Monitoring the structural consequences of Phe12-->D-Phe and Leu15-->Aib substitution in human/rat corticotropin releasing hormone. Implications for design of CRH antagonists.
A new human/rat CRH analogue has been synthesized using the Fmoc/tBu solid-phase synthetic protocol. The sequence of the new peptide differs from the original in two positions, 12 and 15, at which the native amino acids l-phenylalanine 12 and l-leucine 15 have been replaced by the nonprotein amino acids d-phenylalanine and alpha-aminoisobutyric acid (Aib), respectively. The high resolution three-dimensional solution structure of [d-Phe12, Aib15]CRH has been determined by 688 distance constraints (656 meaningful NOE and 32 H-bonds distance limits) and 21 angle constraints. A family of 40 energy-minimized conformers was obtained with average rmsd of 0.39 +/- 0.16 A and 0.99 +/- 0.13 A for backbone and heavy atoms, respectively, and distance penalty functions of 0.42 +/- 0.03 A2. The NMR data acquired in a solvent system of water/trifluoroethanol (34%/66%, v/v) revealed that this 41-polypeptide adopts an almost linear helical structure in solution with helical content which reaches an 84% of the residues. Structural analysis confirmed the existence of two helical peptide fragments. The first was comprised of residues Ile6-Arg16 and the second of residues Glu20-Ile40, forming an angle of 34.2 degrees. The structural differences with respect to the native peptide have been identified in the region d-Phe12-Glu20 where double substitution at positions 12 and 15 seems to perturb the elements of the native 35-residue helix. These structural rearrangements promote non-native intramolecular interactions in the region of the molecule between either the hydrophobic side-chains of d-Phe12, Aib15 and Leu18, or the charged groups of the residue pairs Arg16-Glu20 and His13-Glu17 being responsible for changes in hormonal functionality. This CRH analogue currently exhibits lack of any activity.
Spyroulias
Georgios A
GA
Department of Pharmacy, University of Patras, Greece.
Papazacharias
Spyridon
S
Pairas
George
G
Cordopatis
Paul
P
D000595:Amino Acid Sequence; D000621:Aminoisobutyric Acids / Q000737:chemistry*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry*; D011138:Polytetrafluoroethylene / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry
D000621:Aminoisobutyric Acids; D010455:Peptides; D014867:Water; C100049:2-aminoisobutyric acid; D010649:Phenylalanine; D011138:Polytetrafluoroethylene; D003346:Corticotropin-Releasing Hormone; D007930:Leucine