When a cook drops a live lobster into boiling brine, the lobster’s shell soon changes color from greenish black to orangy red. The molecular actors in this biological redshift have been known for years. Crustacyanin, the colorant in lobster shell, consists of pigment molecules confined in a colorless multiprotein cage. On heating, the proteins denature, releasing their grip on the pigment molecules. Unshackled, the pigment molecules promptly turn red.
Clearly, live lobster’s dark color originates in the way crustacyanin’s proteins hold the pigment molecules. But uncovering the structural basis of this interaction has proved tough. Tipping the molecular scales at 320 kilodaltons, the massive crustacyanin macromolecule has defied attempts to crystallize it—until now. Thanks to a combination of innovative techniques, a multi-institution team from the UK has determined the structure of a key subunit in the pigment–protein complex. 1
The team’s discovery, which accounts for most of the color change, satisfies the curiosity of scientifically minded epicures, but it could also have applications. Astaxanthin, the pigment in lobster shell, is a powerful antioxidant. As such, it could have potential benefits to human health.
Nobel laureate George Wald tackled the lobster shell mystery as long ago as 1948. Others took up the challenge, among them Peter Zagalsky of London’s Royal Holloway college. Zagalsky has worked on lobster pigment for decades, frequenting local fishmongers to bag leftover lobsters for his lab. His painstaking chemical analysis has definitively established crustacyanin’s molecular building blocks.
Properly called α-crustacyanin, the macromolecule that colors lobster shell is made up of eight β-crustacyanin subunits. Each subunit, in turn, is made up of two smaller protein components (apoproteins is the term structural biologists use), along with two astaxanthin molecules. Five closely similar apoproteins pair up in various combinations to form three closely similar variants of β-crustacyanin. In native α-crustacyanin, the three variants occur in the same fixed ratio.
Having isolated and purified α-crustacyanin and its components, Zagalsky needed to find someone who could determine their structure. His 15-year quest ended in 1998, when he heard that Imperial College’s Naomi Chayen had developed several new ways to form crystals.
One of her techniques is an adaptation of the simplest crystallization method, batch processing, in which the protein, buffers, and solute are mixed together like the ingredients of Jello™ and left alone to solidify. In her version of batch processing, Chayen places microliter droplets of the mixture in tiny trays and covers them with a layer of kerosene. The kerosene is crucial. By floating on top of the crystallizing mixture, the kerosene prevents the solution from evaporating, but still lets enough solute seep out to slowly concentrate the solution. The process is indeed slow. Beta-crustacyanin took so long to crystallize—four months—that Chayen nearly despaired and threw away the samples. In the end, her patience was blessed with bright blue crystals.
To determine the crystals’ structure, Chayen sought her longtime collaborator John Helliwell of the University of Manchester and the Daresbury Laboratory in nearby Warrington. Helliwell, in turn, gave the task to his graduate student Michele Cianci. Rather than tackle α- or β-crustacyanin from the get-go, Cianci worked first on one of the apoproteins. But even with this simplifying first step, the structure determination was difficult. The culprit was what crystallographers call the “phase problem.”
To deduce molecular structure from diffraction data, crystallographers need both the intensity of the scattered x rays and their phase. Phase, though, doesn’t appear in a two-dimensional diffraction pattern. In the case of proteins, a way around the phase problem is to swap heavy atoms like molybdenum and selenium for some of the light atoms that make up the bulk of a protein’s mass. Heavy atoms scatter x rays more strongly than light atoms, so they act like tracers. Unfortunately, the usual techniques for solving the phase problem—multiple isomorphous replacement and anomalous dispersion with multiple wavelengths—failed to yield a clear picture.
Two new techniques have saved the day. Helliwell and his University of Manchester colleague Andrzej Olzcak realized that, by increasing the wavelength of the x rays to 2.0 Å from the usual 1.5 Å, they could enhance the scattered signal from the sulfur atoms in the protein’s cysteine amino acid residues. In effect, they used sulfur as a heavy atom without making a substitution. Sulfur is a rather dilute constituent of the crustacyanin proteins, so it couldn’t yield clear enough phase information by itself. To nail down the phase, Cianci and Daresbury’s Pierre Rizkallah infused the crystal with xenon atoms, which attach to the proteins at fixed locations. By happy coincidence, using 2.0-Å x rays also sharpened the xenon signal.
Thanks to sulfur and xenon, and with help from University of Manchester’s James Raftery, Cianci and Helliwell succeeded last year in determining the structure of one of the crustacyanin apoproteins. 2 The apoproteins are similar enough to each other that the researchers could use the apoprotein structure as a template for solving the structure of one of the three variants of β-crustacyanin.
It’s in the twist
The β-crustacyanin structure shows just how astaxanthin is perturbed by the protein cage. In solution, astaxanthin’s two terminal rings do not lie in the same plane. But when astaxanthin is bound to crustacyanin, the molecule’s twist is flattened.
Another key difference between astaxanthin’s free and bound states involves the doubly bonded oxygens, known as keto oxygens, on the terminal rings (see the figure on page 22). Thanks to its symmetry, free astaxanthin lacks electrical polarity. But in bound astaxanthin, the two keto oxygens inhabit different electronic environments: One keto oxygen is next to a polar water molecule that the crustacyanin structure habitually traps in the same position; the other keto oxygen is next to a nonpolar amino acid. The University of Liverpool’s George Britton and his collaborators had deduced the polarizing role of keto oxygens from spectroscopic data. 3 The x-ray structure clinched the case.
As a result of the molecule’s flattening and polarization, the energy gap between the molecule’s highest unoccupied orbital and its lowest occupied orbital narrows. Free astaxanthin’s absorption hits a peak at about 470 nanometers (the value depends on the solvent). Bound in β-crustacyanin, the peak is 100 nm higher. That difference in wavelength doesn’t quite account for cooked lobster’s color change. It’s likely that the remainder of the redshift originates in whatever additional perturbations arise when eight β-crustacyanins form an α-crustacyanin. The team hopes to test this idea by determining α-crustacyanin’s structure, but the crystals produced so far haven’t yielded a clear diffraction pattern.
Astaxanthin belongs to a class of yellow-orange pigments called carotenoids. Salmon, carrots, and autumn leaves all owe their color to a carotenoid of one kind or another. Some carotenoids, such as the ones in leaves, provide more than color. The energy they absorb in a narrow wavelength band helps to drive the transfer of electrons (see “How Nature Harvests Sunlight,” by Xiche Hu and Klaus Schulten, Physics Today, Physics Today 0031-9228 50
In lobster’s astaxanthin, the energy dumped into the molecule by the absorbed photons ends up as vibrational energy. Pigmentation appears to be the molecule’s only role. But that doesn’t limit interest in its properties. Astaxanthin’s string of double bonds offers a rich source of delocalized electrons. These electrons are especially efficient as antioxidants. By intercepting and disarming free radicals before they damage tissue, antioxidants, it is hoped, might mitigate the wear and tear of aging. Other carotenoids, such as tomato’s lycopene, are more powerful antioxidants than astaxanthin, but the lobster pigment may have better disease-fighting properties.
But because of its lack of polarity, astaxanthin is insoluble in water and, by extension, blood. And that’s where crustacyanin—or a macromolecule like it—might be useful. A water-soluble protein wrapper could help deliver the antioxidant to its target. Drug companies are interested.