E2Medicine

Protein folding refers to the process by which a protein assembles itself into its correct native structure by arranging its chains of amino acids into structural motifs and orientating them relative to one another. Although some of you may be thinking along the lines of a kind of glorified origami this field of biochemical research is more complicated than its name might suggest (although it is a valid metaphor, just bear in mind that instead of hands this kind of folding occurs via molecular interractions involving various kinetic factors).

Proteins are synthesised as linear chains of amino acids which are only of biological use once they have folded into a stable specific 3-dimensional conformation. For example, a digestive enzyme such as pepsin would be of little use as a loose chain of amino acids flapping around as they please in the stomach but must instead fold to the correct shape in order to bind to its substrates and break them down. This folding must be precise in order for the protein to be functional.

Protein folding is a spontanteous, ordered and reversible process.

Protein folding has long known to be spontaneous in vitro (Anfinsen 1973) with no other factors required for correct folding in solution. Therefore ultimately all the information required for correct folding exists within the protein itself, in its primary structure (the linear sequence of amino acids that make up the protein, encoded for by DNA). Specifically folding refers to the formation of secondary and tertiary structure. Secondary structure refers to structural motifs such as alpha helices and beta sheets, which can form repeated structural patterns within a protein. Tertiary structure relates to the arrangement of these motifs and overall arrangement of the protein in space, its overall shape often referred to as its fold. The observation that primary structure determines a protein's fold and contains all the information necessarry for the protein to fold correctly has wide ranging implications as it suggests that at some point in the future we may be able to predict a protein's fold from its amino acid sequence, and because we can determine amino acid sequence from gene sequences potentially we may be capable of determining a proteins structure from a gene sequence (see below).

Is folding fast or slow?

Well actually its both. The Levinthal Paradox gives us one perspective on this question. This calculation assumes that in a protein of n amino acids each amino acid has two rotatable bonds (admittedly a gross underestimate considering the long side chains of some amino acid residues) and that each bond has three stable conformations (another underestimate). Therefore there are 3²ⁿ possible conformations in a protein. Now assume that bonds in a protein can reorientate at the same rate as a single bond to find around about 10¹³ conformations a second (almost certainly an overestimate). The time taken for a protein to search all the conformations available to it can thus be expressed as t = 3²ⁿ/10¹³.

Try this for a small protein of 100 amino acids. Can't be bothered? Well lucky I'm here - the answer is around about 2.7 x 10⁸² seconds. To put things in perspective that's longer than the universe is thought to be old. Thus even while applying assumptions grossly innaccurate in favour of quicker than actual folding, a small 100 residue protein would need to be older than the universe to guarantee finding its native conformational state by random folding. In the more complicated reality of protein dynamics folding in this manner by exhaustive random re-orientation of bonds would take even longer. Thus it has been concluded that folding is not a random process but a folding pathway must exist to guide folding.

Protein folding actually takes seconds. However, although the early protein-folding scientists questioned how protein folding could be so fast later studies showing that folding of isolated secondary structure such as alpha helices could take place in nanoseconds questioned why folding of whole proteins was so slow in comparison. The reason for this is that an energy barrier exists in the folding pathway as proteins must acheive an energitically unfavourable transition state on their way to their final conformation. The transition state only lasts for around about a picosecond but the difference in stability between the transition state and unfolded states (i.e. the difference in free energy) represents a barrier that must be overcome in order for folding to proceed.

Protein folding pathways

Since the 1980s there have been two main contrasting proposals for the protein folding pathway:

The framework model - secondary structures are proposed to form first and dock with each other to influence the folding of other parts of the proteins into the native state.

The hydrophobic collapse model - the protein collapses into a compacted state due to the hydrophobic effect and thus limits the conformational search for the native state.

However, experimental evidence does not suggest the presence of either tightly compacted structures without secondary structure or expanded molecules with highly ordered secondary structure. In addition a tightly compacted collapsed state would limit the reorganisation of structure and thus not favour folding while evidence suggests that secondary structure present in unfolded states still requires hydrophobic interractions for stability.

Nucleation condensation - a unifying mechanism for protein folding.

