Why understanding mutations phenomena is mandatory for the application of Protein Engineering?

Basic of Protein Engineering

Protein Engineering………As a blessing to modify protein structure and functional activity according to our needs.

Our mind query about Protein Engineering

Before going to discuss protein engineering. Such types of question must be in our minds about protein engineering.

1. What is Protein Engineering? 
2. What are the aims of protein engineering?
3. What are the applications of protein engineering?

Background of Protein Engineering

As we know that proteins are mostly DNA product (synthesized by ribosome according to the shared information of DNA in the form of codons template known as mRNA). So, this is natural that we must have strong knowledge of DNA as well as RNA. Because we will have to introduce our desired changes in DNA coding sequencing to achieve the planned targets. Any type of sequencing change in DNA building block (i.e., nucleotides) are known as DNA mutations. So, we must have strong background of DNA mutations

In how many types, DNA mutations can be divided?

In this post, I am going to explain little bit about DNA mutations and their types. These mutations occur through different agents they may be physical like UV light and or chemical like Ethidium Bromide. But at this stage we will not discuss about these agents. Because we just want to know about mutation at DNA. Then to produce such types of mutations when we need to modify naturally occurring protein using site directed mutagenesis methods in place of physical and or chemicals agents.

Definition of mutation

Mutation can be simply defined as “any change in a DNA nucleotides sequence that is not present wildly and can be propagated through cellular generations. It may be so small like one bp change or may be few pbs. These mutations can result disruption of function either “function is lost” or more function is gained.

Mutation and its influence on organism/generation 

Mutation are broadly categories into two different groups based on cell types.

Somatic mutation

Somatic mutation that occurs in the body cells which is normally known as “somatic cells”. Somatic mutation affect remains only to the single individual having mutation in his/her somatic cells.

Mutation in Reproductive cells (Germ cell mutation)

In contrast to somatic mutation, mutations can also be occured in DNA present in gametes. Such type of mutation propagates via mating process in individual that consequently affect next generation.

Types of Mutations

Mutations in either somatic or germline can be divided into the following types.

Point Mutation

Point mutation referred to a change in a single base pair. Point mutations can be categories in two groups based on nucleotides. Nucleotides are two types. Purines consist of A and G. Itself purine word is simple but chemical structure of purine nucleotides are complex because it consists of two rings. Pyrimidines consist of C, T (in case of DNA) as well as U (in case of RNA). As the name is difficult when compared with purine but chemical structure is very simple and consist of single ring. So based on these two groups of nucleotides, mutation can be divided into two groups.

1. Transition mutation. In this type of mutation either one purine is exchanged by another purine base pair (A with G or G with A). Similarly, when one pyrimidine base (either C or T) is replaced by another pyrimidine (for example C with T or T with C).
2. Transversion mutation When a purine is exchanged with pyrimidine or pyrimidine is exchanged with purine, such type of mutations are known as “Transversion mutation”. Such as A or G is either replaced by C or T in DNA strand.

Keep in mind that transition mutations are nearly 10 times more frequent than transversions.

Effect of Point Mutation

Let see what happened in case when point mutation occur in amino acid-coding region of a gene? This change in DNA of protein coding gene due to point mutation can lead to alter protein backbone made of amino acids, consequently partial or complete loss of function can be observed at protein level. If the protein is central to cell viability, the cell could die. But in parallel, mutation in the protein-coding region can also result to enhance the protein functions when compared with normal version of protein that is present naturally.

Point Mutation Mediated human diseases

Point mutations are known to cause a wide variety of human diseases. One example is sickle-cell anaemia, resulting from a transversion that produces an amino acid change in haemoglobin. In haemoglobin gene GAG code at position 6 that code for glutamate change into GTG, resulting incorporation of valine amino acid instead of glutamate. Due to this change at amino acid level in haemoglobin, morphology of red blood cell changes and they are unable to carry or transport oxygen properly in human body.

Lethal Effect of Mutations in Cell Cycle and DNA repair associated genes

Cell cycle is the biological process in which parental cell divide into two daughter cells (having same genetic material in case of somatic cells) while in case of germ cell, a single germ cell is divided into four daughter cells (each has half number of genetic material when compared with parental cells. During cell division, DNA is replicated before equal or half distribution among daughter cells in somatic and germ cells respectively. During DNA replication and distribution among daughter cells, when there is some mismatch at nucleotide level with parental DNA molecule or abnormal distribution of chromosomes is occurred, cell cycle is stopped to repair DNA molecule or to distribute right chromosomes in daughter cells. All such type of activities such as DNA replication and repair, DNA organization into chromosomes, distribution of chromosomes among daughter cells, are strictly under control of proteins. For example, some gene product such as proto-oncogenes drive the cell division cycle forward, and in contrast to forward direction, the product of some genes i.e., tumor suppressor genes suppress cell division.

