Secondary structure of a fibrillin-1 eight-cysteine motif.

Abstract

University of Manchester, Wellcome Center For Cell/Matrix Research, 2.205 Stopford, Manchester, MI3 9PT *University of Stirling, Scotland Eight-cysteine motifs occur within the fibrillin family of extracellular proteins which includes fibrillin-I and fibrillin-2, and the latent transforming growth factor-p binding proteins (LTBPs) 1-3 [1,2]. The importance of these motifs is highlighted by identification of mutations in eight-cysteine motifs resulting in Marfan syndrome [3], and the fact that one such module in LTBP-I binds TGF-P latency associated peptide via a disulphide linkage [4]. The signature of the eight-cysteine motifs is the distribution of cysteine residues, three of which are contiguous. While biochemical analysis suggests that all cysteine residues are involved in intramolecular disulphide bonds [5], the structure of the eight-cysteine motifs of fibrillin have not been defined. This report compares computer assisted modelling based on the primary sequence with the secondary structure determined by circular dichroism on recombinant fibrillin-I eight-cysteine motifs. Fibrillin1 cDNA encoding three domains, an eight-cysteine motif flanked by two epidermal growth factor (EGF) motifs, was generated by RT-PCR. The sequence was expressed in COS-1 cells using the mammalian expression vector signal plg tail (Invitrogen), into which an enterokinase site was introduced. The hsion protein contained an Fc tail which allowed purification by protein A-Sepharose affinity chromatography. Enterokinase cleavage allowed release of the recombinant protein. Protein purity was assessed by SDS-PAGE on 15% gels and by mass spectrum determination by MALDI-TOF. Circular dichroism was conducted using a JASCO J-600 with scan rates at 10 nm min -’ with a time constant of 2 seconds. Data were collected from 280-195 run. Fitting ofthe data was conducted using CONTM procedure which fits a-helix and Psheet quantities to spectral data when compared to a structural database.