Untitled

Abstract

Large residual (15)N-(1)H dipolar couplings have been measured in a Src homology II domain aligned at Pf1 bacteriophage concentrations an order of magnitude lower than used for induction of a similar degree of alignment of nucleic acids and highly acidic proteins. An increase in (1) H and (15)N protein linewidths and a decrease in T(2) and T(1)ρ relaxation time constants implicates a binding interaction between the protein and phage as the mechanism of alignment. However, the associated increased linewidth does not preclude the accurate measurement of large dipolar couplings in the aligned protein. A good correlation is observed between measured dipolar couplings and predicted values based on the high resolution NMR structure of the SH2 domain. The observation of binding-induced protein alignment promises to broaden the scope of alignment techniques by extending their applicability to proteins that are able to interact weakly with the alignment medium.

Structure-Free Validation of Residual Dipolar Coupling and Paramagnetic Relaxation Enhancement Measurements of Disordered Proteins

“…Working at high ionic strengths would reduce these perturbations but also increase the correlation between RDCs measured in different alignment media, thus reducing the amount of information that can be extracted from them. Suitable alignment media should, in addition, allow high-resolution spectra to be recorded without extensive line broadening caused by binding or by unresolved 1 H– 1 H dipolar interactions . Strong interactions can be mitigated by using lower concentrations of the alignment media or by screening electrostatic interactions with higher concentrations of salt. , …”

“…To measure RDCs, weak alignment of the protein molecules is induced by the presence of a liquid crystalline alignment medium, , which could alter the protein conformations through long-range interactions or even by direct binding to the surfaces of the alignment media (nematogens) . These perturbations could be expected to be more likely for disordered than for native proteins as their conformations are not stabilized by large numbers of intramolecular contacts that are present in their ordered counterparts.…”

Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold

“…It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions ( Hansen et al, 1998 , 2000 ; Dahlke Ojennus et al, 1999 ) in NMR spectroscopy.…”

Determination of the three-dimensional structure for weakly aligned biomolecules by NMR spectroscopy

“…220 Even when the bacteriophage concentration is abnormally low, these systems are effective. 223 Presumably, in this case, a different orientation mechanism is involved associated with weak binding of the protein under study to the phage. The observed orientation is due to fast exchange between the bound and free states of the protein.…”