Authors: Deepak V Pawar1, Mahesh Mahajan1, Rakesh Kumar Prajapat1, Kishor U Tribhuvan1
1ICAR-NRCPB, I.A.R.I, New Delhi-12
From the time when the first protein sequences and structures have been determined, it has been clear that the position and properties of amino acids are key in determining the biological function of proteins. For example, the first protein structure to be discovered, haemoglobin provided a molecular basis for understanding the basis of genetic disease sickle cell anaemia. A single nucleotide mutation leads to a substitution of glutamate with valine is the basis of disease. The substitution is results in lower solubility of haemoglobin and also causes the molecules to form long fibres within blood cells which leads to the unusual sickle-shaped cells. Haemoglobin is just one of many examples now known where single mutations can have radical consequences on protein structure, function and thereby protein associated phenotype. With the availability of thousands or even millions of DNA and protein sequences now we have knowledge of many mutations, either naturally occurring or artificially induced.
Protein Features Relevant to Amino Acid Behavior
The function of protein is determined by a number of general properties
- Protein Environments
Cells also contain numerous compartments, the organelles, which can also have slightly
different environments from each other. Proteins in the nucleus often interact with DNA, meaning they contain different preferences for amino acids on their surfaces (e.g. positive amino acids or those containing amides most suitable for interacting with the negatively charged sugar-phosphate backbone). Some organelles such as mitochondria or chloroplasts are quite similar to the cytosol, while others, such as lysosomes and Golgi bodies are more alike the extracellular environment. Therefore, it is important to consider the likely cellular location of any protein before considering the consequences of amino acid substitutions.
- Protein Structure
- Protein Evolution
a protein belongs in will generally give insights into the possible function. The processes that give rise to homologous protein families are speciation or duplication. Proteins related by speciation only are referred to as orthologues, these proteins have the same function in different species. Whereas proteins related by duplications are referred to as paralogues. Sequential rounds of speciation and intra-genomic duplication can lead to confusing situations where it becomes difficult to say whether proteins are paralogous or orthologous in nature. To be maintained in a genome over time, paralogous proteins are likely to evolve different functions (or have a dominant negative phenotype and so resist decay by point mutation. Differences in function can range from subtle differences in substrate (e.g. malate versus lactate dehydrogenases), to only weak similarities in molecular function (e.g. hydrolases) to complete differences in cellular location and function (e.g. an intracellular signaling domain homologous to a secreted growth factor (Schoorlemmer and Goldfarb, 2001)). At the other extreme, the molecular function may be identical, but the cellular function may be altered, as in the case of enzymes with differing tissue specificities.
- Protein Function
- Post-translational Modification
How mutations affect the protein function
Several studies have been carried out previously in an attempt to decipher general principles about the association between mutations and protein structure & function. SNPs are the point mutations which are present at a measurable frequency a population. They can occur either in coding or non-coding DNA. They may influence regulatory mechanisms such as promoter activity (gene expression), messenger RNA (mRNA) conformation (stability), and subcellular localization of mRNAs and/or proteins. Coding SNPs can be further being divided into two main categories, synonymous (where there is no change in the amino acid they code for), and non-synonymous. synonymous SNPs tend to occur much more frequently than Non-synonymous SNPs. The main reason for this is the natural selection force which keeps deleterious effect of Non-synonymous mutations in check. Site-directed mutagenesis is a powerful tool for discovering the importance of an amino acid in the function of the protein. Gross changes in amino acid type can reveal sites that are important in maintaining the structure of the protein. Peracchi (2001) has reviewed the use of site-directed mutagenesis to investigate mechanisms of enzyme catalysis.
