Alan Finkelstein, Ph.D.

PROTEIN TRANSLOCATION ASSOCIATED WITH CHANNEL GATING

We study voltage-gated channels formed by the bacterial protein toxins diphtheria, anthrax and colicin Ia.  By investigating these toxins using planar lipid bilayer membranes, we have shown that associated with the opening and closing of both diphtheria toxin channels and colicin Ia channels, there is massive translocation of parts of these proteins back and forth across the membrane.  In the case of diphtheria toxin, this consists of the N-terminal 270 residues bearing the cytotoxic activity.   For colicin Ia, a region of about 70 residues is translocated with channel opening and closing, and inserted foreign epitopes, including an intact, highly charged protein, can be translocated as well.   

Our use of the in vitro bilayer system has, for example, allowed us to dissect the requirements for specific amino acid residues for proper insertion of the diphtheria toxin channels in the membrane or the degree to which anthrax toxin lethal factor must unfold as it passes through the channel formed by heptamers of its protective antigen, one of the components of this important tripartite toxin. 

These molecules appear to be capable of translocating "any" sequence of polar residues. Our research is ultimately directed at deducing the channel structure, identifying the voltage sensor, and determining the mechanism and pathway of protein translocation.

The bilayer system has also been used to gain some understanding of an important family of bacterial outer membrane proteins, the TonB-dependent transporters (TBDT).  These proteins are used by bacteria for the uptake of essential siderophore-bound iron and vitamins, and have also been co-opted by colicins and phages as their receptors.  The TBDT are all b-barrels with a plug formed by the N terminus of the protein, and we have found conditions where response of the plug to its biological substrates can be investigated in the bilayer system.  Some of the TBDTs are also investigated in vivo in our laboratory, using translocation of colicin proteins as a model system to understand what causes these transporters to unplug (open) to allow the uptake of their natural substrates.

Recently, we have also begun investigating African trypanosome parasites and the nature of a blood factor, apolipoprotein L1 (apoL1) that confers resistance to the trypanosomes on some primate species.  We have shown that apoL1 makes channels in planar lipid bilayer membranes.

Thomson R, Finkelstein A. Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: relevance to trypanosome lysis. Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):2894-9. doi: 10.1073/pnas.1421953112. Epub 2015 Feb 17.  PMID: 25730870.

Kienker PK, Wu Z, Finkelstein A. Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel. J Gen Physiol. 2015 Feb;145(2):107-25. doi: 10.1085/jgp.201411326. Epub 2015 Jan 12. PMID: 25582482.

Schiffmiller A, Finkelstein A. Ion Conductance of the Stem of the Anthrax Toxin Channel during Lethal Factor Translocation. J Mol Biol. 2014 Jul 1. pii: S0022-2836(14)00328-3. doi: 10.1016/j.jmb.2014.06.016. [Epub ahead of print] PubMed PMID: 24996036. 

Vargas-Uribe M, Rodnin MV, Kienker P, Finkelstein A, Ladokhin AS. Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state. Biochemistry. 2013 May 21;52(20):3457-63. doi: 10.1021/bi400249f. Epub 2013 May 9. PubMed PMID: 23621842; PubMed Central PMCID: PMC3772984. 

Basilio D, Jennings-Antipov LD, Jakes KS, Finkelstein A. Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins. J Gen Physiol. 2011 Apr;137(4):343-56. doi: 10.1085/jgp.201010578. Epub 2011 Mar 14. PubMed PMID: 21402886; PubMed Central PMCID: PMC3068283. 

Jakes KS, Finkelstein A. The colicin Ia receptor, Cir, is also the translocator for colicin Ia. Mol Microbiol. 2010 Feb;75(3):567-78. doi: 10.1111/j.1365-2958.2009.06966.x. Epub 2009 Nov 17. PubMed PMID: 19919671; PubMed Central PMCID: PMC3493618. 

Rodnin MV, Kyrychenko A, Kienker P, Sharma O, Posokhov YO, Collier RJ, Finkelstein A, Ladokhin AS. Conformational switching of the diphtheria toxin T domain. J Mol Biol. 2010 Sep 10;402(1):1-7. doi: 10.1016/j.jmb.2010.07.024. Epub 2010 Jul 21. PubMed PMID: 20654627; PubMed Central PMCID: PMC3042302. 

Udho E, Jakes KS, Buchanan SK, James KJ, Jiang X, Klebba PE, Finkelstein A. (2009). Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes. Proc Natl Acad Sci U S A. 106(51):21990-5.

Basilio D, Juris SJ, Collier RJ, Finkelstein A. Evidence for a proton-protein symport mechanism in the anthrax toxin channel. J Gen Physiol. 2009 Mar;133(3):307-14. doi: 10.1085/jgp.200810170. Epub 2009 Feb 9. PubMed PMID: 19204186; PubMed Central PMCID: PMC2654084. 

Janowiak BE, Finkelstein A, Collier RJ. (2009). An approach to characterizing single-subunit mutations in multimeric prepores and pores of anthrax protective antigen. Protein Sci. 18(2):348-58.

Finkelstein A. Proton-coupled protein transport through the anthrax toxin channel. Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):209-15. doi: 10.1098/rstb.2008.0126. PubMed PMID: 18957378; PubMed Central PMCID: PMC2674091.

Kienker PK, Jakes KS, Finkelstein A. Identification of channel-lining amino acid residues in the hydrophobic segment of colicin Ia. J Gen Physiol. 2008 Dec;132(6):693-707. doi: 10.1085/jgp.200810042. PubMed PMID: 19029376; PubMed Central PMCID: PMC2585860. 

Slatin SL, Finkelstein A, Kienker PK. (2008). Anomalous proton selectivity in a large channel: colicin A. Biochemistry. 47(6):1778-88.

 Wu Z, Jakes KS, Samelson-Jones BS, Lai B, Zhao G, London E, Finkelstein A. (2006). Protein translocation by bacterial toxin channels: a comparison of diphtheria toxin and colicin Ia. Biophys J. 91(9):3249-56.

Krantz BA, Finkelstein A, Collier RJ. (2006). Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient. J Mol Biol. 355(5):968-79.

Krantz BA, Melnyk RA, Zhang S, Juris SJ, Lacy DB, Wu Z, Finkelstein A, Collier RJ. (2005). A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore. Science. 309(5735):777-81.

Zhang S, Finkelstein A, Collier RJ. (2004). Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel. Proc Natl Acad Sci U S A. 101(48):16756-61.

Zhang S, Udho E, Wu Z, Collier RJ, Finkelstein A. (2004). Protein translocation through anthrax toxin channels formed in planar lipid bilayers. Biophys J. 87(6):3842-9.

Nassi S, Collier RJ, Finkelstein A. (2002). PA₆₃ channel of anthrax toxin: an extended beta-barrel. Biochemistry. 41(5):1445-50.

Kienker PK, Jakes KS, Finkelstein A. (2000).  Protein translocation across planar bilayers by the colicin Ia channel-forming domain: where will it end? J Gen Physiol. 116(4):587-98.

Senzel L, Gordon M, Blaustein RO, Oh KJ, Collier RJ, Finkelstein A. (2000).  Topography of diphtheria Toxin's T domain in the open channel state. J Gen Physiol. 115(4):421-34.