One of the most widely used experimental approaches in virtually every life sciences laboratory, protein expression has witnessed, particularly in recent years, transformative changes that were greatly catalyzed by advances in biotechnology.
Depending on several factors, including individual characteristics and downstream applications, proteins can be generated in various settings, most frequently involving bacterial, eukaryotic, and cell-free expression systems.
Proteins represent one of the major therapeutic targets. Accordingly, the strategies that are used to characterize these targets influence drug discovery and development efforts.
“It is beneficial to use a number of different biophysical approaches to characterize protein structure to be confident that one can obtain relevant structural information that is not adversely influenced by a particular method,” says Brian K. Kobilka, M.D., professor of molecular and cellular physiology and medicine at Stanford University School of Medicine and co-recipient of the 2012 Nobel Prize in Chemistry.
In a recent study, Dr. Kobilka and colleagues used NMR to study the beta 2-adrenergic receptor, a prototypical G-protein-coupled receptor. Approximately 40–50% of the existing therapeutic agents target GPCRs, the most diverse group of eukaryotic membrane receptors, making them occupy a particularly important position in the therapeutic arena.
By using NMR spectroscopy to examine the effect of different drugs on receptor structure, Dr. Kobilka and colleagues unveiled a significant conformational flexibility that exists particularly in the agonist-bound receptor, pointing toward the importance of studying protein dynamics for a better understanding of the events shaping signal transduction. “The most challenging aspect is finding ways to express and prepare labeled G-protein-coupled receptors for NMR studies, so that we can learn about the timescales of conformational changes,” notes Dr. Kobilka.
Structure-function relationship studies are particularly difficult for membrane proteins, which have lagged in terms of X-ray crystallographic characterization, and for which NMR approaches also open challenges. “We learned quite a bit about proteins by using fluorescent techniques even before crystal structures became available, and these studies ultimately informed our approach to generating crystals, but having the crystal structures now helps us interpret fluorescence, NMR, and other biophysical experiments, and they are all powerfully complementing each other,” remarks Dr. Kobilka.
Part of the difficulty in understanding the structure-function relationships of GPCRs stems from their highly dynamic behavior, the complex environment within the phospholipid membrane bilayer where their biology has to be captured, and the associated signaling proteins that are also important for their activity.
“We continue to explore not only NMR but also other approaches, including fluorescence, single-molecule methods, and electron paramagnetic resonance spectroscopy, and one aspect that we are really interested in is understanding the role of lipids,” concludes Dr. Kobilka.