Technical Highlight - November 2012
Short description: Using a series of NMR experiments, weak interactions between membrane proteins can be classified as specific or nonspecific.
NMR has proved to be a valuable tool for investigating protein-protein interactions. It can provide information difficult to obtain via other methods, such as the interface between two proteins, dissociation constants and Weak Interactions.
While the ability to observe Weak Interactions can be extremely useful, it often requires high concentrations of an interacting partner, which can result in nonspecific interactions. In the case of membrane proteins, two proteins can be forced to interact if the number of available micelles is low, a process known as forced cohabitation. So how does one determine whether a weak interaction is specific (i.e., a stoichiometric single-solution structure) or the result of nonspecific interactions? In a recent study, Sanders and colleagues (PSI MPSbyNMR) describe a technique to differentiate between specific and nonspecific Weak Interactions in transmembrane proteins by NMR.
The authors used two transmembrane domains, C99 from the amyloid precursor protein and the C-terminal domain from CD147, as a model system in which to evaluate interactions between membrane proteins. Changes in the chemical shifts of the C99 heteronuclear single-quantum coherence (HSQC) spectrum upon the addition of CD147 at low concentrations of detergent indicated that an interaction occurs between the two proteins. To determine whether this interaction was specific, the authors collected a series of HSQC spectra of 15N-labeled C99 with various concentrations of both unlabeled CD147 and two spin-labeled CD147 constructs as well as a negative control membrane protein, KCNE1.
First, a comparison of the HSQC spectra of C99 with excess CD147 and with a negative control membrane protein, both at low detergent levels, enabled the authors to rule out an interaction between C99 and CD147 due to forced cohabitation. Next, by adding a spin label to either the extracellular or intracellular domains of CD147, the authors determined that the spin label only affected the HSQC peaks for residues on one end of C99—the end on the same side of the membrane as the spin label. These data suggest that the interaction between CD147 and C99 is specific.
This study provides a framework that can be used to assess the interaction between other membrane proteins to determine whether a complex merits further characterization.
T. Zhuang et al. Solution NMR Approaches for Establishing Specificity of Weak Heterodimerization of Membrane Proteins.
J Am Chem Soc. 133, 20571-20580 (2011). doi:10.1021/ja208972h