1. A Genetic Approach to Analyzing Membrane Protein Topology Colin Manoil and Jon Beckwith Science, Vol. 233, 1403-1408, September 26, 1986

2. Question: After reading this article we decided to create some of our very own alkaline phosphatase fusions to investigate the topology of a “fictional” membrane protein we have named, BADH, which we discovered recently from an “unique” bacterium known as B. anseli. Our new protein, like the E. coli Tsr protein, seems to be involved in chemotaxis as a chemoreceptor and so we hypothesize that it may closely resemble Tsr in its membrane protein topology as well. After careful screening of various fusions we identified one, named pBA100, with high alkaline phosphatase activity of 330 units/OD 600 compared to other fusions with varying activities of between 5-9 units/OD 600. Based on the results of the “Beckwith and Manoil” paper, where on the schematic diagram below of the BADH protein, would you expect that the TnphoA inserted itself onto the BADH gene to produce the fusion pBA100 (use arrows to indicate)?

3. Answer:

4. Background The study of the three-dimensional structure of proteins is a key to unlocking the mystery of their interactions in vivo One of the most informative methods for discerning protein structure is x-ray crystallography If a protein can be crystallized, then the diffraction of x-rays by the crystals can be used to determine the position of every atom in the molecule Membrane proteins are difficult to crystallize and structurally analyze Genetic approaches to analyzing membrane protein structure offer a useful alternative to traditional diffraction studies

5. Introduction Part I Integral membrane proteins have easily identified long contiguous stretches of hydrophobic amino acids Structural analyses of photosynthetic reaction center polypeptides have shown that such long hydrophobic sequences correspond to transmembrane alpha-helical stretches of a membrane protein (J. Mol. Biol. 163, 451 (1983)) Using the plot of average hydrophobicity along the sequence of a membrane protein, one can determine possible 2-D membrane topologies for the polypeptide (J. Mol. Biol. 157, 105 (1982)) Previous methods of studying membrane protein topology included: 1. Studying or identifying sites that interact with proteins of known cellular location (Methods Enzymo. 98, 91 (1983)) 2. Reaction with small molecules, proteases or antibodies added from one side or other of the membrane (Methods Enzymo. 125, 453 (1986)) 3. NMR spectroscopy of purified membrane proteins (J. Biol. Chem. 249, 8019 (1974))

6. Introduction Part II In this paper the authors propose a gene fusion approach to studying membrane protein toplogy The chemorececptor protein Tsr, in E. coli, has a tetrameric transmembrane structure, organized in such a way that each polypeptide chain has a periplasmic domain between two transmembrane sequences, and a large cytoplasmic domain at the carboxyl terminus of the protein Alkaline phosphatase needs to be exported to the periplasm in order to show enzymatic activity (J. Bacteriol. 154, 366 (1983)) For this study, fusions of alkaline phosphatase to Tsr protein were made by random insertion of transposon TnphoA into a plasmid carrying the tsr gene and screened on media containing the alkaline phosphatase indicator, 5-bromo-4chloro-3-indolyl phosphate

7. Hypothesis The alkaline phosphatase activity of genetically engineered fusions at different positions on the E. coli Tsr protein should reflect the normal membrane topology of the protein.

8. Figure 1- Scheme for using alkaline phosphatase fusions to identify membrane protein topology

9. Table 1- Properties of tsr-phoA fusion plasmids

10. Figure 2-Fusions of alkaline phosphatase to chemoreceptor proteins

11. Fusions to a Tsr Protein Deletion Mutant Analyzed fusions of alkaline phosphatase to the cytoplasmic end of a mutant Tsr protein lacking second transmembrane domain (tsrΔ1) Without this domain, normally cytoplasmic regions might pass into the periplasm pCM234, pCM235 have high enzymatic activity

12. Figure 3-Fusions of Alkaline Phosphatase to a Deletion Derivative of tsr Protein

13. Fusions to a Tsr Protein Deletion Mutant (cont.) Used Tnpho1 to insert the wild-type second transmembrane sequence back into the plasmids that express fusion proteins with high AP activity These transmembrane seuqences are expected to translocate alkaline phosphatase back into the cytoplasm, and the resulting fusion plasmids should show low activity pCM251, pCM252

