Protein, first... and when it goes awry (Part 2 of 2)

Protein folding and misfolding

The basic structure of a polypeptide is a linear chain of amino acids in the form of a random coil. The amino acid sequence and the chemical and physical properties of the amino acids influence protein folding into a 3-D shape. Folded protein typically has amino acid side chain packing that stabilizes the native state, with hydrophilic (“water-loving”) or polar amino acids located at the surface where they can interact with water molecules in the cell. Minimizing the hydrophobic (“water-fearing”) or nonpolar amino acids exposed to water is an essential driving force in the folding process.

Folding primarily involves the formation of secondary structure and then tertiary structure. Hydrogen bonds contribute to the stability of the folded protein. Formation of disulfide bonds between cysteine residues allows more interactions between other amino acids. The folding process also depends on the temperature and presence of molecular chaperones. Before assuming the more energetically preferred native state, protein molecules pass through an unstable intermediate state. Under certain conditions, the protein may not fold into its biologically functional form. Enzymes which enhance folding, and molecular chaperones transiently interact with the protein. The molecular chaperones either mask the protein so that folding is not disrupted by interactions with other proteins, or they help unfold misfolded protein so that the protein can refold correctly. Molecular chaperone-assisted folding is crucial in the crowded environment in the cell to prevent aggregation of protein molecules.

Failure to fold into the native structure gives rise to non-functional proteins which are toxic. Certain brain disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and Creutzfeldt-Jakob disease, arise from the accumulation of misfolded proteins that form aggregates causing death of brain cells, leading to movement problems, dementia, and various other neurological symptoms. The peptides/proteins involved are beta amyloid and tau protein in Alzheimer’s disease, alpha synuclein in Parkinson’s disease, huntingtin in Huntington’s disease, and prion protein in Creutzfeldt-Jakob disease.

Protein misfolding has been implicated in diabetes mellitus, atherosclerosis, retinitis pigmentosa and osteogenesis imperfecta. A type of diabetes mellitus is due to deficiency of insulin caused by misfolding of proinsulin, precursor of insulin. Atherosclerosis is thickening of arterial wall due to deposition of low density lipoproteins (LDL, or “bad cholesterol”) with misfolded apolipoprotein, and is a risk factor affecting predisposition to heart disease. Retinitis pigmentosa is retinal dystrophy which is caused by misfolding of opsin, photoreceptor protein found in the retina of the eye, resulting in tunnel vision and blindness. Osteogenesis imperfecta or brittle bone disease is due to misfolding of collagen, leading to bone deformity.

‘Test tube’ peptide

For many years, I undertook research on cone snail peptides that included amino acid sequencing, peptide synthesis and folding, among others. Amino acid sequencing is not difficult because nowadays the procedure can be done automatically by an instrument called protein sequenator which was developed by Pehr Victor Edman and Geoffrey Begg. So our team did not have to do sequencing the tedious way that Sanger did for insulin! Chemical synthesis of linear peptide by solid phase method is also not difficult because it is an automated process. But the challenge for us was in folding the synthetic linear peptide.

Our objective was to fold peptide into a 3-D structure that would be identical to the natural peptide so that we could have unlimited amount for further study, and would not have to purify more of the peptide from the cone snails. Folding a peptide with up to six cysteine residues could be accomplished simply by adding chemical oxidants and/or continuously aerating the peptide brew. In each case, we found the proper conditions to get the correctly folded peptide with the correctly matched pairs of cysteines as the major component of the product.

Folding a peptide with less than 50 amino acid residues, including 10 cysteine residues, was really a big challenge. In the correctly folded peptide, the 10 cysteines should form five disulfide bonds, with the correctly matched pairs of cysteines. During the course of the experiment, the presence of precipitate (aggregate) was observed. Analysis of the product of the folding process by chromatographic technique showed several peaks presumably due to partially folded peptides and/or misfolded peptides containing mismatched cysteines. Unfortunately, we were unable to obtain significant amount of the correctly folded peptide. On a good note though, we published in an SCI-indexed journal a paper on the natural peptide derived from a few cone snails even without the synthesis part, by focusing on other novel and interesting aspects of that peptide.

Perhaps it would take another genius with painstaking effort to make in vitro folding an automated process. By then, we would be looking forward to peptides coming out of the peptide synthesizer already properly folded!

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Dr. Elsie C. Jimenez, a member of the Philippine-American Academy of Science and Engineering, is a retired professor of chemistry at the University of the Philippines Baguio. She undertook research on peptides from cone snails, for which she is co-inventor in several US patents on these peptides. She is currently doing research on proteins and toxins of red tide organisms at the Marine Science Institute, UP Diliman. E-mail her at [email protected].