|Sr No.||Amino acid||Substituted by||Amino acid charge||Function in protein|
|1||Alanine (Ala, A)||Other small amino acids||hydrophobic and nonpolar||Play a role in substrate recognition or specificity, particularly in interactions with other non-reactive atoms such as carbon|
|2||Isoleucine (Ile, I)||Other hydrophobic, particularly aliphatic amino acids||Hydrophobic||The isoleucine side chain is very non-reactive and is thus rarely directly involved in protein functions like catalysis, although it can play a role in substrate recognition. In particular, hydrophobic amino acids can be involved in binding/recognition of hydrophobic ligands such as lipids.|
|3||Leucine (Leu, L)||Other hydrophobic, particularly aliphatic amino acids||Hydrophobic||Same as Isoleucine|
|4||Valine (Val, V)||Other hydrophobic, particularly aliphatic amino acids||Hydrophobic||Same as Isoleucine|
|5||Methionine (Met, M)||Other hydrophobic, particularly aliphatic amino acids||Hydrophobic||Binding/recognition of hydrophobic ligands such as lipids. Sulphur atom of Methionine can involve in binding to metal atoms.|
|6||Phenylalanine (Phe, F)||other aromatic or hydrophobic amino acids, prefers to exchange with tyrosine||Hydrophobic||Aromatic residues can also be involved in interactions with non-protein ligands that themselves contain aromatic groups via stacking interactions|
|7||Tryptophan (Trp, W)||other aromatic residues||Hydrophobic||Same as Phenylalanine|
|8||Tyrosine (Tyr, Y)||other aromatic amino acids||partially hydrophobic||Tyrosine contains a reactive hydroxyl group, which helps in interactions with non-carbon atoms. A common role for tyrosines (and serines and threonines) within intracellular proteins is in phosphorylation reactions|
|9||Histidine (His, H)||Being polar amino acid it does not substitute particularly well with any other amino acid||pKa near to that of physiological pH||Most common amino acids in protein active or binding sites. Also very common in metal binding sites (e.g. zinc), often acting together with cysteines|
|10||Arginine (Arg, R)||polar amino acids||amphipathic nature||Arginines are quite frequent in protein active or binding sites. The positive charge helps in interaction with negatively-charged non-protein atoms (e.g. anions or carboxylate groups). Arginine contains a complex guanidinium group on its side chain that has a geometry and charge distribution that is ideal for binding negatively-charged groups on phosphates|
|11||Lysine (Lys, K)||arginine or other polar amino acids||Amphipathic||Lysines are quite frequent in protein active or binding sites. Lysine contains a positively charged amino group on its side chain which helps in forming hydrogen bonds with negatively-charged non-protein atoms (e.g. anions or carboxylate groups)|
|12||Aspartate (Asp, D)||glutamate or other polar amino acids||Polar||Most commonly present in protein active or binding sites. The negative charge means that they can interact with positively-charged non-protein atoms|
|13||Glutamate (Glu, E)||aspartate or other polar amino acids||Polar||Frequently involved in protein active or binding sites of proteases or lipases|
|14||Asparagine (Asn, N)||other polar amino acids, especially aspartate||Polar||Frequently involved in protein active or binding sites. The polar side chain is good for interactions with other polar or charged atoms. Asparagine can play a similar role to aspartate in some proteins. The best example is found in certain cysteine proteases|
|15||Glutamine (Gln, Q)||other polar amino acids, especially glutamate||Polar||Frequently involved in protein active or binding sites. The polar side chain is good for interactions with other polar or charged atoms.|
|16||Serine (Ser, S)||other polar or small amino acids in particular threonine||Polar||Serines are quite common in protein functional centres. The hydroxyl group is fairly reactive, being able to form hydrogen bonds with a variety of polar substrates.|
|17||Threonine (Thr, T)||other polar amino acids, particularly serine||Polar||Threonines are quite common in protein functional centres. The hydroxyl group is fairly reactive, being able to form hydrogen bonds with a variety of polar substrates. Intracellular threonines can also be phosphorylated (see Tyrosine) and in the extracellular environment they can be O-glycosylated (see Serine).|
|18||Cysteine (Cys, C)||substitution with any other amino acid||Polar||Cysteines are also very common in protein active and binding sites. Binding to metals can also be important in enzymatic functions.|
|19||Glycine (Gly, G)||other small amino acids||Hydrophobic||Glycines can play a distinct functional role, such as using its backbone (without a side chain) to bind to phosphates|
|20||Proline (Pro, P)||other small amino acids, although its unique properties does not often substitute well||Hydrophobic||The proline side chain is very non-reactive. This, together with its difficulty in adopting many protein main-chain conformations means that it is very rarely involved in protein active or binding sites|
Hanks SK, Quinn AM, Hunter T. (1988). The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241: 42-52.
Jeffrey PD, Russo AA, Polyak K, Gibbs E, Hurwitz J, Massague J, et al. (1995). Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature 376: 313-320.
Parekh RB, Rohlff C. (1997). Post-translational modification of proteins and the discovery of new medicine. Curr Opin Biotechnol 8: 718-723.
Peracchi A. (2001). Enzyme catalysis: removing chemically 'essential' residues by sitedirected mutagenesis. Trends Biochem Sci 26: 497-503.
Schoorlemmer J, Goldfarb M. (2001). Fibroblast growth factor homologous factors are intracellular signaling proteins. Curr Biol 11 : 793-797.
About Author / Additional Info:
I am PhD research scholar, pursuing PhD at IARI, New Delhi in the discipline of Molecular Biology and Biotechnology. I am working on blast disease resistance in O. sativa