HETCOR Spectra Research Articles

Synergetic impact of MgO on PMMA-ZrO2 hybrid composites: Evaluation of structural, morphological and improved mechanical behavior for dental applications

This work aims to demonstrate the effect of ZrO2 and MgO inclusion into the Poly(methyl methacrylate) (PMMA). To fabricate novel hybrid composites via heat cure method, various composites (PZM2, PZM4 and PZM6) were synthesized in the system [(95-x) PMMA + 5 ZrO2 + x MgO] (x = 2, 4, and 6) respectively. Density of the prepared composites were determined and varying between 1.035–1.152 g/cm3. X-ray Diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) followed by EDAX and mechanical testing were performed to evaluate the fabricated composite properties. Moreover, to explore the structure of the fabricated composites the 13 C CP-MAS SSNMR and 1 H-13 C Phase-Modulated Lee Goldberg (PMLG) HETCOR Spectrum were recorded which clarify chemical shifting and motional dynamics of the composites. Mechanical tests were performed by UTM and the obtained parameters such as compressive strength, Young’s modulus, fracture toughness, brittleness coefficient, flexural strength and flexural modulus are found to be in the range of 91–100 MPa, 0.48–0.51 GPa, 9.122–9.705 MPa.m1/2, 0.66–0.815, 51.03–42.78 MPa and 499–663 MPa respectively. Some more mechanical parameters such as proportional limit, elastic limit, failure strength, modulus of resilience and modulus of toughness were also calculated. Furthermore, tribological properties were also determined and the coefficient of friction (COF) was decreased by 17.4 % and 38 % for composite PZM6 at 20 N and 40 N as compared to the composite PZM2 and the lowest wear volume of 1.55 mm3 was observed for PZM2, whereas the maximum volume loss of 5.64 mm3 is observed for composite PZM6. To check out the biocompatibility, cytotoxicity and genotoxicity of the fabricated composites the Trypan-blue assay was also performed for PZM2 and PZM6 composites. Dissection on the gut of larvae was also performed on the both composites followed by DAPI and DCFH-DA staining. Therefore, these synthesized samples can be used for the fabrication of denture materials.

Organic NMR crystallography: enabling progress for applications to pharmaceuticals and plant cell walls.

The application of NMR crystallography to organic molecules is exemplified by two case studies. For the tosylate salt of the active pharmaceutical ingredient, Ritlectinib, solid-state NMR spectra are presented at a 1H Larmor frequency of 1 GHz and a magic-angle spinning (MAS) frequency of 60 kHz. Specifically, 14N-1H heteronuclear multiple-quantum coherence (HMQC) and 1H-1H double-quantum (DQ) single-quantum (SQ) correlation experiments are powerful probes of hydrogen bonding interactions. A full assignment of the 1H, 13C and 14N/15N chemical shifts is achieved using also 1H-13C cross polarization (CP) HETCOR spectra together with gauge-including projector augmented wave (GIPAW) DFT calculation for the geometry-optimised X-ray diffraction crystal structure that is reported here (CCDC 2352028). In addition, GIPAW calculations are presented for the 13C chemical shifts in the two polymorphs of cellulose for which diffraction structures are available. For both case studies, a focus is on the discrepancy between experiment and GIPAW calculation.

Probing covalent and non-covalent interactions between vanillic acid and starch and their effects on digestibility by solid-state NMR

Intermolecular interactions play a significant role on the physicochemical properties and digestibility of starchy foods. This study investigated the covalent and non-covalent interactions between vanillic acid (VA) and porous starch (PS) as well as their effects on digestibility using solid-state NMR. VA-PS conjugates and mixtures were synthesized and characterized using 1H NMR, FT-IR, SEM and XRD. 13C NMR peaks at 163 ppm and FT-IR signals at 1737 cm−1 indicated the formation of ester bond in VA-PS conjugates. While differences between covalent and non-covalent interactions were also probed by solid-state NMR. The specific binding sites between VA and PS were subsequently identified by 1H13C HETCOR spectra before assessing the impact of covalent and non-covalent interactions on digestibility through an in vitro digestion test. The results revealed 13C chemical shifts of about 2.0 ppm, indicating stronger intermolecular interactions, and reduced mobility of the VA-PS conjugate due to its covalent bonding. Overall, the results showed that the VA-PS conjugate, characterized by stronger covalent interactions, exhibited superior effects in inhibiting starch digestibility compared with non-covalent interactions.

