The role of tryptophan residues in the function and stability of the mechanosensitive channel MscS from Escherichia coli.
Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched in protein surfaces involved in protein docking and are often found in membrane proteins close to the lipid head groups. However, they are usually absent from the membrane domains of mechanosensitive channels. Three Trp residues occur naturally in the Escherichia coli MscS (MscS-Ec) protein: W16 lies in the periplasm, immediately before the first transmembrane span (TM1), whereas W240 and W251 lie at the subunit interfaces that create the cytoplasmic vestibule portals. The role of these residues in MscS function and stability were investigated using site-directed mutagenesis. Functional channels with altered properties were created when any of the Trp residues were replaced by another amino acid, with the greatest retention of function associated with phenylalanine (Phe) substitutions. Analysis of the fluorescence properties of purified mutant MscS proteins containing single Trp residues revealed that W16 and W251 are relatively inaccessible, whereas W240 is accessible to quenching agents. The data point to a significant role for W16 in the gating of MscS, and an essential role for W240 in MscS oligomer stability.
Rasmussen
Akiko
A
School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, UK.
Rasmussen
Tim
T
Edwards
Michelle D
MD
Schauer
Daniela
D
Schumann
Ulrike
U
Miller
Samantha
S
Booth
Ian R
IR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D007473:Ion Channels / Q000235:genetics / Q000502:physiology*; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D029968:Escherichia coli Proteins; D007473:Ion Channels; C469542:MscS protein, E coli; D014364:Tryptophan