How does protein denaturation work?

Question

I was wondering how protein denaturation works.1. Are there covalent bonds, such as disulfide bridges involved, or is it based purely on non-covalent bonds such as hydrogen bonds? Why is denaturation irreversible in most cases if only non-covalent bonds are involved?1. Is it possible to denature protein by rapid changes in electromagnetic field or pressure? (The articles I have read so far mention only stress factors like sudden pH, osmolarity, temperature changes...)1. How can I protect a protein against denaturation? e.g. in PCR we use a heat resistant DNA polymerase, so certain amino acid sequences might protect against heat denaturation, but I need reassurance about this.

Answer 1

When a protein denatures, the quarternary, tertiary and secondary structures are disrupted. Meaning, everything about the protein except its amino acid sequence changes. Changes in temperature, pH, or solute concentration change will disrupt the hydrogen bonding within the protein, but will not affect disulfide bridges. Disulfide bridges are broken when a reducing agent is present, like beta-mercaptoethanol.

You can protect a protein by keeping it in an environment that resembles the natural environment as closely as possible. That is where buffers come into play, to keep the pH in check, or additives like BSA that mimic other proteins present in the cell. If you have a protein function that you can measure, it is helpful to see how much of your protein has remained functional and if the conditions you chose are suitable.

Proteins from the various organisms are very different from each other. There is no universal amino acid sequence that makes a protein more stable in the heat. However, proteins coming from organisms living at higher temperature need to be able to function at higher temperature. Those proteins will have more hydrogen bonds to keep the functional amino acids in the places where they are needed. The overall composition of amino acids in heat-stable proteins needs to provide the stability so that the protein functions there.