ABSTRACT
The fundamental basis of T1 and T2 contrast in brain MRI is not well understood; recent literature contains conflicting views on the nature of relaxation in white matter (WM). We investigated the effects of inversion pulse bandwidth on measurements of T1 and T2 in WM. Hybrid inversion-recovery/Carr-Purcell-Meiboom-Gill experiments with broad or narrow bandwidth inversion pulses were applied to bovine WM in vitro. Data were analysed with the commonly used 1D-non-negative least squares (NNLS) algorithm, a 2D-NNLS algorithm, and a four-pool model which was based upon microscopically distinguishable WM compartments (myelin non-aqueous protons, myelin water, non-myelin non-aqueous protons and intra/extracellular water) and incorporated magnetization exchange between adjacent compartments. 1D-NNLS showed that different T2 components had different T1 behaviours and yielded dissimilar results for the two inversion conditions. 2D-NNLS revealed significantly more complicated T1/T2 distributions for narrow bandwidth than for broad bandwidth inversion pulses. The four-pool model fits allow physical interpretation of the parameters, fit better than the NNLS techniques, and fits results from both inversion conditions using the same parameters. The results demonstrate that exchange cannot be neglected when analysing experimental inversion recovery data from WM, in part because it can introduce exponential components having negative amplitude coefficients that cannot be correctly modeled with nonnegative fitting techniques. While assignment of an individual T1 to one particular pool is not possible, the results suggest that under carefully controlled experimental conditions the amplitude of an apparent short T1 component might be used to quantify myelin water.
Subject(s)
Magnetic Resonance Spectroscopy/methods , White Matter/chemistry , Algorithms , Animals , Body Water/chemistry , Cattle , Extracellular Space/chemistry , Least-Squares Analysis , Magnetic Resonance Spectroscopy/statistics & numerical data , Models, Statistical , Myelin Sheath/chemistryABSTRACT
Rotational-Echo Double-Resonance (REDOR) NMR on strategically 13C and 15N labeled samples is used to study the conformation of the LGXQ (X = S, G, or N) motif in the major ampullate gland dragline silk from the spider Nephila clavipes. A method is described for calculating REDOR dephasing curves suitable for background subtractions, using probability distributions of nitrogen atoms surrounding a given carbon site, which are developed from coordinates in the Brookhaven Protein Data Bank. The validity of the method is established by comparison to dephasings observed from natural abundance 13C peaks for G and A. Straightforward fitting of universal REDOR dephasing curves to the background corrected peaks of interest provides results which are not self-consistent, and a more sophisticated analysis is developed which better accounts for 15N labels which have scrambled from the intended positions. While there is likely some heterogeneity in the structures formed by the LGXQ sequences, the data indicate that they all form compact turn-like structures.
Subject(s)
Fibroins , Magnetic Resonance Spectroscopy/methods , Proteins/chemistry , Spiders/chemistry , Amino Acid Sequence , Animals , Biopolymers/chemistry , Biopolymers/genetics , Carbon Isotopes , Leucine/chemistry , Models, Molecular , Molecular Sequence Data , Nitrogen Isotopes , Protein Conformation , Protein Structure, Secondary , Proteins/genetics , Spiders/geneticsABSTRACT
The molecular origin of the exceptional mechanical properties of spider silk is unclear. This paper presents solid-state 2H nuclear magnetic resonance data from unoriented, oriented, and supercontracted fibers, indicating that the crystalline fraction of dragline silk consists of two types of alanine-rich regions, one that is highly oriented and one that is poorly oriented and less densely packed. A new model for the molecular-level structure of individual silk molecules and their arrangement in the fibers is proposed. These data suggest that it will be necessary to control the secondary structure of individual polymer molecules in order to obtain optimum properties in bio-inspired polymers.
Subject(s)
Fibroins , Insect Proteins , Proteins/chemistry , Spiders/chemistry , Alanine/analysis , Algorithms , Amino Acid Sequence , Animals , Crystallization , Crystallography, X-Ray , Glycine/analysis , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Peptides/analysis , Protein Conformation , Protein Structure, Secondary , SilkABSTRACT
Solid-state 31P-NMR of Nephila clavipes dragline silk indicates the presence of phosphorus in at least two chemically distinct environments. Amino acid analyses of acid-hydrolyzed silk confirm the presence of phosphotyrosine as one of the phosphorus-containing components. The unusual chemical shift (18.9 ppm downfield from 85% H3PO4), proton chemical shift, and acid lability of a second component suggest that it is part of a strained five-membered cyclic phosphate that might be found on a beta-D-ribose. The five-membered cyclic phosphate is not removed from the silk fibers by exhaustive aqueous extraction. It is absent in nascent silk fibroin from the glands, suggesting that its formation is part of the fiber processing that occurs in the ducts leading to the spinnerets. High-resolution NMR spectra of silk dissolved in propionic acid/12 N HCl (50:50 v/v) show five phosphorus sites assigned to phosphorylated tyrosine residues, phosphorylated serine residues, inorganic phosphate, and two hydrolysis products of the cyclic phosphate compound. The observed posttranslational phosphorylation may be important in the processing and modulation of the physical properties of dragline silk.
