Changes in Natural Silk Fibres by Hydration, Tensile Loading and Heating as Studied by 1H NMR: Anisotropy in NMR Relaxation Times.

B. mori silkworm natural silk is a fibrous biopolymer with a block copolymer design containing both hydrophobic and hydrophilic regions. Using 1H NMR relaxation, this work studied B. mori natural silk fibres oriented at 0° and 90° to the static magnetic field B0 to clarify how measured NMR parameters reflect the structure and anisotropic properties of hydrated silk fibres. The FTIR method was applied to monitor the changes in the silk I and ß-sheet conformations. Unloaded B. mori silk fibres at different hydration levels (HL), the silk threads before and after tensile loading in water, and fibres after a stepped increase in temperature have been explored. NMR data discovered two components in T1 and T2 relaxations for both orientations of silk fibres (0° and 90°). For the slower T2 component, the results showed an obvious anisotropic effect with higher relaxation times for the silk fibres oriented at 90° to B0. The T1 component (water protons, HL = 0.11) was sequentially decreased over a range of fibres: 0° oriented, randomly oriented, silk B. mori cocoon, 90° oriented. The degree of anisotropy in T2 relaxation was decreasing with increasing HL. The T2 in silk threads oriented at 0° and 90° also showed anisotropy in increased HL (to 0.42 g H2O/g dry matter), at tensile loading, and at an increasing temperature towards 320 K. The changes in NMR parameters and different relaxation mechanisms affecting water molecular interactions and silk properties have been discussed. The findings provide new insights relating to the water anisotropy in hydrated Bombyx mori silk fibres at tensile loading and under a changing HL and temperature.

Spin noise gradient echoes.

Nuclear spin noise spectroscopy in the absence of radio frequency pulses was studied under the influence of pulsed field gradients (PFGs) on pure and mixed liquids. Under conditions where the radiation-damping-induced line broadening is smaller than the gradient-dependent inhomogeneous broadening, echo responses can be observed in difference spectra between experiments employing pulsed field gradient pairs of the same and opposite signs. These observed spin noise gradient echoes (SNGEs) were analyzed through a simple model to describe the effects of transient phenomena. Experiments performed on high-resolution nuclear magnetic resonance (NMR) probes demonstrate how refocused spin noise behaves and how it can be exploited to determine sample properties. In bulk liquids and their mixtures, transverse relaxation times and translational diffusion constants can be determined from SNGE spectra recorded following tailored sequences of magnetic field gradient pulses.

NMR techniques in studying water in biotechnological systems.

Different NMR methodologies have been considered in studying water as a part of the structure of heterogeneous biosystems. The current work mostly describes NMR techniques to investigate slow translational dynamics of molecules affecting anisotropic properties of polymers and biomaterials. With these approaches, information about organized structures and their stability could be obtained in conditions when external factors affect biomolecules. Such changes might include rearrangement of macromolecular conformations at fabrication of nano-scaffolds for tissue engineering applications. The changes in water-fiber interactions could be mirrored by the magnetic resonance methods in various relaxations, double-quantum filtered (DQF), 1D and 2D translational diffusion experiments. These findings effectively demonstrate the current state of NMR studies in applying these experiments to the various systems with the anisotropic properties. For fibrous materials, it is shown how NMR correlation experiments with two gradients (orthogonal or collinear) encode diffusion coefficients in anisotropic materials and how to estimate the permeability of cell walls. It is considered how the DQF NMR technique discovers anisotropic water in natural polymers with various cross-links. The findings clarify hydration sites, dynamic properties, and binding of macromolecules discovering the role of specific states in improving scaffold characteristics in tissue engineering processes. Showing the results in developing these NMR tools, this review focuses on the ways of extracting information about biophysical properties of biomaterials from the NMR data obtained.

Spin-Noise-Detected Two-Dimensional Nuclear Magnetic Resonance at Triple Sensitivity.

A major breakthrough in speed and sensitivity of 2 D spin-noise-detected NMR is achieved owing to a new acquisition and processing scheme called "double block usage" (DBU) that utilizes each recorded noise block in two independent cross-correlations. The mixing, evolution, and acquisition periods are repeated head-to-tail without any recovery delays and well-known building blocks of multidimensional NMR (constant-time evolution and quadrature detection in the indirect dimension as well as pulsed field gradients) provide further enhancement and artifact suppression. Modified timing of the receiver electronics eliminates spurious random excitation. We achieve a threefold sensitivity increase over the original snHMQC (spin-noise-detected heteronuclear multiple quantum correlation) experiment (K. Chandra et al., J. Phys. Chem. Lett. 2013, 4, 3853) and demonstrate the feasibility of spin-noise-detected long-range correlation.

Nonlinear detection of secondary isotopic chemical shifts in NMR through spin noise.

The detection of minor species in the presence of large amounts of similar main components remains a key challenge in analytical chemistry, for instance, to obtain isotopic fingerprints. As an alternative to the classical NMR scheme based on coherent excitation and detection, here we introduce an approach based on spin-noise detection. Chemical shifts and transverse relaxation rates are determined using only the detection circuit. Thanks to a nonlinear effect in mixtures with small chemical shift dispersion, small signals on top of a larger one can be observed with increased sensitivity as bumps on a dip; the latter being the signature of the main magnetization. Experimental observations are underpinned by an analytical theory: the coupling between the magnetization and the coil provides an amplified detection capability of both small static magnetic field inhomogeneities and small NMR signals. This is illustrated by two-bond 12C/13C isotopic measurements.

The increase in denaturation temperature following cross-linking of collagen is caused by dehydration of the fibres.

Differential scanning calorimetry (DSC) was used to study the thermal stability of native and synthetically cross-linked rat-tail tendon at different levels of hydration, and the results compared with native rat-tail tendon. Three cross-linking agents of different length between functional groups were used: malondialdehyde (MDA), glutaraldehyde and hexamethylene diisocyanate (HMDC). Each yielded the same linear relation between the reciprocal of the denaturation temperature in Kelvin, T(max), and the water volume fraction, epsilon (1/T(max)=0.000731epsilon+0.002451) up to a critical hydration level, the volume fraction of water in the fully hydrated fibre. Thereafter, water was in excess, T(max) was constant and the fibre remained unchanged, no matter how much excess water was added. This T(max) value and the corresponding intrafibrillar volume fraction of water were as follows: 84.1 degrees C and 0.48 for glutaraldehyde treated fibres, 74.1 degrees C and 0.59 for HMDC treated fibres, 69.3 degrees C and 0.64 for MDA treated fibres, and 65.1 degrees C and 0.69 for untreated native fibres. Borohydride reduction of the native enzymic aldimines did not increase the denaturation temperature of the fibres. As all samples yielded the same temperature at the same hydration, the temperature could not be affected by the nature of the cross-link other than through its effect on hydration. Cross-linking therefore caused dehydration of the fibres by drawing the collagen molecules closer together and it was the reduced hydration that caused the increased temperature stability. The cross-linking studied here only reduced the quantity of water between the molecules and did not affect the water in intimate contact with, or bound to, the molecule itself. The enthalpy of denaturation was therefore unaffected by cross-linking. Thus, the "polymer-in-a-box" mechanism of stabilization, previously proposed to explain the effect of dehydration on the thermal properties of native tendon, explained the new data also. In this mechanism, the configurational entropy of the unfolding molecule is reduced by its confinement in the fibre lattice, which shrinks on cross-linking.