A molecular fluorophore in citric acid/ethylenediamine carbon dots identified and quantified by multinuclear solid-state nuclear magnetic resonance.

The composition of fluorescent polymer nanoparticles, commonly referred to as carbon dots, synthesized by microwave-assisted reaction of citric acid and ethylenediamine was investigated by 13 C, 13 C{1 H}, 1 H─13 C, 13 C{14 N}, and 15 N solid-state nuclear magnetic resonance (NMR) experiments. 13 C NMR with spectral editing provided no evidence for significant condensed aromatic or diamondoid carbon phases. 15 N NMR showed that the nanoparticle matrix has been polymerized by amide and some imide formation. Five small, resolved 13 C NMR peaks, including an unusual ═CH signal at 84 ppm (1 H chemical shift of 5.8 ppm) and ═CN2 at 155 ppm, and two distinctive 15 N NMR resonances near 80 and 160 ppm proved the presence of 5-oxo-1,2,3,5-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid (IPCA) or its derivatives. This molecular fluorophore with conjugated double bonds, formed by a double cyclization reaction of citric acid and ethylenediamine as first shown by Y. Song, B. Yang, and coworkers in 2015, accounts for the fluorescence of the carbon dots. Cross-peaks in a 1 H─13 C HETCOR spectrum with brief 1 H spin diffusion proved that IPCA is finely dispersed in the polyamide matrix. From quantitative 13 C and 15 N NMR spectra, a high concentration (18 ± 2 wt%) of IPCA in the carbon dots was determined. A pronounced gradient in 13 C chemical-shift perturbations and peak widths, with the broadest lines near the COO group of IPCA, indicated at least partial transformation of the carboxylic acid of IPCA by amide or ester formation.

Hyperpolarised 1H–13C Benchtop NMR Spectroscopy

Benchtop NMR spectrometers with sub-ppm spectral resolution have opened up new opportunities for performing NMR outside of the standard laboratory environment. However, the relatively weak magnetic fields of these devices (1–2 T) results in low sensitivity and significant peak overlap in 1H NMR spectra. Here, we use hyperpolarised 13C{1H} NMR to overcome these challenges. Specifically, we demonstrate the use of the signal amplification by reversible exchange (SABRE) parahydrogen-based hyperpolarisation technique to enhance the sensitivity of natural abundance 1D and 2D 13C{1H} benchtop NMR spectra. We compare two detection methods for SABRE-enhanced 13C NMR and observe an optimal 13C{1H} signal-to-noise ratio (SNR) for a refocused INEPT approach, where hyperpolarisation is transferred from 1H to 13C. In addition, we exemplify SABRE-enhanced 2D 13C benchtop NMR through the acquisition of a 2D HETCOR spectrum of 260 mM of 4-methylpyridine at natural isotopic abundance in a total experiment time of 69 min. In theory, signal averaging for over 300 days would be required to achieve a comparable SNR for a thermally polarised benchtop NMR spectrum acquired of a sample of the same concentration at natural abundance.

19F Dynamic Nuclear Polarization at Fast Magic Angle Spinning for NMR of HIV-1 Capsid Protein Assemblies.

We report remarkably high, up to 100-fold, signal enhancements in 19F dynamic nuclear polarization (DNP) magic angle spinning (MAS) spectra at 14.1 T on HIV-1 capsid protein (CA) assemblies. These enhancements correspond to absolute sensitivity ratios of 12-29 and are of similar magnitude to those seen for 1H signals in the same samples. At MAS frequencies above 20 kHz, it was possible to record 2D 19F-13C HETCOR spectra, which contain long-range intra- and intermolecular correlations. Such correlations provide unique distance restraints, inaccessible in conventional experiments without DNP, for protein structure determination. Furthermore, systematic quantification of the DNP enhancements as a function of biradical concentration, MAS frequency, temperature, and microwave power is reported. Our work establishes the power of DNP-enhanced 19F MAS NMR spectroscopy for structural characterization of HIV-1 CA assemblies, and this approach is anticipated to be applicable to a wide range of large biomolecular systems.

