Comparative evaluation of the synthesis and purification of transmembrane peptide fragments. Rat bradykinin receptor fragment 64-97 as model.
The 34-residue peptide CTVAEIYLGNLAGADLILASGLPFWAITIANNFD (TM-34), corresponding to the 64-97 sequence of the rat bradykinin, receptor, was selected as a model of hydrophobic transmembrane peptide segment for systematic study of synthesis and purification strategies. Application of conventional Boc/Bzl chemistry resulted in very low yield of the synthesis (around 4%) when DMF was used as the solvent for coupling reactions. As shorter resin-bound fragments of TM-34 showed improved swelling in 80% NMP/DMSO, the synthesis was repeated in this mixed solvent and the yield increased to 12%. A comparative synthesis using optimized Fmoc chemistry and Fmoc-(FmocHmb) derivatives of Ala and Leu to prevent aggregation did not provide any detectable TM-34. Taken together, these results illustrate the synthetic problems associated with hydrophobic sequences, almost regardless of the chemistry used. As expected, the hydrophobicity of TM-34 and of most of its minor fragments made them scarcely soluble in common solvents. Purification could be achieved by loading the crude materials dissolved in 90% AcOH onto a C4 HPLC column and eluting with a TFA/MeCN linear gradient. CD studies of the TM-34 and of the shorter fragment with the 74-97 sequence (TM-24) showed a higher percentage of alpha-helix structure for the latter. This suggests that the shorter sequence may better represent the correct transmembrane region of the second helix of the rat bradykinin receptor.
Oliveira
E
E
Department of Biophysics, Federal University of São Paulo, Brazil.
Miranda
A
A
Albericio
F
F
Andreu
D
D
Paiva
A C
AC
Nakaie
C R
CR
Tominaga
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D051381:Rats; D018002:Receptors, Bradykinin / Q000737:chemistry*
D007202:Indicators and Reagents; D009842:Oligopeptides; D010446:Peptide Fragments; D018002:Receptors, Bradykinin