Showing papers by "James U. Bowie published in 2009"

Methods for measuring the thermodynamic stability of membrane proteins.

Abstract: Learning how amino acid sequences define protein structure has been a major challenge for molecular biology since the first protein structures were determined in the 1960s. In contrast to the staggering progress with soluble proteins, investigations of membrane protein folding have long been hampered by the lack of high-resolution structures and the technical challenges associated with studying the folding process in vitro. In the past decade, however, there has been an explosion of new membrane protein structures and a slower but notable increase in efforts to study the factors that define these structures. Here we review the methods that have been used to evaluate the thermodynamic stability of membrane proteins and provide some salient examples of how the methods have been used to begin to understand the energetics of membrane protein folding.

Similar energetic contributions of packing in the core of membrane and water-soluble proteins.

Abstract: A major driving force for water-soluble protein folding is the hydrophobic effect, but membrane proteins cannot make use of this stabilizing contribution in the apolar core of the bilayer. It has been proposed that membrane proteins compensate by packing more efficiently. We therefore investigated packing contributions experimentally by observing the energetic and structural consequences of cavity creating mutations in the core of a membrane protein. We observed little difference in the packing energetics of water and membrane soluble proteins. Our results imply that other mechanisms are employed to stabilize the structure of membrane proteins.

Genetic selection system for improving recombinant membrane protein expression in E. coli.

Abstract: A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold.

Structural imperatives impose diverse evolutionary constraints on helical membrane proteins

Abstract: The amino acid sequences of transmembrane regions of helical membrane proteins are highly constrained, diverging at slower rates than their extramembrane regions and than water-soluble proteins. Moreover, helical membrane proteins seem to fall into fewer families than water-soluble proteins. The reason for the differential restrictions on sequence remains unexplained. Here, we show that the evolution of transmembrane regions is slowed by a previously unrecognized structural constraint: Transmembrane regions bury more residues than extramembrane regions and soluble proteins. This fundamental feature of membrane protein structure is an important contributor to the differences in evolutionary rate and to an increased susceptibility of the transmembrane regions to disease-causing single-nucleotide polymorphisms.

Identifying polymer-forming SAM domains.

Abstract: Sterile Alpha Motif (SAM) domains are common protein modules in eukaryotic cells. It has not been possible to assign functions to uncharacterized SAM domains because they have been found to participate in diverse functions ranging from protein-protein interactions to RNA binding. Here we computationally identify likely members of the subclass of SAM domains that form polymers. Sequences were virtually threaded onto known polymer structures and then evaluated for compatibility with the polymer. We find that known SAM polymers score better than the vast majority of known non-polymers: 100% (7 of 7) of known polymers and only 8% of known non-polymers (1 of 12) score above a defined threshold value. Of 2901 SAM family members, we find 694 that score above the threshold and are likely polymers, including SAM domains from the proteins Lethal Malignant Brain Tumor, Bicaudal-C, Liprin-β, Adenylate Cyclase and Atherin.

G-protein-coupled receptor structures were not built in a day

Abstract: Among the most exciting recent developments in structural biology is the structure determination of G-protein-coupled receptors (GPCRs), which comprise the largest class of membrane proteins in mammalian cells and have enormous importance for disease and drug development. The GPCR structures are perhaps the most visible examples of a nascent revolution in membrane protein structure determination. Like other major milestones in science, however, such as the sequencing of the human genome, these achievements were built on a hidden foundation of technological developments. Here, we describe some of the methods that are fueling the membrane protein structure revolution and have enabled the determination of the current GPCR structures, along with new techniques that may lead to future structures.

Protein unfolding with a steric trap.

Abstract: The study of protein folding requires a method to drive unfolding, which is typically accomplished by altering solution conditions to favor the denatured state This has the undesirable consequence that the molecular forces responsible for configuring the polypeptide chain are also changed It would therefore be useful to develop methods that can drive unfolding without the need for destabilizing solvent conditions Here we introduce a new method to accomplish this goal, which we call steric trapping In the steric trap method, the target protein is labeled with two biotin tags placed close in space so that both biotin tags can only be bound by streptavidin when the protein unfolds Thus, binding of the second streptavidin is energetically coupled to unfolding of the target protein Testing the method on a model protein, dihydrofolate reductase (DHFR), we find that streptavidin binding can drive unfolding and that the apparent binding affinity reports on changes in DHFR stability Finally, by employing the slow off-rate of wild-type streptavidin, we find that DHFR can be locked in the unfolded state The steric trap method provides a simple method for studying aspects of protein folding and stability in native solvent conditions, could be used to specifically unfold selected domains, and could be applicable to membrane proteins

Chapter 5 Practical Aspects of Membrane Proteins Crystallization in Bicelles

Abstract: Publisher Summary The structure determination of membrane proteins is a significant challenge. One of the important bottlenecks is growing high quality crystals. The crystallization of membrane proteins directly from detergents is by far the most popular approach; although, lipidic cubic phase crystallization is attracting increasing use. This chapter introduces the use of bicelles as an alternative method for the crystallization of membrane proteins. Bicelles are a mixture of a detergent and a lipid and can be described as a compromise between the two media with beneficial aspects from both. Membrane proteins reconstituted in bicelles are maintained in a native like bilayer environment and can be manipulated with almost the same ease as detergent solublized membrane proteins, making it compatible with standard high-throughput screening. An increasing number of recent bicelle crystallization successes support the expanded use of this method, including β 2-adrenergic receptor, the voltage-dependent anion channel, and xanthorhodopsin. This chapter describes the bicelle method, its properties, advantages and disadvantages and considers how to achieve future progress with the bicelle method.