Stereochemical investigation of N-formyl-2,7-diphenyl-1,4-diazepan-5-ones by NMR spectroscopy and single crystal X-ray diffraction studies

A series of N-formyl-2,7-diphenyl-1,4-diazepan-5-ones 11–15 are synthesized in significant yields (86–92%) from their corresponding 2,7-diphenyl-1,4-diazepan-5-ones 6–10 using acetic-formic anhydride. Synthesized compounds 11–15 are characterised by their analytical and spectral (IR, Mass, 1D & 2D NMR) data. All the N–formyl compounds 11–15 show doubling of NMR signals due to the existence of syn and anti rotamers at RT which are unambiguously assigned by HETCOR spectra and ASIS method. The preferred conformation has been arrived by considering the chemical shifts, coupling constants, dihedral angles and destabilizing strain factors. The N-formyl-2,7-diphenyl-1,4-diazepan-5-ones 11–15 are best accommodated in a coplanar orientation of formyl function and flattened boat conformation of the ring where the phenyl groups are occupying pseudo-axial orientations in solution state. Further, dynamic NMR study is carried out for 14 from 28 °C to 116 °C and the calculated average energy barrier is 84.4 kJ mol−1. The single crystal X-ray diffraction study on N-formyl-2,7-diphenyl-1,4-diazepan-5-one 13 shows that the molecule adopts distorted boat conformation with coplanar orientation of formyl group.

Determination of Controlled Self-Assembly of a Paracrystalline Material by Homology Modelling with Hybrid NMR and TEM.

Controlling complexity, flexibility, and functionality of synthetic and biomimetic materials requires insight into how molecular functionalities can be exploited for steering their packing. A fused NDI‐salphen (NDI=naphthalene diimide) prototypic artificial photosynthesis material, DATZnS, is shown to be comprised of a phenazine motif, in which the alignment of electric dipole moments in a P2/c supramolecular scaffold can be modulated with bulky substituents. They can also be switched between parallel stacks of dipoles running antiparallel in the DATZnS‐H compared with parallel stacks of dipoles in polar layers running in opposite directions in the DATZnS(3′‐NMe) parent compound. Spatial correlations obtained from HETCOR spectra, collected with a long cross polarization contact time of 2 ms, reveal an antiparallel stacking for the DATZnS‐H homologue. These constraints and limited data from TEM are used to construct a structural model within the P2/c space group determined by the molecular C 2 symmetry. By using homology modelling, a pseudo octahedral coordination of the Zn is shown to follow the packing‐induced chirality with enantiomeric pairs of the Λ and Δ forms alternating along antiparallel stacks. The model helps to understand how the steric hindrance modulates the self‐assembly in this novel class of fused materials by steric hindrance at the molecular level.

Solid State NMR Techniques Study the Structural Characteristics of As-Synthesized ITQ-33

Multinuclear solid state NMR techniques have been applied to study the structural characteristics of extra-large-pore zeolite ITQ-33. Through analysis of 2D 29Si{19F} HETCOR NMR spectra, the configurations of Ge-D4R units in ITQ-33 can be confirmed to have the most separation between Si and Ge atoms. Because F anions are not in the center of D4R units for shorter Ge–F bond lengths and Ge-D4R units with Ge–F bonds in the crystals are related by mirror symmetries, 29Si NMR signals of D4R units are magnetically inequivalent, therefore two 29Si peaks are observed in 19F–29Si CP/MAS and 19F–29Si HETCOR spectra. The formation of specific D4R configurations proves that Ge atoms and F– anions play important structural directing roles in the formation process of zeolite ITQ-33. 27Al 5QMAS experimental results confirm that a major amount of Al atoms is incorporated into the 3-ring in the framework. 1D 13C–27Al S-RESPDOR experimental results show that Al atoms in the framework are spatially close to the methyl group...

