Role of cysteine residues in structural stability and function of a transmembrane helix bundle.
To study the structural and functional roles of the cysteine residues at positions 36, 41, and 46 in the transmembrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis to prepare alpha-amino-n-butyric acid (Abu)-PLB, the analogue in which all three cysteine residues are replaced by Abu. Whereas previous studies have shown that replacement of the three Cys residues by Ala (producing Ala-PLB) greatly destabilizes the pentameric structure, we hypothesized that replacement of Cys with Abu, which is isosteric to Cys, might preserve the pentameric stability. Therefore, we compared the oligomeric structure (from SDS-polyacrylamide gel electrophoresis) and function (inhibition of the Ca-ATPase in reconstituted membranes) of Abu-PLB with those of synthetic wild-type PLB and Ala-PLB. Molecular modeling provides structural and energetic insight into the different oligomeric stabilities of these molecules. We conclude that 1) the Cys residues of PLB are not necessary for pentamer formation or inhibitory function; 2) the steric properties of cysteine residues in the PLB transmembrane domain contribute substantially to pentameric stability, whereas the polar or chemical properties of the sulfhydryl group play only a minor role; 3) the functional potency of these PLB variants does not correlate with oligomeric stability; and 4) acetylation of the N-terminal methionine has neither a functional nor a structural effect in full-length PLB.
Karim
C B
CB
Departments of Biochemistry, Molecular Biology, and Biophysics, Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA. cbk@ddt.biochem.umn.edu
Paterlini
M G
MG
Reddy
L G
LG
Hunter
G W
GW
Barany
G
G
Thomas
D D
DD
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000613:Aminobutyrates / Q000737:chemistry; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005449:Fluorenes / Q000737:chemistry; D007700:Kinetics; D008245:Lysophospholipase / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D010452:Peptide Biosynthesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D000099211:Phospholamban
D000409:Alanine; D000596:Amino Acids; D000613:Aminobutyrates; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D003545:Cysteine; D003902:Detergents; D005449:Fluorenes; D008245:Lysophospholipase; D000099211:Phospholamban; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; C012223:alpha-aminobutyric acid