PROTEINS are extraordinary molecules. In their countless guises they perform
amazing feats of chemistry, accelerating sluggish reactions to dizzying speeds,
folding into fibres tougher than steel, tightly gripping small molecules one
second and releasing them the next. Now biochemists have uncovered a remarkable
new trick in their repertoire.
This trick is performed by an 鈥渋ntein鈥�, a tiny protein that spends the first
moments of its existence as part of a longer strand of protein. It doesn鈥檛 stay
there long. With a sleight of hand that any magician would be proud of, the
intein cuts itself free. But as it departs, it ties the two protein 鈥渙ffcuts鈥�
together, leaving no trace that it was ever there.
This is an impressive piece of chemistry. Usually, a second molecule such as
an enzyme is needed to cut and paste the building blocks of proteins, amino
acids. But somehow the intein manages this by itself. Henry Paulus, a biochemist
at Harvard School of Medicine in Boston, recalls the incredulity of researchers
when they watched the intein鈥檚 trick for the first time. 鈥淧eople claimed that
proteins just couldn鈥檛 do that without the help of other enzymes,鈥� he says.
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The intein plays other tricks too. Once it has left a protein it can
sometimes behave like a primitive form of parasite, somehow introducing the DNA
that encodes it into genes coding for other proteins. It can then start its
routine all over again.
Weird and wonderful
Researchers can only guess at how this weird protein evolved and what
functions it might fulfil. Whatever the answers, inteins could turn out to be
astonishingly useful tools. Already, biochemists are copying their behaviour to
develop new ways of purifying genetically engineered proteins, and to create
entirely new proteins that are impossible to make using conventional techniques.
In the long term, inteins even offer hope of new cures for deadly infections
such as leprosy and tuberculosis.
Until inteins came along, it was thought that the production of a protein
follows a well-mapped path, starting with a sequence of DNA inside a gene. The
cell uses this sequence as a blueprint to assemble the protein, and the
resulting sequence of amino acids, bar the odd bit of tweaking, stays much the
same until the protein takes its place in the metabolism of the cell. But nine
years ago, two groups鈥攐ne led by Tom Stevens at the University of Oregon,
the other by Yasuhiro Anraku at the University of Tokyo鈥攆ound evidence
that this doesn鈥檛 always hold true.
They were studying the protein produced by a gene called TFP1 in
yeast cells. From its DNA sequence, they estimated that TFP1 coded for
a protein containing more than 1000 amino acids. But instead of one long
protein, the cell created two short proteins, each of about 500 amino acids.
When the researchers looked closely, they realised the protein wasn鈥檛 simply
splitting down the middle鈥攖he process seemed to be far more complex.
Early attempts to unravel the exact mechanism behind the protein鈥檚 strange
behaviour got nowhere: the event occurred so swiftly that researchers simply
couldn鈥檛 follow the details. The breakthrough came in 1992 when Francine Perler
and her colleagues at New England Biolabs (NEB) of Beverly in Massachusetts
discovered similar behaviour in a protein found in thermophilic archaea.
These organisms thrive in the boiling waters of geysers and geothermal vents.
Cool them down, Perler thought, and maybe the process would be slowed
sufficiently to let them follow the chemical choreography in detail. When the
researchers tried this, the results amazed them. A small section of
protein鈥攍ater christened an intein鈥攚as cutting itself out and
鈥減asting over鈥� the hole it left behind
(see Diagram). 鈥淲e were stunned at
just how sophisticated this small protein was,鈥� says Paulus. 鈥淚t was hard to
believe it could carry out such a complex set of manoeuvres all alone.鈥�
Biologists have since learnt that the intein triggers three simple chemical
reactions to cut and paste amino acids. And to date, about 90 inteins have been
discovered in simple, single-celled organisms such as fungi, bacteria and algae.
But how did inteins evolve and what advantages could they offer an organism?
Some clues lie in the fact that most inteins possess two distinct sequences
of amino acids that behave like separate enzymes. One of these sequences, called
the protein-splicing centre, cuts and pastes amino acids. The other sequence,
called a homing endonuclease, cuts DNA. Most researchers believe that these two
activities arose originally in separate proteins, but at some point in history
the two came together as one protein. When combined, they seem to endow inteins
with the characteristics of a parasite.FIG-mg21714901.JPG
After the protein-splicing centre has cut the intein out of the protein, the
homing endonuclease can go to work. It cuts specific sites in strands of
intein-free DNA, and by a recombination mechanism, the DNA that codes for the
intein is inserted at this site. Although the complete mechanism isn鈥檛
understood, it seems that inteins can 鈥渋nfect鈥� the entire genome. This may seem
strange behaviour, but there could be a good reason for it.
Move bits of DNA around the genome and they 鈥渞eshuffle鈥� the genetic code,
disrupting the proteins encoded there. On rare occasions, this reshuffling can
result in a winning hand of cards鈥攁 novel protein that gives the organism
a competitive edge in the survival game. Could this be inteins鈥� role in life?
Introns鈥攕egments of DNA that can also hop from gene to gene鈥攈ave a
similar effect on organisms鈥� genetic code (鈥淢essage in a genome?鈥�, New
杏吧原创, 12 August 1995, p 30).
