Functions of the periplasmic loop of the porin MspA from Mycobacterium smegmatis.
MspA is the major porin of Mycobacterium smegmatis and mediates diffusion of small and hydrophilic solutes across the outer membrane. The octameric structure of MspA, its sharply defined constriction zone, and a large periplasmic loop L6 represent novel structural features. L6 consists of 13 amino acids and is directly adjacent to the constriction zone. Deletion of 3, 5, 7, 9, and 11 amino acids of the L6 loop resulted in functional pores that restored glucose uptake and growth of a porin mutant of M. smegmatis. Lipid bilayer experiments revealed that all mutant channels were noisier than wild type (wt) MspA, indicating that L6 is required for pore stability in vitro. Voltage gating of the Escherichia coli porin OmpF was attributed to loops that collapse into the channel in response to a strong electrical field. Here, we show that deletion mutants Delta7, Delta9, and Delta11 had critical voltages similar to wt MspA. This demonstrated that the L6 loop is not the primary voltage-dependent gating mechanism of MspA. Surprisingly, large deletions in L6 resulted in 3-6-fold less extractable pores, whereas small deletions did not alter expression levels of MspA. Pores with large deletions in L6 were more permissive for glucose than smaller deletion mutants, whereas their single channel conductance was similar to that of wt MspA. These results indicate that translocation of ions through the MspA pore is governed by different mechanisms than that of neutral solutes. This is the first study identifying a molecular determinant of solute translocation in a mycobacterial porin.
Huff
Jason
J
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Pavlenok
Mikhail
M
Sukumaran
Suja
S
Niederweis
Michael
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002463:Cell Membrane Permeability; D004797:Enzyme-Linked Immunosorbent Assay; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000378:metabolism*; D018272:Porins / Q000737:chemistry*; D021381:Protein Transport; D013499:Surface Properties; D013997:Time Factors
D018272:Porins; C121838:mspA protein, Mycobacterium smegmatis; D005947:Glucose