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On the Function and Structure of Synthetically Modified Porins. Angew. Chemie. Int.Ed , 2009, 48, 1-6 Lyndelle LeBruin CHEM 258. Outline. Beta barrels Porins and ion channel engineering Structure and function of OmpF porin Main paper discussion Conclusions. β eta barrels.
Angew. Chemie. Int.Ed, 2009,48,1-6
Sucrose specific porin for Salmonella typhimurium- a canonical beta barrel protein
- create biological channels to control existing function:
structural information of channels, chemical synthesis to obtain hybrid ion channels
- de novo design: use of self assembly to form π & β barrel pores
eases synthetic modifications in the pore interior
- allow flow of ions across the membrane close to diffusion limit in water, high specificity for ion conductance
Secondary structure of OmpF porin monomer:
a: side view of OmpF momomer
b: top view of the OmpF trimer with loop region L3
β1: peptide stretch for native chemical ligation
Lys16: site for attachment of synthetic modulators
- protein semi-synthesis via NCL/click chemistry
- cysteine residue formed via mutation and then S-alkylation
OmpF Hybrid 5
OmpF hybrids 8 & 10
Trimers remain stable after tryptic digestion
Refolding yield (~70%), comparable to that of native OmpF
Not a significant change in I/U values for recombinant OmpF, wild type OmpF by NCL and OmpF hybrid5 51.5 pA vs. 51.2 pA for OmpF
Modulator(319Da) near constriction zone does not alter conductance of OmpF, good method for constructing native-like OmpF polypeptides
Large spread of trimer conductances, seen for hybrid 8,were not observed for refolded wild type OmpF or its mutants
Average specific conductance of 8 decreased by 15% 0.78 nS vs. unmodified OmpF 0.92 nS
Indicates conformational mobility and heterogenity of modulator within the pore
BLM measurements in 5 mM HEPES, pH 7.2; 150 mM KCl
Size of hybrid 5 vs.hybrid 8 (451 vs. 377 Da) not sufficient to determine effect of modulator on channel
I/U for 10 similar to that of hybrid 8:
large spread in trimer conductances, 18% (0.75 nS)conductance reduction vs. 15% (0.78 nS) for hybrid 8, limiting conductances of 0.42 and 1.14 nS
Single trimer events, show reduced conductivity for hybrid 10
Side view of the cross section of 10
Top view of OmpF trimer with dibenzo-crown-6 ether modulator
Fobs - Fcaldifference density map at 3.4 Å
Crystal packing of novel OmpF crystal: quasi-continuous arrangement of trimers along “c” axis
Surface representation for dibenzo crown 6 compound between L3 loop and basic amino acids
- ether crown moiety from Cys16 transverses constriction zone
- distorted conformation of ether moiety in constriction zone
Stretched, inward-oriented conformation of crown-ether required for blockage: partial contact of L3 loop
Crown ether points away form constriction zone : more conformational freedom, no pore blockage
Linker between crown ether and position 16 on β-strand 1 is extremely important in determining efficiency of channel or pore blockage
Blocked (orange) and
loosened (light yellow) conformations
The term “black” bilayer refers to the fact that they are dark in reflected light because the thickness of the membrane is only a few nanometers, so light reflecting off the back face destructively interferes with light reflecting off the front face.
To make a BLM, a small aperture is created in a hydrophobic material such as teflon.
A solution of lipids dissolved in an organic solvent is then applied with a brush or a syringe across the aperture
Electrical characterization has been particularly important in the study of voltage gated ion channels which can be inserted into a BLM by coating them with a detergent and mixing them into the solution surrounding the BLM.