As with many directly opposing scientific theories the answer appears to lie somewhere inbetween the two extremes. The nucleation condensation theory arose in the 1990s and has been developed recently as proteins such as chymostrypsin inhibitor 2 were found to fold without folding intermediates (described as having two-state kinetics) while phi-value analysis of the transition state (an unstable conformation that lasts for a picosecond) showed that secondary and tertiary structure forms in parallel as the protein undergoes a general collapse. Molecular dynamic simulations of unfolding have provided furthur atomic resolution to support this experimental work which is also in agreement with general kinetic models. In brief this model suggests that patches of residual structure in the unfolded state such as hydrophobic clusters and short alpha helices interract with each other through long range contacts (in terms of sequence) to form a nucleus of native-like structure in the transition state. Nucleus formation is the rate-determining step of folding representing the energy barrier discussed previously. Secondary structure and tertiary structures formed in the nucleus aids the formation of furthur native structure through furthur long range contacts, via side-chain interractions for example, referred to as contact-assisted structure formation. Multi-domain proteins can use this mechanism to fold each domain separately in a localised manner. Although the sequence of nuclei are not conserved from protein to protein their secondary structural motifs do appear to be conserved. Because secondary structure can be predicted from amino acid sequence by methods of varying accuraccy there is potential for identification of the structure of folding nuclei from which overall structure prediction may eventually be possible.

A Helping Hand

Chaperones such as the Hsp70 family, are protein molecules required for efficient folding in vivo. They have no influence on how a protein folds but are important in allowing folding to occur in the intracellular environment. They bind to highly hydrophobic sequences and thus recognise unfolded proteins which would otherwise have such sequences buried in the core of the protein. Because proteins are synthesised as a chain of amino acids emerging from a ribosome it is important that a chaperone binds to the emerging chain to prevent premature folding before all the "information" (i.e. the complete amino acid sequence) is present. In addition the cell is a highly crowded environment (~300g/l protein and other macromolecules) which increases the possibility of unfolded protein structures associating through hydrophobic interractions and forming aggregates. It is the role of chaperones to sequester proteins and prevent this type of "clumping". Chaperones bind and stabilise proteins in unstable states and through regulated binding and release facilitate their correct folding.

So why is this all important?

Well you've read this far (or skipped right to the end), I guess you might want to know what implications this has in the "real world". As mentioned previously, proteins need to fold properly to be functional. So unsurprisingly diseases exist due to misfolding of proteins. As proteins aggregate due to misfolding these aggrergates adsorb other important macromolecules which damages and kills cells. Protein aggregates released from dead cells can in extreme cases damage tissues such as the brain, which is particularly vulnerable due to its highly organised network of nerve cells necessarry for function. Thus misfolding diseases such as Alzheimer's disease, Huntingdon's disease and prion diseases (such as Creutzfeldt-Jacob disease) manifest themselves in neurodegeneration and dementia. Possible treatment of these diseases could exploit detailed knowledge of protein folding and the prevention of abnormal folding.

The potential of prediction of protein structure from DNA/amino acid sequence as mentioned before will rely on a detailed and complex understanding of how proteins fold. Prediction of protein fold and thus protein structure would mean that in conjunction with the Human Genome Project (or the sequence of any other organism's genome for that matter) the structure of proteins encoded for by genes of unknown function could have a structure determined without actually having to isolate the protein itself. Such determined structures are likely to suggest the function (or at least a class of function) of the protein encoded and perhaps lead to the identification of the roles and modes of action of genes for which there is currently little information.

Despite its simplistic name, protein folding is a very complex and wide-reaching field of study providing many exciting prospects for the future of biology, biochemistry and molecular biology.

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References:

Daggett V and Fersht A R, Is there a unifying mechanism for protein folding?, Trends in Biochemical Sciences, Vol.28 No.1 January 2003: pp 18-25,

Agashe V R and Hartl F-U, Roles of molecular chaperones in cytoplasmic protein folding, Seminars in Cell & Developmental Biology, Vol 11, 2000: pp. 15-25

Anfinsen CB, Principles that govern the folding of protein chains, Science 181:223-230 1973

Alberts et al., Molecular Biology of the Cell (4th edition), Garland Sciences, USA, (2002)

Voet D and Voet J G, Biochemistry (2nd edition), John Wiley & Sons, Inc., 1995.

Lectures from Dr S E Radford at the University of Leeds

My life began in a little-known part of the human liver named the left lobe. My parents were two very attractive sporozoites that came over the Pacific in a mosquito named Phil. My parents were injected into a guy named John, whose liver we inhabited.

John was, above all things, a drinker. He was addicted to potato vodka. All the bartenders knew his name, even though he only frequented one bar. His liver was in terrible shape for a 23 year old.

My parents and I were living in John’s liver for about three years. What John didn’t know is that I have millions of brother and sister cells, all moving about and metastasizing to other organs. He finally started to show signs of Malaria.

Malaria normally presents with a long period of chills and fever. John was semi-immune, so that’s why it took two years for symptoms to appear. To put it shortly, John went untreated for too long. He passed away some time later partly from cirrhosis and partly from his liver arteries being blocked. However, a mosquito picked us up and transported us to Kevin.

Kevin took very good care of himself, in that he didn’t drink, smoke, or eat junk food. However, we were in a bad place, because Kevin was, and is, a paranoid doctor. He does a multi-test on his blood every 3 days. Crazy, huh?

