Biomolecules of the Cell - Proteins

Proteins are the biomolecules which are polymers of amino acids covalently linked through a peptide bond into a chain. Before understanding the structure of proteins, lets clarify some of the terms in this context.

1. Peptide bond:
A peptide bond is the covalent bond that is formed between the carboxyl group of one amino acid and the amino group of the next amino acid. The resulting C(O)NH bond is called the peptide bond (as can be seen in the figure).  The peptide bond formation occurs as a result of the condensation reaction involving loss of a water molecule.

2. Peptide:
What is a peptide? Amino acids are covalently linked to each other with the help of the above mentioned peptide bonds and form a chain. When the length of this chain is very short, say less than 40 amino acids, it is called peptide. The shortest peptide is dipeptide (consisting of 2 amino acids joined by a single peptide bond); then there can be tripeptides (consisting of 3 amino acids with 2 peptide bonds); tetrapeptides (consisting of 4 amino acids with three peptide bonds) and so on.

3. Polypeptide:
Is peptide and polypeptide one and the same thing? NO. Polypeptide is a long chain of amino acids having around more than 40 amino acids. It is primarily a single, long, continuous, linear and unbranched peptide chain. For example, Angiotensin II is a peptide hormone (that causes vasoconstriction and increase in blood pressure) that contains only 8 amino acids (octapeptide). Another example of peptide hormone is mammalian hormone, oxytocin, which is made up of only 9 amino acids (nonapeptide).

4. Protein:
Proteins are made up of one or more polypeptides. The proteins are considered to be the 'workhorses of the cell' as they include structural and motor elements in the cell and are responsible for serving as the catalysts for literally every biochemical reaction that occur in living things. We will have a look at the structure of proteins now.

Structure of Proteins:

As mentioned above, proteins being workhorses of the cell and to understand their functions, it is necessary to determine their structure. Proteins tend to fold into one or more specific conformations which are driven by a number of interactions which we are going to see below. There are four distinct levels into which we can divide the structure of proteins as follows:

a. Primary Structure:
The linear sequence of amino acids in a protein is considered to be the primary structure of the protein. This amino acid sequence is ultimately responsible for the folding and intermolecular bonding of the linear amino acid chain which thus determines the protein’s 3-D shape. The primary structure is mainly held by peptide bonds which have been made during the process of protein biosynthesis.

b. Secondary Structure:
Secondary structure refers to the areas of local stable folding (or coiling) within a protein. These mainly include α-helix and β-sheets.

i. α-helix:  In an α-helix, the protein chain is coiled like a loosely-coiled spring (just like the telephone cable). Why it is called “alpha”? Alpha measn that if you look down the length of the spring, the coiling happens in a clockwise direction as it goes away from you. An α-helix is stabilized by hydrogen bonds between the (backbone) amino and carbonyl groups. The H-atom of amide group forms the bond with the OH atoms of carbonyl group. There is regularity in the arrangement of hydrogen atoms. All the N-H groups point upwards as against the C=O groups that point downwards (as in the figure). In an α-helix, the R-groups (side chains) of amino acids protrude out from the helically coiled polypeptide backbone. Also, each complete turn of the spiral has approximately 3.6 amino acids residues in it and the distance between two turns is 0.54nm.

ii. β-sheet: Another common secondary structure is β-sheet. In a β-sheet, strands of protein lie adjacent to one another and interact via H-bonds between backbone carbonyl oxygen and amino H atoms. There are two main kinds of arrangements as parallel and antiparallel. In parallel arrangement, all the N-termini of successive strands are oriented in the same direction whereas in an antiparallel arrangement, the successive β-strands alternate directions such that the N-terminus of one strand is adjacent to the C-terminus of the next strand (it will be more clear from the diagrams below).

c. Tertiary Structure: 

The tertiary structure refers to the three-dimensional structure of a protein. The α-helices and β-sheets are folded into compact globular structures. This results from a large number of interactions between amino acids such as:

i. Ionic Interactions: Some amino acids are basic (positively charged) while some are acidic (negatively charged). Hence, ionic bond forms between the negative and positive group if the chains folded in such a way that they are close to each other.

ii. Hydrogen Bonds: Here, we are talking about the H-bonds between the side-groups not between the groups actually in the backbone of the chain. Some amino acids contain –OH group in the side chain (eg. Serine). A H-bond can form between two different serine residues in different parts of a folded chain.

iii. Van der Waal’s forces: Van der Waal’s interaction is transient, wee electrical attraction of one atom to another. It provides an important component of protein structure. Most of the atoms in a protein are so sufficiently close to be involved in such kind of transient interaction.

iv. Disulfide bonds: Cysteine is the sole amino acids whose side-chain can form covalent bonds. When two cysteine residues in a protein comes close to one another, then the R-groups of both interacts with each other yielding disulphide bridges.