14. Table 1- Properties of tsr-phoA fusion plasmids.

15. Activation of a Low Activity Fusion Protein Hypothesized that any manipulation involving the deletion of the second transmembrane sequence increases activity pCM211 Expect plasmids without RV 1 -RV 3 and RV 2 -RV 3 sequences to show high activity

16. Figure 4-Activation of the Alkaline Phosphatase in a Cytoplasmic Domain Hybrid Protein

17. Activation of a Low Activity Fusion Protein (cont.) Most transformed colonies were Pho- Examined Pho- and Pho+ colonies through restriction analysis Pho- colonies had precise RV 1 -RV 3 deletions (ex. Plasmid pw1) Pho+ colonies had: 1)RV 2 -RV 3 deletions (ex. plasmid pb1) 2)RV 1 -RV 3 deletions with a small loss of DNA (ex. plasmid pb4)

18. Sequence of tsr Protein in This Study Differs From Published Sequence Precise RV 1 -RV 3 deletions lead to TnphoA sequence being out of frame and low enzymatic activity Comparison of the sequences of pb1 and pb4 with pw1 identified a difference in the translational reading frame of EcoRV 1 between the sequence of this plasmid and the published tsr sequence Any precise deletions involving RV 1 would put the phoA DNA out of frame and lead to low AP activity

19. Fractionation of tsr-phoA Hybrid Proteins Grow cells with labeled amino acids Osmotically shock cells and centrifuge SupernatantPeriplasmic fraction Resuspend pellet and incubate cells with lysozyme CentrifugeSupernatant Pellet Cytoplasmic fraction Membrane fraction Fractions analyzed via SDS gel electrophoresis

20. Fractionation of tsr-phoA Hybrid Proteins β-lactamase – alkaline phosphatase hybrids secreted to periplasm give fragments the size of alkaline phosphatase 1 Full-length hybrid proteins fractionated primarily with the membrane Hybrid proteins degraded to size of alkaline phosphatase in periplasmic fractions (ex. pCM203 and pCM235), consistent with a periplasmic location of alkaline phosphatase in these fusions 1 Proc. Natl. Acad. Sci. U.S.A. 82, 5107 (1985)

21. Table 2-Fractionation of tsr-phoA Hybrid Proteins

22. Potential Problems Potential problems with this approach identified by the authors: The protein sequence carboxyl terminal to the fusion junction must be nonessential to protein localization Alkaline phosphatase must not dominate localization of the hybrid Need to combine traditional crystallographic techniques with this approach Can’t apply this technique if alkaline phosphatase is toxic to the host cell Need to show a negative control for alkaline phosphatase activity

23. Conclusion The transmembrane sequences of tsr are responsible for how the protein inserts into the membrane In-frame fusion proteins lacking the second tsr transmembrane domain show decreased alkaline phosphatase activity Replacement of the second transmembrane domain, recovers the alkaline phosphatase activity TnphoA insertion into the periplasmic domain of Tsr protein results in high alkaline phosphatase activity whereas insertion into the cytoplasmic domain results in low alkaline phosphatase activity

24. Topology prediction  PSORT - Prediction of protein subcellular localization PSORT  TargetP - Prediction of subcellular location TargetP  DAS - Prediction of transmembrane regions in prokaryotes using the Dense Alignment Surface method (Stockholm University) DAS  HMMTOP - Prediction of transmembrane helices and topology of proteins (Hungarian Academy of Sciences) HMMTOP  PredictProtein - Prediction of transmembrane helix location and topology (Columbia University) PredictProtein  SOSUI - Prediction of transmembrane regions (TUAT; Tokyo Univ. of Agriculture & Technology) SOSUI  TMAP - Transmembrane detection based on multiple sequence alignment (Karolinska Institut; Sweden) TMAP  TMHMM - Prediction of transmembrane helices in proteins (CBS; Denmark) TMHMM  TMpred - Prediction of transmembrane regions and protein orientation (EMBnet-CH) TMpred  TopPred 2 - Topology prediction of membrane proteins (Stockholm University) TopPred 2 Current Trends For more fun with proteins, visit http://us.expasy.org/!http://us.expasy.org/ Protein Secondary & Tertiary Structure Predictions Online 3-D Model of Tsr