Well, as I was older than most plasmodium, I mutated into a drug-resistant version of myself. My parents, however, were not so lucky. Kevin treated himself with proteolytic enzymes after he started getting chills. It would suck to be Kevin if we were any other parasite, as PEs are the equivalent of shooting a BB gun at a freight train.

I took great offence when Kevin killed off my parents, so I got all buggy on his pampered, medical self. I metastasized to his colon and gave him polyps. How's that for revenge?! Now he has to have one of his peers check him. Mwahahahahahah!!!! (For those of you reading this that are looking for medical accuracy, look elsewhere.)

Kevin, as I said, is paranoid. He is also quite brilliant and mad as a hatter.

He went into a closet and started talking to someone that I could only assume to be me.

The conversation went somewhat like this: "All right, you little vermin, if you don't hitch a ride on one of the mosquitoes I'm going to bathe in, I'm sending in the Vancomiacin." I was horrified at this, as Vancomiacin has been described as sending liquid fire through the veins. Kevin walked to a jar and opened it. Mosquitoes poured out. I was scared of the Vanco, but, I figured that Vanco can't hurt me. So I didn't leave.

The bordering psychotic took the Vanco out of the cabinet and swiftly injected it into his carotid. I never swam so fast in my life. I found out that Kevin had a stomach ulcer. I entered the ulcer and prayed the Vanco wouldn't go to the ulcer. It didn't. And so, now I sit, in Kevin's liver. And I'm not planning on moving.

Pectus excavatum (PE) is a deformity of the sternum that ranges from mild to severe. The chest caves inwards in the center (place a ball on something like a pillow and you'll see what it looks like). Two common names are "funnel chest" and "sunken chest". The result will range from cosmetic problems for mild cases to smaller lung capacity and problems with heart displacement for severe cases. Scoliosis is common among all cases. Approximately 1 in 500 people have PE, and it is more common in males. It is something that can be inherited.

The cause of PE varies from person to person, but here is a list of conditions that have been associated with it:

• Rickets
• Turner's syndrome
• Noonan syndrome
• Marfan's syndrome
• Aarskog syndrome
• Homocystinuria
• Osteogenesis Imperfecta
• Multiple Lentigines Syndrome
• Mitral Valve Prolapse
• Neurofibromatosis
• Ehlers-Danlos syndrome
• Congenital pectus (present at birth without any other conditions)

There are three known ways of correcting PE: the Ravitch, Nuss, and Leonard procedure. For mild cases, certain exercises and better posture have been known to help. Some correction methods are easier to do when younger because the bones will be softer.

I'm surprised by how few people know about this deformity, even though it's not that rare. My childhood doctor didn't even know what it was. Many doctors are known to tell patients to just shrug it off because they think it's only a cosmetic problem. That's not a very good thing to do because it can get worse with age and make treating it more difficult.

Osteogenesis Imperfecta, also called Brittle Bone Disease, means literally "imperfect bone formation". It can be the result of direct genetics - two parents who are carriers of the gene - or of a random genetic mutation, which accounts for a surprising number of cases. Bones are brittle and weak and tend not to grow straight - many people with OI have extremely curved spines which limit mobility. People with the more severe forms of OI (type III, mostly) tend to have stunted growth - 3'5" would be considered tall. The shortest man and shortest woman in the Guinness Book of World Records - both at just over 2 feet tall - both suffer from OI. The harsh curvature of the spine tends to make the already diminished height seem even less. Babies with OI are often born with blue-ish teeth and eyes (the whites of the eyes, not the iris). Hearing loss is very common in adulthood, generally beginning in the person's late 20s. Fractures and breaks are very common, especially in babyhood and childhood, while bones are still forming. Because of this, many children with milder forms of OI, where the disability is not as visible but where the weak bones and thus many breakages nevertheless exist, are suspected of being abused, and some have been seized from their parents. Back pain is also very common. OI does not cause infertility, but for most OI women giving birth to a normal sized baby would be a very dangerous proposition with a high risk of shattering their pelvis. Children of people with OI would have a 50% chance of being born with OI themselves.

OI is very rare, and thus not the subject of major research, but some developments have been made in treatment. A new drug recently developed has been tentatively shown to help straighten children's bones in development, avoiding the need for surgery. Common treatments include surgery to straighten bones by breaking and re-placing bones or inserting metal rods; surgery to insert a metal rod along the spine; cosmetic whitening of blue teeth; physiotherapy to develop exercise regimens and living skills for unusual body shapes. Most people with a severe form of OI require an electric wheelchair or at least braces and/or a manual wheelchair to move around. Some have trouble typing because of their small hands. Most require adapted office furniture and homes to accomodate their small stature.

OI does not in any way affect the intellect.

All that needs to be said.