Probing Surface Hydrogen Bonding and Dynamics by Natural Abundance, Multidimensional, 17O DNP-NMR Spectroscopy

Dynamic nuclear polarization (DNP)-enhanced solid-state nuclear magnetic resonance (SSNMR) spectroscopy is increasingly being used as a tool for the atomic-level characterization of surface sites. DNP surface-enhanced SSNMR spectroscopy of materials has, however, been limited to studying relatively receptive nuclei, and the particularly rare 17O nuclide, which is of great interest for materials science, has not been utilized. We demonstrate that advanced 17O SSNMR experiments can be performed on surface species at natural isotopic abundance using DNP. We use 17O DNP surface-enhanced 2D SSNMR to measure 17O{1H} HETCOR spectra as well as dipolar oscillations on a series of thermally treated mesoporous silica nanoparticle samples having different pore diameters. These experiments allow for a nonintrusive and unambiguous characterization of hydrogen bonding and dynamics at the surface of the material; no other single experiment can give such details about the interactions at the surface. Our data show that, u...

Discrimination of Surface and Bulk Structure of Crystalline Hydroxyapatite Nanoparticles by NMR

Solid state 1H and 31P NMR spectroscopy was used to characterize well-crystallized hydroxyapatite samples of different stoichiometry prepared by a precipitation route. The aim of the paper was to investigate the bulk structural features of samples with different stoichiometry and to discriminate signals related to the surface from those related to the bulk. Thanks to the implementation of (i) in situ thermal pretreatment at 623 K, (ii) filling of the NMR rotor in a controlled atmosphere, (iii) relative proton enrichment of the surface performed under controlled isotopic H-D exchanges, and (iv) specific NMR sequences including inversion recovery measurements, two-dimensional HETCOR and DQSQ spectra, new resolved NMR signals originating from the surface and from the bulk were identified alongside already reported signals associated with adsorbed water, structural phosphates, and OH groups. In particular, considering the influence of the stoichiometry, it was possible to identify a specific signature associa...

Fast experiments for structure elucidation of small molecules: Hadamard NMR with multiple receivers.

We propose several significant improvements to the PANSY (Parallel NMR SpectroscopY) experiments-PANSY COSY and PANSY-TOCSY. The improved versions of these experiments provide sufficient spectral information for structure elucidation of small organic molecules from just two 2D experiments. The PANSY-TOCSY-Q experiment has been modified to allow for simultaneous acquisition of three different types of NMR spectra-1D C-13 of non-protonated carbon sites, 2D TOCSY and multiplicity edited 2D HETCOR. In addition the J-filtered 2D PANSY-gCOSY experiment records a 2D HH gCOSY spectrum in parallel with a (1) J-filtered HC long-range HETCOR spectrum as well as offers a simplified data processing. In addition to parallel acquisition, further time savings are feasible because of significantly smaller F1 spectral windows as compared to the indirect detection experiments. Use of cryoprobes and multiple receivers can significantly alleviate the sensitivity issues that are usually associated with the so called direct detection experiments. In cases where experiments are sampling limited rather than sensitivity limited further reduction of experiment time is achieved by using Hadamard encoding. In favorable cases the total recording time for the two PANSY experiments can be reduced to just 40 s. The proposed PANSY experiments provide sufficient information to allow the CMCse software package (Bruker) to solve structures of small organic molecules.

Solid-state NMR as a probe of anion binding: molecular dynamics and associations in a [5]polynorbornane bisurea host complexed with terephthalate.

A range of solid-state NMR techniques is used to characterise a molecular host:guest complex consisting of a [5]polynorbornane bisurea host binding a terephthalate dianion guest. Detailed information is obtained on the molecular dynamics and associations from the point of view of both the host and guest molecules. The formation of the complex in the solid state is confirmed using (1)H 2D exchange NMR, and the 180° flipping of the (2)H-labelled terephthalate guest and its eventual expulsion from the complex at elevated temperatures are quantified using variable-temperature (2)H spin-echo experiments. Two-dimensional (1)H-(13)C HETCOR spectra obtained under fast magic angle spinning conditions (60 kHz) show a high resolution despite the poor crystallinity of the solid complex, and clearly reveal changes in the rigidity of the host molecule when complexed. Short-range intra- and intermolecular (1)H-(1)H proximities are also detected using 2D SQ-DQ correlation methods, providing insight into the molecular packing in the solid phase.

