Domains 17-27 of tropoelastin contain key regions of contact for coacervation and contain an unusual turn-containing crosslinking domain.
The central region of tropoelastin including domains 19-25 of human tropoelastin forms a hot-spot for contacts during the inter-molecular association of tropoelastin by coacervation [Wise, S.G., Mithieux, S.M., Raftery, M.J. and Weiss, A.S (2005). "Specificity in the coacervation of tropoelastin: solvent exposed lysines." Journal of Structural Biology 149: 273-81.]. We explored the physical properties of this central region using a sub-fragment bordered by domains 17-27 of human tropoelastin (SHEL 17-27) and identified the intra- and inter-molecular contacts it forms during coacervation. A homobifunctional amine reactive crosslinker (with a maximum reach of 11 A, corresponding to approximately 7 residues in an extended polypeptide chain) was used to capture these contacts and crosslinked regions were identified after protease cleavage and mass spectrometry (MS) with MS/MS verification. An intermolecular crosslink formed between the lysines at positions 353 of each strand of tropoelastin at the lowest of crosslinker concentrations and was observed in all samples tested, suggesting that this residue forms an important initial contact during coacervation. At higher crosslinker concentrations, residues K425 and K437 showed the highest levels of involvement in crosslinks. An intramolecular crosslink between these K425 and K437, separated by 11 residues, indicated that a structural bend must serve to bring these residues into close proximity. These studies were complemented by small angle X-ray scattering studies that confirmed a bend in this important subfragment of the tropoelastin molecule.
Dyksterhuis
L B
LB
School of Molecular and Microbial Biosciences, University of Sydney, NSW, Australia.
Baldock
C
C
Lammie
D
D
Wess
T J
TJ
Weiss
A S
AS
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009391:Nephelometry and Turbidimetry; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics; D053719:Tandem Mass Spectrometry; D013696:Temperature; D014333:Tropoelastin / Q000235:genetics* / Q000378:metabolism*
D003432:Cross-Linking Reagents; D014333:Tropoelastin