Question

How to know when your research has been going terrible wrong

Answer 1

Me and my research team set up to analyze the structural influence of lysozyme in the presence of calcium ions. The idea for the project was derived as we found out that equine lysozyme was able to bind calcium ions. In order to investigate this we choose to study the stability upon meta coordination. Multiple readings using fluorescence spectroscopy gave the image that the stability remained the same if coordinated or not. This was highly interesting and it was hypothesized that could perhaps be that the confrontational change was to strengthen the hydrophobic core of lyzozyme. In order to investigate this we have been doing a near-UV CD spectrum fingerprint of apo-lysozyme and lysozyme in the presence of calcium ions. The results showed that the CD fingerprint was almost identical indicating that the tertiary structure has remained unchanged. In a final hope we choose to do NMR but with egg white lysozyme in apo-form and under the presence of calcium ions, furthermore we this time choose to add equine lysozyme under same conditions which my follow students as been purifying. The NMR experiment shows no change in chemical shift in the egg white lysozyme experiment, but equine experiment shows a change in chemical shift concentrating on the residues Asp-85, Asp-90, Asp-91, Lys-82 and Asn-87.
Using bioinformatics I studied these data. The next post will show the (un)surprising and stupid results .

Answer 2

Using bioinformatics and doing my homework I choose to study the two lysozymes in structure by comparison. A structural alignment reviled that the structure of both lysozymes are highly the same with a RMSD on 0.96. I choose further to investigate the results from the NMR experiment by looking at the specific residues that gave rise to a change in chemical shift. the results showed that these specific amino acids were substituted with no conservation what so ever. Further bioinformatics showed that these residues needs to be completely conserved, in order for calcium to bind, in other proteins closely related to equine lysozyme.

Answer 3

So there you have it: How to waist 2-3 weeks of research and money