Contents 1. Introduction 3519 2. Origin of Residual Dipolar Couplings and Complementary Observables 3520 2.1. Residual Dipolar Couplings (RDCs) 3520 2.2. Chemical Shift Anisotropy (CSA) 3521 2.3. Pseudocontact Shifts in Paramagnetic Systems 3522 2.4. Cross-Correlated Relaxation 3522 3. Alignment of Samples 3523 3.1. Bicelles 3523 3.2. Bacteriophage 3523 3.3. Polyacrylamide Gels 3524 3.4. Other Media 3524 3.5. Practical and Theoretical Considerations 3524 4. RDC Data Acquisition 3526 4.1. One-Bond H N −N and C−H RDCs 3526 4.2. Other Protein Backbone RDCs 3528 4.3. One-Bond C−C and C−H RDCs in Protein Side-Chains 3531 4.4. Proton−Proton RDCs 3531 5. Structural Interpretation of RDCs 3532 5.1. Additional Constraints in Structure Determination and Refinement 3533 5.2. Direct Structure Determination 3533 5.3. A Tool for Structure Validation or Homology Searches 3535 5.4. Orientational Relationships between Components 3535 6. Limitations and Future Directions 3537 7. Abbreviations 3537 8. Acknowledgments 3538 9. References 3538

For Abstract see ChemInform Abstract in Full Text.

No abstract

Solid-state NMR techniques were used to study two different types of spider silk from two Australian orb-web spider species, Nephila edulis and Argiope keyserlingi. A comparison of (13)C-T(1) and (1)H-T(1rho) solid-state NMR relaxation data of the Ala Calpha, Ala Cbeta, Gly Calpha, and carbonyl resonances revealed subtle differences between dragline and cocoon silk. (13)C-T(1rho) and (1)H-T(1) relaxation experiments showed significant differences between silks of the two species with possible structural variations. Comparison of our data to previous (13)C-T(1) relaxation studies of silk from Nephila clavipes (A. Simmons et al., Macromolecules, 1994, Vol. 27, pp. 5235-5237) also supports the finding that differences in molecular mobility of dragline silk exist between species. Interspecies differences in silk structure may be due to different functional properties. Relaxation studies performed on wet (supercontracted) and dry silks showed that the degree of hydration affects relaxation properties, and hence changes in molecular mobility are correlated with functional properties of silk.

A simple solid-state NMR method was used to study the structure of 13 C-and 15 Nenriched silk from two Australian orb-web spider species, Nephila edulis and Argiope keyserlingi. Carbon-13 and 15 N spectra from alanine-or glycine-labeled oriented dragline silks were acquired with the fiber axis aligned parallel or perpendicular to the magnetic field. The fraction of oriented component was determined from each amino acid, alanine and glycine, using each nucleus independently, and attributed to the ordered crystalline domains in the silk. The relative fraction of ordered alanine was found to be higher than the fraction of ordered glycine, akin to the observation of alanine-rich domains in silk-worm (Bombyx mori) silk. A higher degree of crystallinity was observed in the dragline silk of N. edulis compared with A. keyserlingi, which correlates with the superior mechanical properties of the former #

For many biological molecules, determining their geometry as they exist in a membrane environment is a crucial step in understanding their function. Variable angle sample spinning (VASS) NMR provides a new route to obtaining geometry information on membrane-associating molecules; it has been used here to scale and separate anisotropic contributions to phosphorus chemical shifts in NMR spectra of phosphatidylinositol phosphates. The procedure allows spectral assignment via correlation with isotropic chemical shifts and determination of a family of probable headgroup orientations via interpretation of anisotropic shift contributions. The molecules studied include phosphtidylinositol-4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). A membrane-like environment is provided by a dispersion of alkyl-poly(ethylene) glycols and n-alcohols that forms a field-orienting liquid crystal with a director that can be manipulated by varying the sample spinning axis. The experiments presented indicate that the variable angle sample spinning method will provide a direct approach for assignment and extraction of structural information from membrane-associating biomolecules labeled with a wider variety of NMR active isotopes.

Analysis of the 13C isotopic labeling patterns of nucleoside monophosphates (NMPs) extracted from Escherichia coli grown in a mixture of C-1 and C-2 glucose is presented. By comparing our results to previous observations on amino acids grown in similar media, we have been able to rationalize the labeling pattern based on the well-known biochemistry of nucleotide biosynthesis. Except for a few notable absences of label (C4 in purines and C3′ in ribose) and one highly enriched site (C1′ in ribose), most carbons are randomly enriched at a low level (an average of 13%). These sparsely labeled NMPs give less complex NMR spectra than their fully isotopically labeled analogs due to the elimination of most 13C–13C scalar couplings. The spectral simplicity is particularly advantageous when working in ordered systems, as illustrated with guanosine diphosphate (GDP) bound to ADP ribosylation factor 1 (ARF1) aligned in a liquid crystalline medium. In this system, the absence of scalar couplings and additional long-range dipolar couplings significantly enhances signal to noise and resolution.