Unnatural Amino Acids and The Schultz Laboratory

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Amino acids are one of the most vital components of all living organisms on Earth, with 22 amino acids occurring naturally, of which 20 are encoded by the genetic code. All 22 can be found in prokaryotes, while only 21 of these are present in eukaryotes. These amino acids that can be naturally assembled into polypeptides are referred to as proteinogentic, or standard amino acids. Proteinogentic amino acids are found in proteins. Aside from these 22 amino acids, there are a large number of others referred to as 'non-proteinogenic', which can be defined as non-standard. These amino acids are either not found in proteins or are not made directly and in isolation. The non-proteinogenic amino acids that are found in proteins will have formed as a result of post-translational modification.

The image (figure 1) [S Grinstead; 2009] on the right shows the basic structure of an amino acid. They consist of carboxylic acid, an amine group and a functional side chain (indicated as R on the diagram.) They have a number of important roles, including being the structural components of proteins [Creighton, Thomas H, 1993]. The different combinations of the amino acids allow the creation of many proteins that have numerous different functions in every creature in existence, including in movement, i.e. contraction proteins actin and myosin, in defence, i.e. specialised proteins called antibodies, that destroy harmful antigens and in transport i.e. transport proteins are found throughout the body. An example of a transport protein is haemoglobin which transports oxygen throughout the blood. Amino acids may also be taken up in the human diet and used to synthesise other molecules, or are oxidised to carbon dioxide and are used as an energy source. Amino acids that exist as in isolation, rather than as structural components of protein, also have a number of vital functions, such as in metabolism, or as synthesisers of a number of neurotransmitters; for example, tyrosine is the precursor of the neurotransmitter dopamine [R.Kuczenski, D.S. Segal; 2006].

However, recently a number of researchers and laboratories have been working to genetically code and synthesise new amino acids, beside the standard twenty two and the non- proteinogenic ones in prokaryotic and eukaryotic organisms. These novel amino acids are termed unnatural amino acids , and aren t produced directly in the standard cellular fashion or found anywhere in nature. Researchers are particularly interested in the development of unnatural amino acids as they possess potentially limitless possibility. Unnatural amino acids may be utilised as building blocks of larger molecules, molecular scaffolds or as active pharmaceutical products, to name just a few of their possible uses [Sigma Aldrich; 2011]. It is possible that new, novel amino acids may be biologically beneficial to single organisms, or even to the whole human race, for example through the biomedical possibility of drug development. Incorporation of unnatural amino acids has taken place both in test tubes and in living cells, offering potentially great amounts of utility. While the production of unnatural amino acids is still in its infancy, a large number of discoveries have been made.

An example of a research laboratory that invests great amounts of time, money and labour into the development of unnatural amino acids is the Schultz Laboratory, run by Peter G Schultz. The Schultz Laboratory is part of the Scripts Research Institute, based in California. Peter Schultz has been researching unnatural amino acids as a part of the Scripts Research Institute for over two decades and has worked towards a number of groundbreaking scientific discoveries and publications. The main interest and focal point of the research undertaken by the Laboratory is why only 22 amino acids occur naturally, when there is clearly a requirement for further functional groups, as shown by the post-transitional changes that occur to all proteins. Another main focal point for the Laboratory is in to what potential differences unnatural amino acids would create. A large part of the Schultz Laboratory's research is into whether additional amino acids would produce new proteins, and what properties these proteins would display. Could it be possible that the 22 naturally occurring amino acids are optimum for organism s growth, structure of general functioning? Or could it be that further amino acids would produce enriched and improved properties and purposes?

To gain insight in to these questions, the Schultz Laboratory has developed a pioneering method that allows novel amino acids to be encoded. This involves generating a unique t-RNA codon. It is imperative that the t-RNA codon does not code for any natural amino acid. A unique t-RNA synthetase is then created that recognises this t-RNA codon only, and esterification can then occur. Esterification refers to a chemical reaction in which two reactants form an ester as a reaction product. The substrate of the t-RNA synthetase is designed to specifically distinguish the amino acid to be made and the related t-RNA, and no others. This method has proven effective in adding large numbers of novel amino acids into proteins and polypeptides in prokaryotic cells such as E.coli, and eukaryotic cells such as mammalian cells [The Schultz Laboratory; 2011]. These amino acids have numerous functions, for example introducing a large number of unnatural functional groups and probes into proteins, and moderating electron transfer within proteins, to name just a few [The Schultz Laboratory; 2011].

The diagram (figure 2) [The Schultz Laboratory; 2011] on the left details the scheme for evolving aminoacyl t-RNA syntheses with novel specificities. This methodology is also used by the Schultz Laboratory. Aminoacyl t-RNA is a form of t-RNA synthetases which allows its specific amino acid to be adhered. A t-RNA synthesis is an enzyme that interprets the RNA code and attaches the required amino acid to the t-RNA with the corresponding anticodons. Aminoacyl t-RNA synthetase will therefore be able to ensure the amino acid that is attached to the t-RNA will be unnatural. Aminoacyl t-RNA synthetase is also able to transport the amino acid to a ribosome where it will added to a polypeptide chain that is being produced. If any natural amino acids are produced, barnase will cause them to be eradicated. Barnase is a bacterial protein that is lethal to cells when not with its inhibitor, barstar [InterPro, 2011]. This assures that any amino acids produced will definitely be unnatural ones.

