Membrane protein structure and assembly



Since membrane proteins are particularly difficult to crystallize and, thus, difficult to structurally analyze, we have developed genetic techniques to determine aspects of their structure. First, we presented a gene fusion technique that allows one to describe the topology of a membrane protein. This approach generates a model for the arrangement of the protein in the membrane including which hydrophilic domains are in the cytoplasm and which are in the extracytoplasmic space (see picture).We have also developed a reporter system that permits detection of interaction between transmembrane segments of proteins. For instance, it can detect those transmembrane segments that are capable of homodimerization. We are also interested in the mechanism of assembly of proteins into membranes. A genetic screeing procedure for defects in membrane protein insertion has revealed mutants in a bacterial homologue of a component of the eukaryotic signal recognition particle and we are analyzing them using a sensitive reporter system. Finally, we have developed a computer program that allows one to take sequences of genomes of organisms and estimate how many membrane proteins that organism is capable of encoding.


Seung Hyun Cho

Disulfide bond isomerization in bacterial periplasm is mediated by DsbC. The reduced state of DsbC is necessary for its activity and maintained by DsbD. DsbD consists of two periplasmic domains and a membrane-embedded one, and transfers electrons from the cytoplasm to the periplasm. The transmembrane domain interacts with the cytoplasmic thioredoxin-1 and its periplasmic thioredoxin-fold domain, catalyzing electron transfer between them. By structural and functional study, we showed that the two redox-active cysteines of transmembrane domain are exposed to both compartments and the structure of it shows an hourglass-like shape. We are further characterizing the structural features of this unusual transmembrane domain. Especially, we are trying to get high-resolution structure of DsbD or a homologue.


People currently involved in this project: Dana Boyd, Mark Gonzalez, Seung Hyun Cho, and Markus Eser

Recent Publications:

Ye J, Cho SH, Fuselier J, Li W, Beckwith J, Rapoport TA. Cystal structure of an unusual thioredoxin protein with a zinc finger domain. J Biol Chem. 2007 Oct 3;

Cho SH, Porat A, Ye J, Beckwith J. Redox-active cysteines of a membrane electron transporter DsbD show dual compartment accessibility. EMBO J. 2007 Aug 8;26(15):3509-20

Buddelmeijer N, Beckwith J. A complex of the Escherichia coli cell division proteins FtsL, FtsB and FtsQ forms independently of its localization to the septal region. (2004). Abstract. Paper.

Katzen F, Beckwith J. Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD. (2003). Abstract. Paper.

Tian H, Beckwith J. Genetic Screen Yields Mutations in Genes Encoding All Known Components of the Escherichia coli Signal Recognition Particle Pathway. J Bacteriol. 184:111-118. (2002). Abstract. Paper.

Leeds JA, Boyd D, Huber DR, Sonoda GK, Luu HT, Engelman HT, Beckwith J. Genetic selection for and molecular dynamic modeling of a protein transmembrane domain multimerization motif from a random Escherichia coli genomic library. J. Mol. Biol. 313:181-5. (2001). Abstract. Paper.

Leeds, JA and Beckwith, J. A gene fusion method for assaying interactions of protein transmembrane segments in vivo. Methods Enzymol. 327:164-75. (2000). No abstract available.

Tian H, Boyd D, Beckwith J. A mutant hunt for defects in membrane protein assembly yields mutations affecting the bacterial signal recognition particle and Sec machinery. Proc. Natl. Acad. Sci. USA. 97:4730-4735. (2000). Abstract. Paper.

Boyd, D., Schierle, C., and Beckwith, J. How many membrane proteins are there? Protein Sci. 7:201-205 (1998). Abstract.

Leeds, J.A., and Beckwith, J. Lambda repressor N-terminal DNA binding domain as an assay for protein transmembrane segment interactions in vivo. J. Mol. Biol. 280:799-810 (1998). Abstract.

Prinz, W.A., Boyd, D.H., Ehrmann, M., and Beckwith, J. The protein translocation apparatus contributes to determining the topology of an integral membrane protein in Escherichia coli. J. Biol. Chem. 273:8419-8424 (1998). AbstractPaper.

Manoil, C., and J. Beckwith. A genetic approach to analyzing membrane protein topology. Science 233: 1403-1408 (1986). Abstract.