Work

From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

Year: 2016

Type: article

Abstract: Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, α-helical and β-barrel. While α-helical membrane proteins can be found in nearly all cellular membranes, β-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite β-barrel outer membrane protein (OMP), general protocols are presented for the production of target β-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

Source: Journal of Visualized Experiments

DOI: 10.3791/53245

Authors: Nicholas Noinaj, Stephen Mayclin, Ann M. Stanley, Christine C. Jao, Susan K. Buchanan

Institutions: Purdue University West Lafayette, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of General Medical Sciences

Language: English

Topic: Enzyme Structure and Function

Subfield: Materials Chemistry

Field: Materials Science

Domain: Physical Sciences

Open Access status: bronze