The molecular basis of the coloration mechanism in lobster shell: β-Crustacyanin at 3.2-Å resolution

Abstract

The binding of the carotenoid astaxanthin (AXT) in the protein multimacromolecular complex crustacyanin (CR) is responsible for the blue coloration of lobster shell. The structural basis of the bathochromic shift mechanism has long been elusive. A change in color occurs from the orange red of the unbound dilute AXT (max 472 nm in hexane), the well-known color of cooked lobster, to slate blue in the protein-bound live lobster state (max 632 nm in CR). Intriguingly, extracted CR becomes red on dehydration and on rehydration goes back to blue. Recently, the innovative use of softer x-rays and xenon derivatization yielded the threedimensional structure of the A 1 apoprotein subunit of CR, confirming it as a member of the lipocalin superfamily. That work provided the molecular replacement search model for a crystal form of the ␤-CR holo complex, that is an A1 with A3 subunit assembly including two bound AXT molecules. We have thereby determined the structure of the A3 molecule de novo. Lobster has clearly evolved an intricate structural mechanism for the coloration of its shell using AXT and a bathochromic shift. Blue͞purple AXT proteins are ubiquitous among invertebrate marine animals, particularly the Crustacea. The three-dimensional structure of ␤-CR has identified the protein contacts and structural alterations needed for the AXT color regulation mechanism.T he bathochromic shift of the absorption by astaxanthin (AXT) (3,3Ј-dihydroxy-␤,␤Ј-carotene-4,4Ј-dione; Fig. 1) in lobster crustacyanin (CR) has intrigued scientists for over 50 years (1-4). The visible absorption spectra of free dilute AXT, of ␤-and ␣-CR peak at 472 nm in hexane or 492 nm in pyridine, 580 and 632 nm, respectively (2). Detailed chemical studies involving reconstitution with natural and chemically synthesized carotenoids (4) have established the essential chemical characteristics required for this bathochromic shift effect, which arises from a reduced energy gap between ground and excited states. Keto groups are needed at positions 4 and 4Ј, conjugated with the polyene chain; methyl groups are needed at the central positions C20 and C20Ј; only E (all trans) isomers are bound to the protein; and no great variation in overall shape and size of the carotenoid is tolerated. The visible CD spectrum of ␤-CR, attributed to helical twisting of the chromophore, shows exciton splitting indicating that the two AXTs are proximal (2, 4). The lowered CϭC of AXT in the resonance Raman spectra of the CRs, indicative of increased electron delocalization in the electronic ground state, has been attributed to polarization of the -electron system by proximal charge groups or hydrogen bonds (3). These spectra argue against a large protein-induced distortion of the polyene chain, although minor changes to its geometry would be required to explain the spectra (3, 4). 13 C magic angle spinning NMR, together with Stark spectroscopy, gives support to an essentially symmetrical polarization of the AXT in CR with some asymmetry superimposed on the two halves of the chromo...

Vertebrate and invertebrate carotenoid-binding proteins

“…It was proposed that α-crustacyanin consists of two groups of homologous proteins (CRTC, containing apoproteins C 1 , C 2 and A 1 , and CRTA, containing apoproteins A 2 and A 3 ) [43]. Cianci et al 2002 further reported the structural basis of the bathochromic shift and binding behavior based on subunits A 1 and A 3 of curstacyanin protein and several different perturbations of the carotenoid molecules [44].…”

“…The absorption spectrum of the carotenoid astaxanthin along with the β-crustacyanins displays a bathochromic shift of 100 nm (λ max = 580 nm [40]. It was proposed that extension of conjugation by coplanarization of the β-ionone rings with the polyene chain and polarization resulting from hydrogen bonding at the C(4) and C(4′) keto groups of astaxanthin may be mainly responsible for the bathochromic shift [30,44].…”

Water-soluble carotenoid proteins of cyanobacteria

“…A paradigm for a modular assembly of carotenoid-containing subunits into distinct holoproteins occurs in crustacyanin, isolated from lobster carapace. Various combinations of five b-crustacyanin subunits (each 20 kDa) combine to form dimers in which two carotenoids span the interface [31].…”

New Class of Blue Animal Pigments Based on Frizzled and Kringle Protein Domains

“…For example, carotenoprotein crustacyanin, a well studied blue pigment from the lobster shell, contains orange carotenoid astaxanthin, which demonstrates a strong bathochromic shift due to noncovalent binding to a protein of lipocalin family (2).…”