Proline — the maverick amino acid
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Proline may become therapeutically useful against neuro-degenerative diseases
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HYDROGEN FACTOR: Chemical structure of proline (left) and leucine.
SOME YEARS ago, during visits to the Indian Institute of Science at Bangalore, I would see some biology students sporting T-shirts with the word Proline written on them. At first glance, I assumed this to be an advertisement for the garment firm named Pro-Line. Then it dawned on me that they were, in a way, paying tribute to the amino acid called proline.
This tribute was indeed appropriate since it was the realisation of the importance of proline that led the great structural biologist, the late Professor G.N. Ramachandran, to delineate the shape of the protein collagen, a vital ingredient of our bodies.
He was struck by the fact that in the long string of amino acids that make up the collagen molecule, proline occurs regularly, in every third position in the chain. While twenty different amino acids are available to put together the collagen polymer, why this alliteration using proline?
Why this refrain, as in a musical chant? His genius lay in realising that it has to do with the unusual chemical structure of proline. This led him to describe the three-stranded spiral staircase shape of collagen, sometimes called the Madras Helix.
In shape is the structure
The chemical structure of proline is depicted in the picture along with that of another (typical) amino acid, leucine, for comparison.
We notice right away that unlike the latter, proline is not a linear or open-chain molecule but a cyclic one. Next, we see that its nitrogen atom does not carry two hydrogens as leucine does, but one. Chemists do not therefore call it an amino acid, but an imino acid. When proline forms part of a protein chain, it uses up even this single hydrogen atom while making the peptide bond with another amino acid `upstream' in the polymer chain.
The nitrogen in other amino acids loses one hydrogen atom in the process, but carries the second intact. This second hydrogen comes in handy to hold up the protein chain in a helical or sheet shape, through the weak force called the hydrogen bond.
Proline cannot do so. But its cyclic structure `rigidifies' its shape unlike other amino acids, which have some freedom of motion or flexibility around their `backbone' bonds. This cardboard rigidity is of use in its shape.
Ramachandran wondered what shape would result if one makes a simple chain made up entirely of proline alone, the poly-proline molecule. He found that while there are no hydrogen bonds to hold up the chain, the rigidity of the proline ring suffices. Polyproline too adopts a helical or screw shape like other poly-amino acids, but of a different type. From here to move to the collagen helix with the regular proline repeat, was natural. The mystery of the maverick imino acid was cracked.
Proline as a stress-buster
Since the 1960s when Ramachandran did this remarkable work, we have found further surprising properties of this maverick molecule.
Even when it occurs in the free (and not attached in the protein chain) form, it displays useful properties. One of the earliest detected has been its ability to act as a compatible solute.
Biological materials experience stress when they are placed in an environment they are not used to. This could be a rise in temperature or the presence of high amounts of salt. A common response is to lose water, as happens when a grape turns into a raisin or a healthy round mango becomes all wrinkled up when pickled in salt water. This is caused by the process called osmosis, wherein the water inside the cells tends to leak out.
Cells tend to fight this osmotic stress by switching on the productions of significant amounts of certain compounds within.
This build-up tends to equalise the osmotic pressure from within and allows the re-entry of osmotically expelled water, and restores equilibrium. These compounds, which make the cell compatible to the external stress are called compatible solutes or osmolytes.
High solubility in water
The hero of our article, proline, is one such. It appears to be able to do so, thanks to its high solubility in water (indeed, proline tops the list of the amino acids in this property). Incidentally, Dr. J. Gowrishankar of the Centre for DNA Fingerprinting & Diagnostics, Hyderabad, has done notable work in studying the genetic basis of salt-stress response in bacterial cells.
A litre of water can dissolve as much as 800 grams of proline. This leads to another interesting property of proline at high concentrations, called hydrotropy.
Here, molecules of proline tend to come together to form a loosely organised assembly. Dr. Volety Srinivas of the Centre for Cellular & Molecular Biology, Hyderabad, has studied the special properties of this hydrotropic aggregate of proline, and showed that it stabilises the structure of some proteins. In a paper in 1995 (Langmuir, 11, 2830, 1995) he has termed proline as a protein-compatible hydrotrope. Eleven years later, Zoya Ignatova and Lila Gierasch of the University of Massachusetts showed that proline can do even better. It inhibits the unwanted aggregation of proteins in cells. Such aggregation of proteins occurs when their natural folding pattern is even slightly perturbed, and causes them to clump up.
These clumps of misfolded proteins can cause serious physiological ill effects. Diseases such as Alzheimer's, Parkinson's and the infamous Scrapie in sheep and the Mad Cow Disease have been traced to such aggregation of misfolded proteins.
In a recent report (PNAS 103, 13357, 2006), Ignatova and Gierasch chose the well-characterised protein P39A CRAB, and studied its stability and self-aggregation when it was introduced into live cells.
When proline was added, it was found to destabilise partially folded or misfolded forms of the protein and also inhibit its aggregation.
Furthermore, it solubilises the properly folded, native state. This effect of proline in inhibiting misfolds and aggregates, and stabilising and solubilising native ones might be more general and not restricted to P39ACRAB alone. These results open up the possibility of small molecule osmolytes such as proline becoming therapeutically useful candidates against neuro-degenerative diseases.
D. BALASUBRAMANIAN
dbala@lvpei.org
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