The Schultz Laboratory has also proven that it is possible to create a bacterium that is not only able to encode an unnatural amino acid, but is also able to synthesise this novel amino acid in to carbon sources. This was the first example of an organism that is able to encode twenty-one amino acids. This allows research into its interactions with different environmental stimuli, such as different nutritional conditions, in comparison to a twenty amino acid bacterium.

The laboratory has also developed a strategy whereby orthogonal t-RNA- synthetase base pairs can encode unnatural amino acids in bacterial in response to base pairs [L. Wang, PG. Schultz 2001]. This method was used to incorporate unnatural amino acids into proteins in E. coli. This research found that two orthogonal t-RNAs were excellent synthetases for the addition of unnatural amino acids into E. coli proteins. It is possible that these findings could lead to further research into the interactions between t-RNAs and their cognate synthetases.

The research that the Schultz Laboratory undertakes provides a vast number of possible outcomes, including many that could be used beneficially, in a biomedical sense. At present, the Laboratory is specifically working on applying its personal, original methodologies to studies of amino acid and polypeptide assembly and purpose, both in-vivo and in-vitro. Active research projects that the Schultz Laboratory is working on include studies in to the creation and implementation of means for adding new segments to the genetic codes of prokaryotic and eukaryotic organisms. The Schultz Laboratory is also working on further studies into proteins with non- proteinogenic amino acids and novel properties, such as therapeutic proteins and vaccines [The Schultz Laboratory; 2011].

Examples of the ways the Laboratory use unnatural amino acids in developments include using immunogenic novel amino acids to develop cancer vaccines. This was done by reducing immune tolerance to self-proteins, which can be defined as proteins which are produced directly by the certain organism. Another development using non- proteinogenic amino acids includes creating antibodies with a good propensity for viral and cancer targets using phage display libraries encompassing of unnatural amino acids [The Schultz Laboratory; 2011]. Phage display is a study of protein, peptide, and DNA interactions using bacteriophages to link proteins with the genetic information that encrypts them [Smith, GP; 1985]. A bacteriophage is a virus that infects bacteria. By using phage display libraries and the activity of bacteriophages, the Schultz Laboratory is able to recognise the interactions between proteins (and therefore unnatural amino acids) and viral and cancer targets. They are also working towards being able to use novel amino acids to create specialised DNA binding and cleaving proteins [The Schultz Laboratory; 2011]. Finally, the Laboratory is using its technologies to research whether post-transitional modifications are the ulterior causes for a number of diseases, including Type 1 Diabetes and Rheumatoid arthritis.

The image overleaf (figure 3) [The Schultz Laboratory; 2011] indicates the vast scale of unnatural amino acids produced by the Schultz Laboratory to be encoded in prokaryotic and eukaryotic organisms. The sheer scale of new amino acids produced is very impressive, especially when considering the possibilities and potential that can be derived from them. When finding out which amino acids needs to be used in a specific practice (i.e. DNA cleaving proteins, antibodies with high affinity for cancer targets, etc.), the method the Schultz Laboratory uses to assess which is the optimum amino acid for the practice is trial and error. The Laboratory investigates the various outcomes when a number of different amino acids are used, before finalising the methodology with the perfect novel amino acid for the procedure being performed.

The Schultz Laboratory is currently undertaking some very interesting research, which will potentially be ground breaking. This includes using their innovative unnatural amino acid encoding method in multicellular organisms including attempting to create a 21 amino acid transgenic mouse [The Schultz Laboratory; 2011]. The Laboratory is also attempting to remove any redundancy in the genetic code of yeast by the addition of new, novel unnatural amino acids.

Peter Schultz and his Laboratory also do a great amount of research in to new medicine which has links to novel amino acids. The Schultz Laboratory has recently started using chemical libraries along with phenotypic data to identify small molecules with novel biological activities. The libraries are checked for any molecules that control both adult and embryonic stem-cells and self-renewal in both adult and embryonic cells. This allows insight into biomedical applications for bone marrow transplant and genetic blood diseases. Large DNA libraries are also scanned and unnatural amino acids gene products are identified, including ones involved in oncogenesis and cell reprogramming. This research allows the Schultz Laboratory to investigate the effect that novel amino acids and novel genes have on the development of diseases, and potentially make discoveries that will alter and perhaps improve the lives of many individuals.

However, while it appears to many people that the already discovered outcomes, as well as any potential consequences, of the use and implementation of novel amino acids are positive, they remain a controversial issue. A number of people could argue that if there were any true use in unnatural amino acids, they would have naturally evolved into existence through the process of natural selection. Other people may argue that the creation and insertion of novel amino acids into organisms is interfering with nature. Detractors argue that, after all, they are referred to as unnatural for a reason. A number of concerns have also been raised about the potential consequences if any unnatural amino acids were to be accidently released into the wild, and problems this could cause. Problems suggested include new, super-bugs, microbial mutation and possibly eventual change to the ecosystem. It could be believed that possible exposure to unnatural amino acids could in fact do the opposite of what they are intended to do, and potentially make an ill individual s condition worse. However, the likelihood of this would be reduced through the clinical trial procedure.

In considering the implications of the research performed by the Schultz Laboratory, it is very clear that it is both pioneering and possesses great potential, in terms of both biomedical and cellular applications. For example, the research into the creation of antibodies with a proclivity for viral and cancer targets has such a wide range of positive possible uses that could improve the lives of many individuals. The extensive research that the Schultz Laboratory performs gives a substantial insight into the huge possibilities that could be obtained through the creation and implementation of novel amino acids, and definitely assists in questioning whether 22 amino acids is the perfect number.