v. Hydrophobic Interactions: Certain amino acid residues possess the hydrophobic or non-polar (water-repelling) side chains. The hydrophobic amino acids are generally at the centre of the protein as against the hydrophilic amino acids which are towards the edges. Not all hydrophobic amino acids are in the interior of proteins, however.

d. Quaternary Structure: It refers to the non-covalent interactions that bind multiple polypeptides into a single, larger protein. The protein may be composed of identical polypeptides or may include different polypeptides as well. Being non-covalent interactions, quaternary structure is mainly stabilized by weak interactions between residues exposed on the surface of the polypeptides within the complex. Hemoglobin is a very good example of quaternary structure which consists of two α-polypeptide chains, two β-polypeptide chains and a prosthetic heme group (see diagram).
Here, we end the structure of proteins.

Biomolecules of Cell - Proteins (Amino Acids)

Introduction:
Before going ahead and understanding proteins - the biomolecules, it is important to first understand amino acids. Amino acids are the organic compounds mainly bonded to a hydrogen atom, a carboxyl group (COO^-) and an amine group (NH₂) along with a side chain (represented as R in the diagram) which is specific to every amino acid. There are about 500 amino acids known. The two broad groups into which these amino acids can be distributed are: proteinogenic amino acids and non-proteinogenic amino acids. The word ‘proteinogenic’ means ‘protein building’. Interesting to note is that of these 500 amino acids, only 23 naturally occurring amino acids come under proteinogenic amino acids i.e.; these amino acids are precursors to proteins. Of these 23, 20 proteinogenic amino acids are encoded by codons (triplet) in genetic code and are called ‘standard’ amino acids. The other three which are ‘non-standard’ amino acids are pyrrolysine, selenocysteine and N-formylmethionine. The pyrrolysine is found in methanogenic organisms and other eukaryotes. Selenocysteine is present in non-eukaryotes as well as in some eukaryotes. While N-formylmethionine, as we all know takes part in protein synthesis initiation in bacteria and also in protein synthesis that takes place in organelles (chloroplast and mitochondria).Here, in this post, we will discuss about the standard 20 amino acids.

Each amino acid has a distinctive 3-letter code and 1-letter code which is mostly use to represent them. Here is the list of 20 standard amino acids with their 3-letter code and 1-letter code.

Glycine (Gly, G)	Alanine (Ala, A)	Valine (Val, V)	Leucine (Leu, L)	Isoleucine (Ile, I)	Proline (Pro, P)
Phenylalanine (Phe, F)	Tyrosine (Tyr, Y)	Tryptophan (Trp, W)	Serine (Ser, S)	Cysteine (Cys, C)	Threonine (Thr, T)
Methionine (Met, M)	Aspartic Acid (Asp, D)	Aspargine (Asn, N)	Glutamic Acid (Glu, E)	Glutamine (Gln, Q)	Histidine (His, H)
Lysine (Lys, K)	Arginine (Arg, R)

Characteristics of Amino Acids:

Since in all amino acids, the carboxyl and amine groups are attached to primary or α-carbon atom, these amino acids are known as α-amino acids. All amino acids except glycine exists as enantiomers (mirror images of each other that are non-superimposable) called as L or D amino acids. Remember that L and D configuration of amino acid does not refer to the optical activity of the amino acid itself. In fact, it refers to the optical activity of isomer of glyceraldehyde (from which all amino acids can be synthesized theoretically). All proteinogenic amino acids exist as L-stereoisomers (meaning left-handed isomers).

The two functional groups of amino acids as carboxyl group and amine group make them amphiprotic meaning it can act as an acid or base.  Carboxylic acid (-COOH) groups can be deprotonated to form carboxylate (COO^- ) whereas amino groups (NH₂) can be protonated to from positively charged ammonia (NH₃⁺) groups. This form when the net charge on the amino acid is zero is called a zwitterion. Remember that, when the pH of any amino acid is greater than the pKa of the carboxylic group of that amino acid, the negative carboxylate ion predominates i.e.; at higher pH, the net charge on amino acid is negative. At pH values lower than the pKa of the alpha-ammonium group, the nitrogen is protonated because of positively charged alpha ammonium group meaning at low pH, the net charge on the amino acid will be negative.

Essential Amino Acids:
Nine of the 20 standard amino acids are essential amino acids meaning that they are not synthesized or created from another compound inside the body and hence has to be taken externally in the form of food. The remaining amino acids are non-essential meaning they need not be taken from the outside.
The nine essential amino acids are arginine (essential only in growing children where they have to obtain from their diet), histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. Is it difficult to remember these 9 amino acids? Here are three ways by which you can make it mnemonic:
1. PVT TIM HALL (like ‘private tim hall’ is someone’s name) OR
2. MAT VIL PhLY (pronounce as if ‘matw ill fly’ where, Ph is for phenylalanine, Y is for tyrosine)
2. By remembering this sentence: Any Help In Learning These Little Molecules Proves Truly Valuable.

