Disulfide-rich structural proteins that are assembled into matrices, fibers, and envelopes are important in a wide range of biological settings – from the keratin-associated protein matrices in differentiating keratinocytes, to disulfide-rich structures formed during spermatogenesis.  We are currently studying a disulfide-rich biomaterial that is both commonplace and cryptic.  Under the shell of a chicken egg is parchment-like film assembled from two layers of proteinaceous fibers (depicted schematically at the left, and in the confocal image at the right).

Surprisingly, the major protein(s) from which these fibers are constructed have remained uncertain because lysine-derived crosslinks between the individual proteins render the material intractable to the standard tools of the protein chemist.  We have recently discovered a new disulfide-rich protein (CREMP: cysteine-rich eggshell membrane protein) in these membranes [Ref].  CREMP shows a striking repetition of disulfide-containing modules.  The regularity of cysteine spacing, and the remarkable conservation of intervening amino acids between modules, severely complicates the assembly of full-length sequences from genomic databases.

As an example, the figure below shows a small fragment of a translated avian EST sequence with an alternating pattern of a and b modules.   In addition to (ab)_n patterns, we have also observed (abb)_n and (a)_n repeats in bird and reptile contigs.

These protein fragments bear a distant evolutionary relationship to spore coat proteins from slime molds.  However the CREMP fragments we have uncovered show a much more regular sequence and, consequently, display a much clearer modularity. We know that avian egg-shell membranes are highly ordered by solid state NMR, and that an abab 4-module segment, expressed in Escherichi coli, is also highly structured (both NMR approaches in collaboration with the Polenova laboratory at UD [Ref]).

Many outstanding questions remain.  Here are a few:

•  what is the sequence of a full-length CREMP protein?
•  do multiple CREMP proteins exist in birds and egg-laying reptiles?
•  how are these novel disulfide-rich proteins incorporated in the fibers?
•  while CREMP seem to be major constituents of eggshell membranes, what are the roles of the other protein components?
•  and how does a chicken assemble these fibers over gloopy egg white anyway!?