[wiggins.ricky]

hello im new to this forum and hope to be an active member for a long while im a graduate biochemistry student

for my first problem i would like to ask related to the alpha (α) helix secondary structure of proteins:

is there a way to design an experiment to whereby i can relatively tell the extent and the number of internal hydrogen bonding (between amino acids of the protein) compared to that of the external (with the surrounding environment and solution)??

thanks in advance its just that this problem has been taking a lot of my time and i haven't been able to reach an assuring end as i dont know whether what im thinking of is right

[wiggins.ricky]

Hello Sorry it seems i posted my question on a different subforum here (the organic chemistry) and didnt get an answer and now noticed the biochemistry one which is what im asking about, excuse me for the inconvenience

but the problem i would like to ask related to the alpha (α) helix secondary structure of proteins:

is there a way to design an experiment to whereby i can relatively tell the extent and the number of internal hydrogen bonding (between amino acids of the protein) compared to that of the external (with the surrounding environment and solution)?? basically this procedure would allow me to compare the strength of the H bonds that make up the alpha helix and the H bonds that occur between it and the external environment

i have been struggling deeply with this and would be very grateful for some guiding or help as it has consumed a lot of my time
Thanks

[salteen]

Are you looking to determine the extent of internal hydrogen bonding in a peptide for the purpose of predicting secondary structure?  Circular dichroism would help with that. 

[wiggins.ricky]

Are you looking to determine the extent of internal hydrogen bonding in a peptide for the purpose of predicting secondary structure?  Circular dichroism would help with that.

yes technically... mostly im concerned with comparing strength of the H bond between those forming the secondary structure to those that interact with the solvent and external environment (water for example)... so i need to know the strength of both or be able to compare them

would circular dichroism do that?

[salteen]

No, CD will only give you an idea of the relative amount of α-helices vs β-sheets in a protein.

I'm not familiar with experimental methods to do what you are proposing.  However, if you have a crystal structure of the protein, I do know there are programs which can calculate hydrogen bond energies with reasonable accuracy by measuring distance/angle of bonding and nearby electrostatic effects.  That would help quantify your internal hydrogen bonds.  The external hydrogen bonds are another matter all together - I imagine this would be quite difficult to calculate since interactions with the solvent and protein surface are very fluid and dynamic....

[Babcock_Hall]

One general rule is that you should show us your attempt before we help you.

Crystal structures of proteins have uncertainties in atomic positions of several tenths of an angstrom, unless it is a very high resolution structure.  I would question whether a calculation under those circumstances is feasible.

[Yggdrasil]

You probably would want to perform an experiment using double mutant cycles (some of the first papers describing the use of double mutant cycles to measure thermodynamic contributions of certain intermolecular interaction to protein structure and stability were done in the '80s and '90s by Alan Fersht, I believe, so you'll probably want to look up some of those papers).  Here's a more recent citation from a colleague of mine that measures the strength of hydrogen bonds inside of a transmembrane protein:

Joh et al 2008. Modest stabilization by most hydrogen-bonded side-chain interactions in membrane proteins. Nature 453: 1266 PMCID:2734483

Double mutant cycle analysis requires the assumption that the mutations you introduce do not introduce any compensatory interactions, so in addition to performing the unfolding experiments on the mutants, the authors of the paper also had to solve the crystal structures of the mutants in order to see that, after breaking a hydrogen bond by mutating one of the residues involve, that the other residue did not change its position significantly in order to interact with another residue in the protein.

Now, the paper I cited measures the strength of hydrogen bonds formed between amino acid side chains.  You seem to be interested in hydrogen bonds involved in forming secondary structure which is more difficult because they involve the peptide backbone and not the amino acid side chains which can easily be broken by mutation.  I'm not sure if there is a way to adapt the method for studying the energetic contributions of hydrogen bonds between backbone atoms (other than perhaps trying unnatural amino acid mutagenesis).