Location of a constriction in the lumen of a transmembrane pore by targeted covalent attachment of polymer molecules

Few methods exist for obtaining the internal dimensions of transmembrane po res for which 3-D structures are lacking or for showing that structures det ermined by crystallography reflect the internal dimensions of pores in lipi d bilayers. Several approaches, involving polymer penetration and transport , have revealed limiting diameters for various pores. But, in general, thes e approaches do not indicate the locations of constrictions in the channel lumen. Here, we combine cysteine mutagenesis and chemical modification with sulfhydryl-reactive polymers to locate the constriction in the lumen of th e staphylococcal alpha -hemolysin pore, a model protein of known structure. The rates of reaction of each of four polymeric reagents (MePEG-OPSS) of d ifferent masses towards individual single cysteine mutants, comprising a se t with cysteines distributed over the length of the lumen of the pore, were determined by macroscopic current recording. The rates for the three large r polymers (1.8, 2.5, and 5.0 kD) were normalized with respect to the rates of reaction with a 1.0-kD polymer for each of the seven positions in the l umen. The rate of reaction of the 5.0-kD polymer dropped dramatically at th e centrally located Cys-111 residue and positions distal to Cys-111, whethe r the reagent was applied from the trans or the cis side of the bilayer. Th is semi-quantitative analysis sufficed to demonstrate that a constriction i s located at the midpoint of the pore lumen, as predicted by the crystal st ructure, and although the constriction allows a 2.5-kD polymer to pass, tra nsport of a 5.0-kD molecule is greatly restricted. In addition, PEG chains gave greater reductions in pore conductance when covalently attached to the narrower regions of the lumen, permitting further definition of the interi or of the pore. The procedures described here should be applicable to other pores and to related structures such as the vestibules of ion channels.