Types of Amino Acids Depending on the Properties of Side Chain:
There are certain unique chemical properties of each amino acid due to its side chains which we will discuss here.  The amino acids can be grouped into four broad categories according to the properties of side chains.

1. Non-Polar Amino Acids or Hydrophobic Amino Acids: Ten amino acids have non-polar side chains meaning that they do not interact with water. Glycine is the simplest amino acid. alanine, valine, leucine and isoleucine have up to four carbon atoms and their side chains are hydrophobic and hence these amino acids are located in the interior of the protein where they are unable to interact with water. Proline also has a hydrophobic side chain. It is interesting to note that it is unique such that the side chain is bonded to nitrogen atom of amine as well as α-carbon thereby forming a cyclic structure. Thus, proline is an imino acid instead of amino acid. The amino acids cysteine and methionine have sulphur in their side chains. Methionine is quite hydrophobic but cysteine is less as it has sulfhydryl group (SH) group. Cysteine plays an interesting role as it can form disulphide bonds between two cysteine residues of side chains of two proteins. The two amino acids, phenylalanine and tryptophan contain hydrophobic aromatic rings.

2. Polar Amino Acids or Hydrophilic Amino Acids: Five amino acids have polar side chains but are uncharged. Serine, threonine and tyrosine have hydroxyl groups in their side chains. Aspargine and glutamine have polar amide (O=C=NH2). These proteins can interact with water to form hydrogen bonds and hence are hydrophilic and are located on the outside of the proteins.


3. Positively Charged Amino Acids/ Basic Amino Acids:  Three amino acids have side chains with charged basic or positive groups. Lysine, arginine and histidine come under this category of basic amino acids.


4. Negatively charged amino acids/Acidic amino acids: Remaining two amino acids as aspartic acid and glutamic acid have side chains which have carboxyl groups and hence are acidic in nature. These are hydrophilic and hence are located on the outer surface of the proteins.

Here, we end with the basics of amino acids. In the next post, we will see about the proteins.

Signal Transduction Pathway - NF-κB Signaling

NF-κB is another example of signaling pathway where there is direct targeting of a specific family of transcription factors. NF-κB stands for nuclear factor kappa beta. It is a transcription factor that plays an important role in the immune systems and in inflammation as well as in the regulation of proliferation and survival of various animal cells. In response to ligation of many receptors, NF-κB is known to regulate the expression of various cytokines, cyclo-oxygenase (COX-2), growth factors, inhibitors of apoptosis etc.

The activation of NF-κB is mainly by two signaling pathways - the canonical pathway (which is the classical pathway) and the non-canonical pathway (which is also called the alternative pathway). Lets discuss about each of the pathway in detail. But before going ahead, it is important to note the common regulatory step of both the cascades is the activation of IκBkinase (IKK) complex. In unstimulated cells, the inhibitor, NF-κB proteins are bound to IκB protein that maintains NF-κB in an inactive state. Hence, when IκB is activated i.e.; phosphorylated, it gets ubiquitinated and is degraded by proteasome. The IKK complex consists of kinase subunits that are catalytic (IKKα and/or IKKβ) and non-enzymatic protein that is regulatory in nature - known as NEMO which stands for NF-κB essential modulator, also known as IKKγ.

Canonical NF-κB Pathway:

The ligand binds to a cell surface receptor such as the members of the Toll-like receptor superfamily. This binding leads to the recruitment of adapters (such as TRAF) to the cytoplasmic domain of the receptor. These adapters then recruit the IKK complex which comprises of IKKα and/or IKKβ and NEMO or IKKγ subunits. The IKK complex then phosphorylates and consequently degrades IκB inhibitor by ubiquitination. NF-κB translocates to the nucleus to activate the target genes. The canonical pathway activates NF-κB dimers that comprises of Rel-A, c-Rel, RelB and p50.

Non-canonical NF-κB Pathway:

The non-canonical pathway is another arm of NF-κB pathway that predominantly targets the activation of p100/RelB complexes that occurs during the development of lymphoid organs which are responsible for the generation of B and T lymphocytes. The stimuli that activate NF-κB via this pathway are very few which includes lymphotoxin B and b-cell activating factor (BAFF). Note that this pathway does not utilize NEMO/IKKγ.  Here, the ligand binding activates the NF-κB-inducing kinase (NIK). This NIK phosphorylates and activates IKK complex (IKKα and IKKβ complex) which in turn phosphorylates p100. This leads to the liberation of p52/RelB heterodimer. This heterodimer then translocates to nucleus to activate the target genes.

Here ends the explanation of this pathway. Any doubts are welcome!