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 (λ _max 472 nm in hexane), the well-known color of cooked lobster, to slate blue in the protein-bound live lobster state (λ _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 three-dimensional 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 β-CR holo complex, that is an A ₁ with A ₃ subunit assembly including two bound AXT molecules. We have thereby determined the structure of the A ₃ molecule de novo . Lobster has clearly evolved an intricate structural mechanism for the coloration of its shell using AXT and a bathochromic shift. Blue/purple AXT proteins are ubiquitous among invertebrate marine animals, particularly the Crustacea. The three-dimensional structure of β-CR has identified the protein contacts and structural alterations needed for the AXT color regulation mechanism.

Crystallographic and computational methods have been used to study the binding of two monosaccharides (glucoside and mannoside) to concanavalin-A. The 2 Å structure of glucoside bound concanavalin-A is reported and compared with the 2 Å structure of the mannoside complex. The interaction energies of the substrate in each crystallographic subunit were calculated by molecular mechanics and found to be essentially the same for both sugars. Further energy minimisation of the active site region of the subunits did not alter this conclusion. Information from crystallographic B-factors was interpreted in terms of mobility of the sugars in the combining site. Molecular dynamics (MD) was employed to investigate mobility of the ligands at the binding sites. Switching between different binding states was observed for mannoside over the ensemble in line with the crystallographic B-factors. A calculated average interaction energy was found to be more favourable for mannoside than glucoside, by 4.9±3.6 kcal mol^-1 (comparable with the experimentally determined binding energy difference of 1.6±0.3 kcal mol^-1). However, on consideration of all terms contributing to the binding enthalpy a difference is not found. This work demonstrates the difficulty in relating structure to thermodynamic properties, but suggests that dynamic models are needed to provide a more complete picture of ligand–receptor interactions.

The structure of concanavalin A, a saccharide-binding metalloprotein of subunit molecular weight 25000 Da§, is reported with precision and accuracy equivalent to those of small-molecule crystal structures. An X-ray data-to-parameter ratio of 6.1:1 has allowed the first unrestrained anisotropic refinement of a protein structure and, indeed, one of large molecular weight. This has been achieved by the combined power of synchrotron radiation, a highly sensitive CCD-based area detector and cryogenic techniques on crystals of exceptional quality. The high precision of the atomic coordinates and the direct observation of many hydrogen atom positions, on both the protein and bound solvent, provide new insights into the protein structure, including precise geometry of the metal-binding sites, the role of water molecules in the extended saccharide-binding site, the structure and dynamics of bound and buried water, and direct evidence that proximal carboxylic acid side chains can exist as carboxyl–carboxylate pairs. The biophysical chemistry of this protein is thereby elucidated in detail in this and the companion paper.