Bioinspired Insights into Silicic Acid Stabilization Mechanisms: The Dominant Role of Polyethylene Glycol-Induced Hydrogen Bonding

Mono- and disilicic acids were stabilized by uncharged polyethylene glycols (PEGs) in silica-supersaturated solutions (the starting solution contained 500 ppm/8.3 mM sodium orthosilicate, Na2SiO3·5H2O, expressed as SiO2) at pH = 7, most likely by hydrogen bonding between the silanol groups and -CH2-CH2-O-ether moieties. The stabilization was monitored by measuring molybdate-reactive silica and also by a combination of liquid- and solid-state (29)Si NMR spectroscopy. It depends on PEG concentration (20-100 ppm) and molecular weight (1550-20,000 Da). Two narrow (29)Si NMR signals characteristic for monosilicic acid (Q(0)) and disilicic acid (Q(1)) can be observed in (29)Si NMR spectra of solutions containing PEG 10000 with intensities distinctly higher than the control, that is, in the absence of PEG. Silica-containing precipitates are observed in the presence of PEG, in contrast to the gel formed in the absence of PEG. These precipitates exhibit similar degrees of silica polycondensation as found in the gel as can be seen from the (29)Si MAS NMR spectra. However, the (2)D HETCOR spectra show different (1)H NMR signal shifts: The signal due to H-bonded SiOH/H2O, which is found at 6 ppm in the control, is shifted to ~7 ppm in the PEG-containing precipitate. This indicates the formation of slightly stronger H-bonds than in the control sample, most likely between PEG and the silica species. The presence of PEG in these precipitates is unequivocally proven by (13)C CP MAS NMR spectroscopy. The (13)C signal of PEG significantly shifts and is much narrower in the precipitates as compared to the pristine PEG, indicating that PEG is embedded into the silica or at least bound to its surface (or both), and not phase separated. FT-IR spectra corroborate the above arguments. The H-bonding between silanol and ethereal O perturbs the band positions attributed to vibrations involving the O atom. This work may invoke an alternative way to envision silica species stabilization (prior to biosilica formation) in diatoms by investigating possible scenarios of uncharged biomacromolecules playing a role in biosilica synthesis.

Indirectly detected heteronuclear correlation solid-state NMR spectroscopy of naturally abundant 15N nuclei

Two-dimensional indirectly detected through-space and through-bond 1H{15N} solid-state NMR experiments utilizing fast magic angle spinning (MAS) and homonuclear multipulse 1H decoupling are evaluated. Remarkable efficiency of polarization transfer can be achieved at a MAS rate of 40kHz by both cross-polarization and INEPT, which makes these methods applicable for routine characterizations of natural abundance solids. The first measurement of 2D 1H{15N} HETCOR spectrum of natural abundance surface species is also reported.

Identifying Selective Host–Guest Interactions Based on Hydrogen Bond Donor–Acceptor Pattern in Functionalized Al-MIL-53 Metal–Organic Frameworks

We present a study analyzing the selectivity of host–guest interactions in a series of functionalized Al-MIL-53-X metal–organic frameworks with X = H, NH2, and NHCHO using acetone, ethanol, and water as probe molecules. While the amino group introduces additional hydrogen bond donor centers the NHCHO anchors function as donor and acceptor. The guests were chosen due to their ability to act solely as an acceptor in the case of acetone, whereas ethanol and water provide acceptor and donor qualities with a gradual decrease of the acceptor strength toward ethanol. The characterization of the host–guest interactions includes a comprehensive solid-state NMR spectroscopic study based on a full assignment of 1H and 13C high-resolution spectra using CRAMPS decoupling schemes to enhance 1H resolution combined with advanced 2D HETCOR (1H–13C, 1H–27Al, and 1H–14N) spectra at high magnetic fields. In spite of a pronounced dynamical disorder of the guests, we could identify a preferred binding of the acetone via a NH··...

