Precise Measurement of RDCs in Water and DMSO Based Gels Using a Silicone Rubber Tube for Tunable Stretching

Abstract

Residual dipolar couplings (RDCs) are of great interest for the structure determination of biomacromolecules as well as of organic molecules like synthetic or natural products. Their accurate measurement requires a proper degree of alignment for the molecule under investigation. As has been shown recently, a stretching device based on a flexible sili- cone rubber tube provides an easy, rapid, and reversible variation of alignment strength. We show in this article that such an apparatus is not limited to gelatin, but can also be used with covalently cross-linked hydrogels and even gels with polar organic solvents like DMSO. Using sucrose and a cyclic hexapeptide, we were able to demonstrate that the approximately linear relation of alignment with the extension factor of the stretched gel allows the measurement of RDCs with high pre- cision if corresponding spectra are acquired at various alignment strengths. The method seems to be widely applicable to the structure determination of molecules of arbitrary size using standard 5 mm NMR equipment. For verification of the alignment properties of the stretch- ing apparatus, we prepared a 40% (w/v) sample of porcine gelatin with an approximate Bloomgrade of 250 dissolved at 50°C in D2O. As a solute molecule for measuring RDCs we added 43 mg of sucrose to a final concentration of approxi- mately 500 mM. Gelatin is a special gel which can be re- casted inside the rubber tube by a simple heating and recool- ing cycle, as it is cross-linked via hydrogen bonds only. In order to test how far the apparatus is applicable to conven- tional polymer gels with covalent cross-linking, we prepared several gel samples using published protocols with gel di- ameters adjusted to accomodate for the dimensions of the stretching apparatus. The results presented in this article were obtained using the following gel preparations: The poly(acrylamide) (PAA) gel was prepared from a solution of 8.55% (w/v) acrylamide and 0.45% (w/v) N,N'- methylenebis(acrylamide) with 0.1% (w/v) ammonium per- sulfate and 0.1% (v/v) N,N,N',N'-tetramethylethylendiamin for cross-linking (5). Radical polymerization was allowed for 1 h within glass tubes of 2.4 mm inner diameter. Gels were washed three times with water to remove free acrylamide before they were dried at room temperature.