Activity and structural comparisons of solution associating and monomeric channel-forming peptides derived from the glycine receptor m2 segment.
A number of channel-forming peptides derived from the second transmembrane (TM) segment (M2) of the glycine receptor alpha(1) subunit (M2GlyR), including the 22-residue sequence NK(4)-M2GlyR p22 wild type (WT) (KKKKPARVGLGITTVLTMTTQS), induce anion permeation across epithelial cell monolayers. In vitro assays suggest that this peptide or related sequences might function as a candidate for ion channel replacement therapy in treating channelopathies such as cystic fibrosis (CF). The wild-type sequence forms soluble associations in water that diminish its efficacy. Introduction of a single substitution S22W at the C-terminus, NK(4)-M2GlyR p22 S22W, eliminates the formation of higher molecular weight associations in solution. The S22W peptide also reduces the concentration of peptide required for half-maximal anion transport induced across Madin-Darby canine kidney cells (MDCK) monolayers. A combination of 2D double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) data were recorded for both the associating WT and nonassociating S22W peptides and used to compare the primary structures and to assign the secondary structures. High-resolution structural studies were recorded in the solvent system (40% 2,2,2-Trifluoroethanol (TFE)/water), which gave the largest structural difference between the two peptides. Nuclear Overhauser effect crosspeak intensity provided interproton distances and the torsion angles were measured by spin-spin coupling constants. These constraints were put into the DYANA modeling program to generate a group of structures. These studies yielded energy-minimized structures for this mixed solvent environment. Structure for both peptides is confined to the 15-residue transmembrane segments. The energy-minimized structure for the WT peptide shows a partially helical extended structure. The S22W peptide adopts a bent conformation forming a hydrophobic pocket by hydrophobic interactions.
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Gabriel A
GA
Cook
Om
O
Prakash
Ke
K
Zhang
Lalida P
LP
Shank
Wade A
WA
Takeguchi
Ashley
A
Robbins
Yu-Xi
YX
Gong
Takeo
T
Iwamoto
Bruce D
BD
Schultz
John M
JM
Tomich
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000502:physiology*; D003198:Computer Simulation; D004285:Dogs; D007473:Ion Channels / Q000502:physiology*; D007668:Kidney / Q000737:chemistry / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D018009:Receptors, Glycine / Q000737:chemistry* / Q000502:physiology*; D020539:Sequence Analysis, Protein / Q000379:methods*; D012996:Solutions; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014867:Water / Q000737:chemistry
D007473:Ion Channels; D018009:Receptors, Glycine; D012996:Solutions; D014867:Water; D014364:Tryptophan