Determining the depth of insertion of dynamically invisible membrane peptides by gel-phase 1H spin diffusion heteronuclear correlation NMR

Solid-state NMR determination of the depth of insertion of membrane peptides and proteins has so far utilized (1)H spin diffusion and paramagnetic relaxation enhancement experiments, which are typically conducted in the liquid-crystalline phase of the lipid bilayer. For membrane proteins or peptide assemblies that undergo intermediate-timescale motion in the liquid-crystalline membrane, these approaches are no longer applicable because the protein signals are broadened beyond detection. Here we show that the rigid-solid HETCOR experiment, with an additional spin diffusion period, can be used to determine the depth of proteins in gel-phase lipid membranes, where the proteins are immobilized to give high-intensity solid-state NMR spectra. Demonstration on two membrane peptides with known insertion depths shows that well-inserted peptides give rise to high lipid cross peak intensities and low water cross peaks within a modest spin diffusion mixing time, while surface-bound peptides have higher water than lipid cross peaks. Furthermore, well-inserted membrane peptides have nearly identical (1)H cross sections as the lipid chains, indicating equilibration of the peptide and lipid magnetization. Using this approach, we measured the membrane topology of the α-helical fusion peptide of the paramyxovirus, PIV5, in the anionic POPC/POPG membrane, in which the peptide undergoes intermediate-timescale motion at physiological temperature. The gel-phase HETCOR spectra indicate that the α-helical fusion peptide is well inserted into the POPC/POPG bilayer, spanning both leaflets. This insertion motif gives insight into the functional role of the α-helical PIV5 fusion peptide in virus-cell membrane fusion.

Dynamic Nuclear Polarization Enhanced Solid‐State NMR Spectroscopy of Functionalized Metal–Organic Frameworks

Dynamic nuclear polarization (DNP) is applied to enhance the signal of solid-state NMR spectra of metal-organic framework (MOF) materials. The signal enhancement enables the acquisition of high-quality 1D 13C solid-state NMR spectra, 2D 1H-13C dipolar HETCOR and 1D 15N solid-state NMR spectra with natural isotopic abundance in experiment times on the order of minutes.

Characterization of heterogeneities and domains in aquatic and sedimentary organic matter by 1H spin diffusion: Potential for elucidating the formation mechanisms

Although the information on domains and heterogeneities of natural organic matter (NOM) can provide insights into its formation mechanisms, the appropriate solid‐state NMR technique for measuring them is still lacking. The traditional technique requires mobility differences in NOM whereas NOM components are primarily rigid. We introduced a new 1H spin diffusion technique, 1H‐13C two‐dimensional heteronuclear correlation (2D HETCOR) NMR with 1H spin diffusion, for characterization of domains and heterogeneities in aquatic and sedimentary organic matter. It was achieved by collecting a series of 2D HETCOR spectra with a variable mixing time, tm, and monitoring the transfer of magnetization from one component to another. The rate of magnetization transfer provided the information on domains and heterogeneities because the magnetization of small domains or heterogeneities equilibrated faster than that of larger ones. Three samples, International Humic Substances Society (IHSS) Suwannee River NOM, IHSS Suwannee River humic acid (HA), and a sedimentary HA, were used. Two model polymers, a random copolymer poly(styrene‐n‐butyl methacrylate) with 0.6‐nm heterogeneity and a block copolymer polystyrene‐b‐poly(methyl methacrylate) with a domain size of 5 nm, were included for calibration. Within tm < 100 µs, half equilibration was reached for all three NOM samples and poly(styrene‐n‐butyl methacrylate), indicating that they were heterogeneous. In contrast, the spin diffusion of polystyrene‐b‐poly(methyl methacrylate) with 5‐nm domain was much slower. Unlike the traditional spin diffusion technique, this technique did not require the differential mobility in NOM and was suitable for investigating the domains and heterogeneities of NOM, which are mostly rigid.

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

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.