Removing acoustic ringing baseline curvature in 13 C NMR spectra for quantitative analyses.

13 C nuclear magnetic resonance (NMR) is traditionally considered an insensitive technique, requiring long acquisition times to measure dilute functionalities on large polymers. With the introduction of cryoprobes and better electronics, sensitivity has improved in a way that allows measurements to take less than 1/20th the time that they previously did. Unfortunately, a high Q-factor with cryoprobes creates baseline curvature related to acoustic ringing that affects quantitative NMR analyses. Manual baseline correction is commonly used to compensate for the baseline roll, but it is a time-intensive process. The outcome of manual baseline correction can vary depending on processing parameters, especially for complicated spectra. Additionally, it can be challenging to distinguish between broad peaks and baseline rolls. A new anti-ring pulse sequence (zgig_pisp) was previously reported to improve on the incumbent single pulse experiment (zgig). The original report presented limited comparison data with 13 C NMR, but a thorough validation is needed before broader implementation can be considered. In this work, we report the round-robin testing and comparison of zgig_pisp and zgig pulse sequences. During the testing phase, we found that zgig_pisp is practically equivalent to zgig to ±2% for the majority of integrals examined. Additionally, a short broadband inversion pulse (BIP) was demonstrated as an alternative to the originally reported adiabatic CHIRP shaped pulse. The zgig_pisp pulse sequence code for Bruker spectrometers is also simplified.

Effects of pH and calcium ions on the conformational transitions in silk fibroin using 2D Raman correlation spectroscopy and 13C solid-state NMR.

Silk fibroin exists in a number of different states, such as silk I and silk II, with different properties largely defined by differences in secondary structure composition. Numerous attempts have been made to control the transitions from silk I to silk II in vitro to produce high-performance materials. Of all the factors influencing the structural compositions, pH and some metal ions play important roles. This paper focuses on the influence of pH and Ca(2+) ions on the conformational transition from silk I to silk II in regenerated (redissolved) Bombyx mori fibroin. One- and two-dimensional correlation Raman spectroscopy was used to describe qualitatively the transitions in secondary structure in silk I, silk II, and their intermediates as pH and Ca(2+) ion concentration were changed, while (13)C cross polarization magic angle spinning (CP/MAS) solid-state NMR was used to quantify these changes. We showed that conditions (low pH, pH 5.2; a defined range of Ca(2+) ion concentrations; gradual water removal) that mimic natural silk spinning promote the formations of beta-sheet and distorted beta-sheet characteristic of silk II or silk II-related intermediate. In contrast, higher pH (pH 6.9-8.0) and higher Ca(2+) ion concentrations maintain "random coil" conformations typical of silk I or silk I-related intermediate. These results help to explain why the natural silk spinning process is attended by a reduction in pH from 6.9 to 4.8 and a change in the Ca(2+) ion concentration in the gland lumen as fibroin passes from the posterior division through the secretory pathway to the anterior division.

Effect of pH and copper(II) on the conformation transitions of silk fibroin based on EPR, NMR, and Raman spectroscopy.

Much attention has been paid to the natural mechanism of silkworm spinning due to the impressive mechanical properties of the natural fibers. Our results in the present work show that the fractional changes of the conformational components in regenerated silk fibroin (SF) extracted from Bombyx mori fibers is remarkably pH- and Cu(II)-dependent as demonstrated by Cu(II) EPR, (13)C NMR, and Raman spectroscopy. Cu(II) coordination atoms in SF are changed from four nitrogens to two nitrogens and two oxygens as well as to one nitrogen and three oxygens when the pH is lowered from 8.0 to 4.0. The addition of a given amount of Cu(II) into a SF solution could induce efficiently the SF conformational fractional change from silk I, a soluble helical conformation, to silk II, an insoluble beta-sheet conformation. This behavior is strikingly similar to that seen in prion protein and amyloid beta-peptide. On the basis of the similarity in the relevant sequence in SF to the octapeptide PHGGGWGQ in PrP, we suggest that at basic and neutral pH polypeptide AHGGYSGY in SF may form a 1:1 complex with Cu(II) by coordination of imidazole N(pi) of His together with two deprotonated main-chain nitrogens from two glycine residues and one nitrogen or oxygen from serine. Such a type of coordination may make the interaction between two adjacent beta-form polypeptide chains more difficult, thereby leading to an amorphous structure. Under weakly acidic conditions, however, Cu(II)-amide linkages may be broken and Cu(II) may switch to bind two N(tau) from two histidines in adjacent peptide chains, forming an intermolecular His(N(tau))-Cu(II)-His(N(tau)) bridge. This type of coordination may induce beta-sheet formation and aggregation, leading to a crystalline structure.