.…”

Section: Discussionsupporting

confidence: 90%

“…The nonfrozen hydration shell presumably leaves ubiquitin and the non-ice-binding surface of AFP III in a similar environment as in solution, explaining the negligible 13 C chemical shift differences we observed for these proteins between solution and frozen solution. (25). However, as soon as the bulk solution freezes, they stop tumbling isotropically, become arrested inside the ice, and can be observed using dipolar-based solid-state NMR methods.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, the hydration shell of soluble proteins does not freeze below the freezing temperature of the bulk solution (23,24). For frozen ubiquitin solutions, Tompa and coworkers showed that the hydration layer starts to freeze at about −50°C (25). These results about protein hydration shells raise the following questions regarding antifreeze proteins: If the hydration shell of proteins does not freeze, how can AFPs bind to ice?…”

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confidence: 99%

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Protein–ice interaction of an antifreeze protein observed with solid-state NMR

Siemer

Huang

McDermott

2010

Proc. Natl. Acad. Sci. U.S.A.

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NMR on frozen solutions is an ideal method to study fundamental questions of macromolecular hydration, because the hydration shell of many biomolecules does not freeze together with bulk solvent. In the present study, we present previously undescribed NMR methods to study the interactions of proteins with their hydration shell and the ice lattice in frozen solution. We applied these methods to compare solvent interaction of an ice-binding type III antifreeze protein (AFP III) and ubiquitin a non-ice-binding protein in frozen solution. We measured 1 H-1 H cross-saturation and cross-relaxation to provide evidence for a molecular contact surface between ice and AFP III at moderate freezing temperatures of −35°C. This phenomenon is potentially unique for AFPs because ubiquitin shows no such cross relaxation or cross saturation with ice. On the other hand, we detected liquid hydration water and strong water-AFP III and water-ubiquitin cross peaks in frozen solution using relaxation filtered 2 H and HETCOR spectra with additional 1 H-1 H mixing. These results are consistent with the idea that ubiquitin is surrounded by a hydration shell, which separates it from the bulk ice. For AFP III, the water cross peaks indicate that only a portion of its hydration shell (i.e., at the ice-binding surface) is in contact with the ice lattice. The rest of AFP III's hydration shell behaves similarly to the hydration shell of non-ice-interacting proteins such as ubiquitin and does not freeze together with the bulk water.hydration shell | type III AFP | ice binding | water solute interaction A ntifreeze proteins (AFPs) can be found in a variety of coldadapted organisms including bacteria, plants, insects, and fish (1). AFPs lower the freezing point of a given solution below its melting point, a process called thermal hysteresis. This thermal hysteresis is thought to result from the strong binding of the AFP to the surface of ice crystals making their further growth energetically unfavorable (2, 3). One of the most intensely studied classes of AFPs is the type III AFPs from the ocean pound. In particular the HPLC-12 isoform (in the following just termed AFP III), was studied with X-ray crystallography (4), NMR (5, 6), mutagenesis (4,7,8), and molecular dynamics (9). These studies showed that the ice-binding site of AFP III is a flat surface formed by predominantly nonpolar residues (5, 8), which is remarkable because the solvent accessible surface of other soluble proteins like ubiquitin are predominantly polar. There have been limited opportunities to study the water molecules at the ice-binding surface of AFPs, although recent computational and sub-angstrom X-ray structures indicate ordered water molecules at the ice-binding surface (9, 10).Ice, with its very slow 1 H spin-lattice relaxation rate (R 1 ) and very fast spin-spin relaxation rate (R 2 ), is relatively difficult to study with NMR. Measurements of relaxation rates of ice showed that R 2 is on the order of 10 6 s −1 and R 1 on the order of 10at a temperatures of about 260 K...

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Protein–ice interaction of an antifreeze protein observed with solid-state NMR

Siemer

Huang

McDermott

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…[17]. We compared and related these findings to similar studies on globular Ubiqitin [18], further IDPs, like ERD14 and p53TAD, and members of the casein family (unpublished results). These latter examples are in full agreement with the general conclusions drawn.…”

Section: Water Molecules Map the Interfacial Spacementioning

confidence: 92%

“…These latter examples are in full agreement with the general conclusions drawn. The reasons of selecting the particular proteins, and the details of the applied experimental methods were outlined earlier [16][17][18]8,9]. It is important to note that the NMR measurements were done in the direction of heating, to avoid the problems caused by hysteresis.…”

Section: Water Molecules Map the Interfacial Spacementioning

confidence: 99%

Water rotation barriers on protein molecular surfaces

et al. 2015

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The experimental characterization of hindered-rotation barriers and mapping the energetic heterogeneity of water molecules bound to the molecular "surface" of proteins is critical for understanding the functional interaction of proteins with their environment. Here, we show how to achieve this goal by an original wide-line NMR procedure, which is based on the spectral motional narrowing phenomenon following the melting (thawing) process of interfacial ice. The procedure highlights the differences between globular and intrinsically disordered proteins and it enables to delineate the effect of solvent on protein structure, making a distinction between point mutants, monomeric and oligomeric states, and characterizing the molecular interactions taking part in different cellular processes. We put this unique experimental approach introducing novel physical quantities and quantifying the heterogeneous distribution of motional activation energy of water in the interfacial landscape into a historical perspective, demonstrating its utility through a variety of globular and disordered proteins.

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