Influence of proline upon the folding and geometry of the WALP19 transmembrane peptide.
The orientations, geometries, and lipid interactions of designed transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because the amino acid proline will introduce a known discontinuity into an alpha helix, we have sought to measure the extent of helix kinking caused by a single proline within the isolated TM helical domain of WALP19. For this purpose, we synthesized acetyl-GWWLALALAP(10)ALALALWWA-ethanolamide and included pairs of deuterated alanines by using 60-100% Fmoc-l-Ala-d(4) at selected sequence positions. Solid-state deuterium ((2)H) magnetic resonance spectra from oriented, hydrated samples (1/40, peptide/lipid; using several lipids) reveal signals from many of the alanine backbone C(alpha) deuterons as well as the alanine side-chain C(beta) methyl groups, whereas signals from C(alpha) deuterons generally have not been observed for similar peptides without proline. It is conceivable that altered peptide dynamics may be responsible for the apparent "unmasking" of the backbone resonances in the presence of the proline. Data analysis using the geometric analysis of labeled alanines (GALA) method reveals that the peptide helix is distorted due to the presence of the proline. To provide additional data points for evaluating the segmental tilt angles of the two halves of the peptide, we substituted selected leucines with l-Ala-d(4). Using this approach, we were able to deduce that the apparent average tilt of the C-terminal increases from approximately 4 degrees to approximately 12 degrees when Pro(10) is introduced. The segment N-terminal to proline is more complex and possibly is more dynamically flexible; Leu to Ala mutations within the N-terminal segment alter the average orientations of alanines in both segments. Nevertheless, in DOPC, we could estimate an apparent kink angle of approximately 19 degrees . Together, the results suggest that the central proline influences not only the geometry but also the dynamics of the membrane-spanning peptide. The results make up an important basis for understanding the functional role of proline in several families of membrane proteins.
Thomas
Rachel
R
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Vostrikov
Vitaly V
VV
Greathouse
Denise V
DV
Koeppe
Roger E
RE
D000595:Amino Acid Sequence; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry; D011392:Proline / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D008051:Lipid Bilayers; D008565:Membrane Proteins; D010455:Peptides; D010713:Phosphatidylcholines; D011392:Proline; C017251:1,2-oleoylphosphatidylcholine