And what is it you do again?

You “Great, so you work with proteins…you and a million other scientists.”
I “Considering the number of proteins out there, that’s a nice rough estimate. You should major in statistics”
I ” Before you reply, did you know a major in statistics is, by far, one of the least popular majors around? I heard this from an MIT student.”

You “…”

So what is it I do again?

Simple!

Centrin, a calcium binding protein, regulated by phosphorylation and calcium is found to be essential in centrosomal duplication during cell division. Centrin undergoes structural changes that occur upon phosphorylation of the protein and its subsequent interaction with other proteins such as Sfi1.

So what’s the overall importance of this fact?

Well, it turns out that hcentrin proteins (short for human centrin) have been found to manifest on a larger scale during cell division of non-viable cells (cancerous cells in this particular case).
So why do we see such an increase in the number of hcentrin proteins, as compared to the small amount present for viable cell division?

Good question…
We do not know, but we’re terribly excited to find out!
Where do we start from though?

Well, Our research is primarily focused on protein structure and dynamic studies. We wish to perform biophysical analyses on our proteins of interest in order to elucidate the interactions these have with other proteins such as hsfi1, which we’ve found interacts with hcentrin. But besides understanding how this interaction takes place, we’re also interested in observing those conformational changes these proteins undertake. This could serve useful for future drug design experiments, as a reference tool for other centrosomal proteins studies, etc…

Convincing, maybe?
I’ll endeavor to by the end of this year

I’ve recently been purifying hsfi1 protein pellet samples that are expressed in the lab. For this particular protein I use an affinity column. The idea behind this procedure consists of the following:

The protein is expressed in a fermentor (you know…you take the vector [plasmid] and you insert that gene of interest you wish your E. Coli cell to express. In this case, it would obviously be hsfi1. Once the cell density increases inside the fermentor, and a satisfying amount of protein is estimated to have been expressed, these pellets end up in my hands and I take care of actually digging inside these pellets on a hunt for protein. But wait! These proteins in particular are GST tagged (this term will make sense in a minute). Consequently, when we transfer our sonicated pellet through the affinity column, this one contains a matrix which active composition I cannot recall at the moment, that binds GST tagged hsfi1 to the column. It separates it from the rest of the protein soup, none of it which contains any GST and can not therefore bind to the affinity column (remember this protein came right our of a cell, filled with everything from a golgi apparatus, to an endoplasmic reticulum, lipids, etc…multiply all those organs by a couple of thousands, a large number of proteins, and quite a bigger number of everything else a cell could possibly hold, and you got yourself quite a job). Once separated, you will most likely use other purification methods that might include other chromatographic columns (must not forget to cut that GST tag out of the protein once done with it..hooray for enzymes!).

Anything else?

I’ve recently been working with two synthetic hsfi1 protein domains (p10, p21). I’m currently using circular dichroism to measure their optical activity (in other words, an analysis of their secondary structure). By measuring them independently, and also interacting with centrin (protein-protein complexes), I can see any structural changes from either protein (random coiled, alfa helix, beta sheet, etc…) We also use calorimeters (DSC, ITC), Infra-red, etc. I have yet to work with these other instruments though. I believe I might start with DSC and infrared shortly. We shall see.

I think I work at an amazing lab. The research we do is absolutely incredible! I’ll let you in a little on protein cristallyzation (did I spell that right?) at our lab soon. A group is non-existent without its leader. I think I did the right thing in choosing my leader (Prof. Belinda Pastrana).