Many tumor suppressors and tumor promoter genes are growth factor, transcription factors that regulate the expression of genes that drive the cell cycle. The transcription factor p53 and the retinoblastoma protein are examples of tumor suppressors that are mutated in many types of cancer.

Mutations are not always harmful

Interestingly, when a nucleotide changes in a codon but the new codon also codes for the same amino acid as it was by previous codon. A change can be observed at DNA level but at protein level there will be no change. Such type of mutation is referred as “Silent Mutation”. This type of mutation can occur in those codons which code for same amino acid. First two nucleotides of these codons are same and third one is normally different in both codons. For example, Glutamate is either coded by GAA or GAG. For example, in a DNA molecule, GAA codon is present, point mutation occur at position number third. Due to this mutation GAA is converted into GAG. Hence both GAA or GAG code for glutamate. This type of point mutation is present at DNA level but not at protein level. A missense mutation is a nucleotide change that results in a different amino acid, such as a change from glutamate (GAA) to glutamine (CAA).

Nonsense and Missense Mutation

In nonsense mutation an amino acid coding codon is converted into a codon that does not code for any amino acid. These types of codons are three (UAA, UAG and UGA) they are responsible for protein polypeptide chain termination. So, any nonsense mutation in protein coding gene can result early termination of protein polypeptide synthesis and consequently premature proteins are synthesized. While in case of Missense mutation any codon of protein coding gene is changed with codon that codes for different amino acids. For example when a codon GAC, which codes for aspartic acid, is changed into GAC, that codes for glutamic acid is known as “Missense Mutation”

Keep in mind that amino acids are divided into different groups based on chemical properties such as acidic, basic, aliphatic, aromatic, S-containing amino acids etc.

Neutral nonsynonymous mutation

Any point mutation that results in changing of an amino acid with similar property amino acid is known as Neutral nonsynonymous mutation. For example, glutamic acid is changed into aspartic acid. Both amino acids have similar chemical properties. In such situation protein function is mostly remain unaltered.

Ways of point mutations

From this discussion, it can be abstracted that basically mutations are errors in codons that are naturally not present in the other organism of the same species. We should know the way how these errors can be occurred at DNA level. There are two ways.

1. Base substitution, in base substitution, as the name denotes one base is substituted with another base. Therefore base substitution associated mutations change only one amino acid in the native protein. This also led to termination when amino acid codon is substituted with termination codons.
2. Insertion or Deletion, while in case of insertion or deletion, either a base is incorrectly inserted or deleted from a codon. Irrespective of base substitution, insertions, and deletions, also known as frameshift mutations, change every amino acid coded for amino acids after the mutated codon. So, the whole frame is changed after the mutation spot. Such type of mutations that are divisible by 3, maintain the open reading frame (ORFs). While mutation that are not divisible by 3 disrupts the ORFs

Both missense and nonsense mutations result in altered structure of protein either due to change in amino acid or due to a shorter polypeptide.

Why Biotechnologists love Protein Engineering?

Simply Protein engineering is a combination of some techniques that are applied to modify the protein structure so that it acquires specific desired properties. Such as enhanced or new functions when compared with wild type of protein.

How it possible to modify protein structure according to our required and/or desired properties?

We have solid knowledge of central dogma that result protein synthesis via transcription of DNA and then translational of mRNA into polypeptide/protein.

So, if we make some changes at DNA level, which is a stable molecule as compared to RNA, then we would be able to observe these changes at polypeptide level. For this purpose, we need to have a sufficient theoretical knowledge and good skills of genetic engineering. What is Genetic Engineering? The alteration of the genome of an organism by laboratory techniques is known as Genetic Engineering. Genetic Engineering is used as a tool in Protein Engineering.

Aims of Protein Engineering

To search a particular protein having some desired properties of specific environment and then analyze that protein at molecular level (sequence, expression, structure) to apply protein engineering tools to modify our natural environment protein at lab. And then to study the new properties of that protein in comparison with specific environment. 

Naturally occurring protein, that are modified through protein engineering can be used at various industries like pharmaceuticals, agriculture and enzyme associated bioprocessing industries etc.