point mutation


Fire, by Giuseppe Arcimboldo. 1566 Oil on wood, 67 x 51 cm Kunsthistorisches Museum, Vienna

Fire, by Giuseppe Arcimboldo.
Oil on wood, 67 x 51 cm
Kunsthistorisches Museum, Vienna
The allegory of Fire combines objects that are more or less directly related to fire in a bizarre profile head. The cheek is formed by a large firestone, the neck and chin are formed by a burning candle and an oil lamp, the nose and ear are contoured by firesteels; a blond moustache is formed by a crossed bundle of wood shavings for kindling, the eye is an extinguished candle stub, the forehead area is a wound-up fuse, the hair of the head forms a crown of blazing logs. The breast is composed of fire weapons: mortar and canon barrels together with the respective gunpowder shovel and a pistol barrel.

In protein engineering studies, mutating a residue to increase thermostability without affecting the activity of the protein/enzyme is a major consideration for researchers. The laborious method is list the number of possible mutations and then finding out the stability and activity for each mutant, one after another.

This method becomes more time consuming if the protein is a membrane proteins and especially determining their 3D structure. I like to call membrane proteins as “diva” proteins. The reason being that they are high maintenance and tend to be picky about what conditions require for them to crystallize. It has been reported that when thermostability is introduced in membrane proteins, their solubility increases, thus increasing the chances of getting a good crystal for diffraction. [1]

Not everyone could avail high-throughput mutation experiments to screen for thermostable membrane proteins. Here is where Bioinformatics based analysis comes to help in faster screening and selecting a few mutants among the hundreds that can be tested experimentally. In the recent issue of Biophysical Journal, Sauer et al have come up with two methods to identify potential “thermoadaptive” sequences. [2]

The first method or global method, involves generating a heatmap of amino acid frequency differences between the thermophilic and mesophilic sequences. So, residues that are either most represented or less represented are identified.

The second method or pairwise method, involves pairwise comparison of thermophilic and mesophilic sequences and identify the differences.

A unique observation was that the the selected list of amino acids did not overlap from either of the methods and the global method identified potential mutants in the N-terminal domain of the test case and the pairwise method identified the potential C-terminal mutants only. This could be a case of thermostabilization for the protein tested, i.e., BsTetL – Tetracycline transporter from Bacillus subtilis.

The caveat is that there should be enough available sequences for identification of potential mutants, in any protein family. This does not, on the outset, seem like a limitation. Since, we have abundant protein sequences available and steadily increasing.

The main selling point is the speed of identifying the mutations given a particular target sequence, and its applicability in membrane protein crystallization. However, their success rate was 26-30%. Here, success indicates both thermostable mutant and maintaining the tetracycline resistance activity.


  1. Mancusso R, Karpowich NK, Czyzewski BK, & Wang DN (2011). Simple screening method for improving membrane protein thermostability. Methods (San Diego, Calif.), 55 (4), 324-9 PMID: 21840396
  2. Sauer DB, Karpowich NK, Song JM, & Wang DN (2015). Rapid Bioinformatic Identification of Thermostabilizing Mutations. Biophysical journal, 109 (7), 1420-8 PMID: 26445442

Image reproduced under Creative Commons licence. Source: Wikimedia commons

The Cellular Prion Protein (PrPc) like Dr. Jekyll converts into PrPSc , a fatal conformational form, like Mr. Hyde, and is responsible for a variety of neurodegenrative disorders. Unlike the use of a potion, this molecular Jekyll and Hyde undergoes conformational change in low pH environment, such as in endosomes. While, there has been many studies done in the past of how this conformational change happens,  a recent paper has tried to list the structural and dynamic properties using Molecular Dynamics.

ResearchBlogging.orgTo list these properties,three structures were taken into consideration; one NMR structure (PDB id: 1QLX) and two X-ray structures (PDB id: 2W9E and 3HAK). Interestingly the 3HAK structure is from a SNP variant of human PrPc, where the Met129 is replaced by Val129. Furthermore, those who genetically have this variant are less susceptible to Prion diseases!

Structural alignment of 1QLX (blue), 2W9E (red), and 3HAK (orange) with Met129/Val129 shown as sticks.

Structural alignment of 1QLX (blue), 2W9E (red), and 3HAK (orange) with Met129/Val129 shown as sticks. Image made using PyMOL

Using an in-house MD package called in lucem molecular mechanicsilmm for short, Chen et al simulated the three structures under two different pH conditions (pH 5 and pH 7) and under two different temperatures (298K/25C and 310K/37C), totaling for about 3.6 microseconds of simulation. (For each structure under each condition the MD simulation was performed in triplicates.)

Analyzing the MD results they found that at 37C and low pH the C-terminal globular domain had significant destabilization effects.

