How guanidinium unfolds proteins

Molecular spectroscopy shows: by breaking salt bridges

7 December 2015

Researchers of the Van ‘t Hoff Institute for Molecular Sciences have discovered that the substance guanidinium chloride unfolds proteins by breaking the electrical contacts that keep a protein molecule together. The discovery is important for a better understanding of the forces that keep a protein in the folded, i.e. healthy state. The discovery is published this week in the leading chemistry journal 'Angewandte Chemie International Edition'.

Protein molecules are long chains of amino acids. Such a chain is usually neatly ‘folded’ in a well-defined, compact structure. Only in this compact form are proteins biologically active. Sometimes, certain proteins unfold spontaneously. This plays an important role in many diseases. It is suspected that such spontaneous unfolding is the first step in the development of geriatric diseases such as Alzheimer’s and Parkinson’s.

To be able to investigate the effect of unfolding proteins, researchers in a biochemistry lab often add guanidinium chloride (named after guano, bird droppings, from which the substance was first prepared in the 19th century). The guanidinium ions (charged molecules) in this substance are very efficient at unfolding protein molecules.

How guanidinium ions do this is still largely unknown. ERC PhD student Heleen Meuzelaar and FOM PhD student Matthijs Panman (now postdoc in Gothenburg) discovered that the guanidinium ions displace other positively charged atoms. This discovery is important for a better understanding of the forces that protect proteins from spontaneous unfolding. 

Salt bridges

The compact structure of proteins is kept together by various forces. One of these is the electrical force: most proteins contain positively and negatively charged atoms in the amino acid chains, arranged in such a way that in the folded state, pairs of positive and negative charges are located opposite each other. The attractive electrical force between the opposite charges in the pair neatly keeps the protein in the folded structure.

Such an electrical contact between two opposite charges in the protein is called a salt bridge (because this is also how salt crystals are held together: think about the opposite charges of sodium and chloride ions in table salt).

How guanidinium defolds a mini protein

Left: Without guanidinium, the folded structure – in this case a helix – is kept together by the attractive electrical force between the negatively charged oxygen atoms (red) and the positively charged nitrogen atoms (blue): the salt bridges. Guanidinium (green) is positively charged and displaces the nitrogen atoms, as a result of which the salt bridges are broken and the mini-protein unfolds. Image: HIMS

Meuzelaar and Panman investigated the effect of guanidinium on the salt bridges. In a series of biochemical experiments, they first examined the effect of guanidinium on a series of different peptides, which can be considered mini-proteins. Each time, they compared peptides identical in terms of composition and size, but with different positions of the oppositely charged atoms in the chain. A small change in the position of the charged atoms in the chain was found to have a huge effect on how well the guanidinium unfolded the peptides. In some mini-proteins, guanidinium even facilitated the folding of the protein instead of its unfolding.

Ions replace positive atoms

Using infrared laser techniques, the researchers could see what guanidinium does with the salt bridges. It seems that the guanidinium ions, which are positively charged, replace the positive atoms in the salt bridges: they displace the positive atoms, as a result of which the salt bridges break and the protein unfolds. A new salt bridge is then formed: no longer between the two oppositely charged atoms of the protein, but between the positively charged guanidinium and the negatively charged atom of the protein.

Article

Meuzelaar, H., Panman, M. R. and Woutersen, S. (2015), Guanidinium-Induced Denaturation by Breaking of Salt Bridges. Angew. Chem. Int. Ed., 54: 15255–15259. doi:10.1002/anie.201508601

Published by  HIMS