Dan Leahy at Johns Hopkins School of Medicine in Baltimore has found another
clue. He discovered that inteins and 鈥淗edgehog proteins鈥�, which are found in
everything from fruit flies to humans, contain matching sequences of DNA.
Hedgehog proteins act as biological timekeepers, coordinating the growth and
development of the embryo into the adult (New 杏吧原创, Science, 16
September 1995, p 16). They are vital for life. The intein-like DNA seems to
control the position of the hedgehog protein. But what is the link between
inteins and hedgehog proteins? Paulus suspects that inteins evolved first,
becoming incorporated into hedgehog proteins later, but no one is sure.
Wherever inteins originated, biochemists are learning to mimic their
behaviour to build proteins that would otherwise be impossible to make.
Inteins鈥� ability to 鈥渢rans-splice鈥� two separate proteins by joining them
together with intein-like units has proved particularly useful. Each of the two
proteins is given an intein-like 鈥渟ticky鈥� end. When the proteins are brought
together they fuse to form a protein-intein-protein sandwich. A chemical, such
as urea, is then applied to persuade the intein to perform its vanishing trick
and the two proteins are linked to form a new protein
(see Diagram). One of the
advantages of this technique is that biochemists can use it to knit together
large proteins, a feat that presents a challenge for traditional protein
chemistry. 鈥淚nteins are terribly powerful tools for protein engineers,鈥� says
Paulus.
NEB was quick to spot the potential applications for inteins in protein
engineering. Last year, it produced a simple kit that offers a new way to purify
tiny amounts of precious proteins from a crude mixture. First, the target
protein is fixed to an artificial intein, attached to a tag that sticks strongly
to molecules of chitin. The mixture is then poured down a column that is
scattered with tiny chitin 鈥渇ishing hooks鈥�. The hooks catch the chitin-loving
tags, extracting the intein-linked proteins from the mix.
Waging war on TB
Then comes the clever part: the protein-intein-chitin complex hanging
precariously from the column is treated with a chemical that triggers the intein
to cut the protein off. This releases the precious protein. A deliberately
introduced mutation in the intein prevents it pasting together the protein and
the tag so the protein can be washed from the column. The advantage of this
approach, says Perler, is that you don鈥檛 need to use powerful enzymes. 鈥淚t
reduces the chance of accidentally digesting the protein you鈥檝e worked hard to
purify,鈥� she says.
In future, she believes, intein technology could be used to incorporate
radio-labelled or fluorescent amino acids into proteins. This will help
biochemists to investigate the structure and function of important proteins,
throwing light on how they actually work inside the secret world of living
cells.
Inteins also offer biochemists a new way to fight deadly infections. In 1992,
Elaine Davis and her colleagues at the National Institute for Medical Research
in Mill Hill, London, discovered an intein in mycobacteria, a group of bacteria
that cause tuberculosis and leprosy.
TB is a highly contagious lung disease and continues to kill around 3 million
people each year worldwide. Vaccination programmes almost eradicated TB in
Europe and North America, but the disease鈥檚 incidence has risen as the TB
bacterium has acquired resistance to antibiotics.
Inteins are found in dangerous species of mycobacteria that cause TB and
leprosy, but not in their harmless relatives. So Davis has proposed that these
bacteria may owe their virulence to inteins.
When a person becomes infected with TB, their immune system is instructed to
seek and destroy the bacteria. Chemicals released by immune cells damage the
bacterial DNA and this halts the organism鈥檚 growth. But the TB bacterium fights
back. It calls up a protein called RecA that repairs the damaged DNA. However,
to do its work, RecA must first release an intein鈥攚hich then splices the
two sections of protein together to leave the active RecA. Could this be TB鈥檚
Achilles heel?
杏吧原创s remain split on the point, since removing the intein DNA from the
RecA gene does not render TB harmless. However, Paulus and Marlene
Belfort, a geneticist at the New York State Department of Health in Albany, are
looking to see if they can disable the RecA protein by preventing the intein鈥檚
departure. 鈥淲e can dissect out the part of the intein that carries out the
splicing act and use it to develop drugs with anti-splicing potential,鈥� says
Paulus. In theory at least, anti-splicing agents might offer a new approach to
developing treatments for TB.
If inteins play such an important role in some organisms, why has none yet
been found in humans? Paul Liu and his team at Dalhousie University in Nova
Scotia believe they have the answer.
In blue-green algae, the DNA coding for an enzyme called DNA polymerase comes
in two pieces, located in different parts of the genome. Each section codes for
a different protein, and at one end of each of these sections is a sequence of
DNA similar to that coding for an intein. Liu has found that when the blue-green
algae produce the proteins, the intein-like segments paste the proteins together
to create an active enzyme.
This could explain why whole inteins have not been discovered in the human
genome. 鈥淚f inteins can be split within the genome,鈥� says Paulus, 鈥渢hen perhaps
they are present in many more species than we thought鈥攅ven humans.鈥� And
who can guess what roles they may play? 鈥淚t鈥檚 terribly exciting,鈥� says Paulus.
鈥淲e should all be prepared for the unexpected.鈥