  • The helix HA and its neighboring loop S1-HA for the SNP variant was higher compared to other two structures at 37C and low pH. It is interesting to note that the S1-HA loop becomes a strand during the prion’s conversion.
  • At low pH, another helix HB destabilizes, where the His187 becomes solvent exposed, leading to partial unfolding of the C-terminus.
  • Two residues, Phe198 and Met134, converting from being part of the hydrophobic core to being exposed to the solvent may be involved in partial unfolding and might possibly provide aggregation sites.
  • The X-loop in the Val129 SNP variant’s structure took a different conformation that was not populated by the other two structures.
  • Formation of new secondary structures of the N-terminus region to either alpha and beta strands is spontaneous. While, in all two structures both alpha and beta strands formation was seen, in the SNP variant alpha strands were rarely formed. (This N-terminus region is missing from the solved structures and hence was modeled and in each starting structure this region was unstructured.)

These results give more insights into the conversion of the benign form of human Prion to the infectious form.


  1. Chen, W., van der Kamp, M., & Daggett, V. (2014). Structural and Dynamic Properties of the Human Prion Protein Biophysical Journal, 106 (5), 1152-1163 DOI: 10.1016/j.bpj.2013.12.053

Listen to the classic song while reading the post!

If you were born in the 1960’s and if you happen to do The Twist with your partner your heart would of course be racing! Thanks to G protein-coupled inwardly-rectifying potassium channels (GIRKs) your heart can beat back to normal levels. Ironically, the protein does a “twist” to slow down the heart. Go Figure!

GIRK is basically a potassium ion-transporter and found in cardiac cells. It regulates the membrane voltage after the GPCR activated G-beta and G-gamma bind to the transporter.

In this groundbreaking work, three structures were used to understand the dynamics of the transporter.

  • The normal GIRK transporter
  • The GIRK bound with G-gamma, and
  • A GIRK mutant (R201A) that is always in the open conformation.

GIRK wildtype (201 position shown as magenta spheres) ions as spheres. PDB id: 3SYO

The mechanism of transporting the K+ ion across the membrane starts after the G-beta and G-gamma bind to GIRK, and thus getting a twist of 4 degrees clockwise (looking from inside the cell). This is observed as a highly energetic, but stable conformation. In other words, a “meta-stable” form. It is just waiting for a “spark” (binding of Na+ ion), due to which it twists further to open the channel to a wider 9 degrees, thereby allowing the K+ to be transported across the membrane. 

This twisting and untwisting continues till the resting potential (Nernst K+) is reached, thus slowing the firing frequency in pacemaker cardiac cells, resulting in slower heart rate.

The coordinates of the GIRK-G-gamma complex will be released soon. LINK


  1. Whorton, M., & MacKinnon, R. (2013). X-ray structure of the mammalian GIRK2–βγ G-protein complex Nature DOI: 10.1038/nature12241

A point mutation in a gene leads to a phenomenal effect on the phenotype. It is a classic Biochemistry textbook case study, Sickle Cell Anemia. The mutant hemoglobin has a Valine instead of the Glutamic acid. The change is highly observable in the form of a debilitating condition. But, not all point mutations in the protein sequence are debilitating, and sometimes they give rise to something spectacular. One such example is the White Tiger, frequently mistaken as an albino. The recent publication in Current Biology [1] has been picked up by many newspapers and is creating news. See here, here, and here. There are two reasons as to why this research has become news. For one, it is about the endangered species and Tiger conservation. Second, it is precisely pinpointing to a molecular level change that can now be understood.

In sickle cell anemia, the longer and charged Glutamic acid changes to a uncharged Valine, thereby wrecking havoc.

Screen Shot 2013-05-24 at 12.14.27 PMResearchBlogging.orgThe gene in limelight is SLC45A2, which is present in many vertebrates including Humans. (Uniprot id: Q9UMX9) In humans this “pigmentation related gene in humans, whose polymorphisms are associated with light skin color in modern Europeans and pathogenic mutations known to cause oculocutaneous albinism type 4“. From a structural bioinformatics perspective, the paper provides as to how this mutation can be understood towards its function in the mutant. It is a membrane protein and the solved structure is not available, but the homology modeled structure is available in ModBase [2]. See below the modeled protein structure from ModBase, with the position 477 highlighted. Figure 3 of the paper shows a different way of showing the multiple sequence alignment.

Human SLC45A2 highlighting the Alanine 477

Human SLC45A2 highlighting the Alanine 477


The residue 477 (Alanine is conserved among many vertebrates) is in the 11th transmembrane helix, and the change from Alanine to Valine, is possibly thought to prevent melanin synthesis therefore the while tiger! In the case of Sickle cell Anemia, one theory is that the mutation could have some evolutionary pressure, that is due to high prevalence of malaria. It so happens that people suffering from sickle cell anemia, do not get malaria, they are resistant to it.

In Tigers, the orange coloration of the fur, helps it to camouflage among the grasslands and forests. But, in the case of white tiger, what could have been the evolutionary pressure? This question remains in my mind after reading this paper.


  1. Xu, X., Dong, G., Hu, X., Miao, L., Zhang, X., Zhang, D., Yang, H., Zhang, T., Zou, Z., Zhang, T., Zhuang, Y., Bhak, J., Cho, Y., Dai, W., Jiang, T., Xie, C., Li, R., & Luo, S. (2013). The Genetic Basis of White Tigers Current Biology DOI: 10.1016/j.cub.2013.04.054