Anfinsen's conclusion - that protein structures are encoded within their sequence - is still the main hypothesis for how proteins fold.

Some proteins do not fold (intrinsically disordered proteins), and this has been shown to be due to a bias in amino acid composition towards charged residues, proline and glycine (Ward 2004).

In the cell, some proteins require chaperones to fold correctly, but this is often to avoid aggregation with the high concentrations of intracellular proteins. In the test tube, these proteins can often fold on their own, and therefore follow Anfinsen behaviour (there are some exceptions).

In terms of the native state being the free energy minima, that is correct for a monomeric protein. However, research by Chris Dobson has suggested that the most stable state (lowest free energy) for all proteins is as an oligomeric amyloid.

See Dobson 2003 Nature (https://www.nature.com/articles/nature02261) for a great review on all of the above.

Dear Renata,

Sorry for being blunt. Neither your question not supporting answer of Michael are accurate nor anytime valid. Many new developments happened since the mid 1960's. Unfortunately Science development is about marketing. Below you find information that not necessarily finds its way to the marketing workplace.

The original formulation of Anfinsen for which he got a Nobel prize was that the global free energy miniumum of a particular sequence in a particular environment determines the folding - not that sequence alone determines the shape (fold). This cannot possibly be correct because it would exclude a possibility of life; which is synonymous with change. So even if you admit thermodynamic fluctuations, life would be impossible in an extremely rugged (deep minima) energy landscape nor in extremely flat one because of instability (like in liquids). Besides even Anfinsen's experiments contradicted his original notion as cold and warm unfolding experiments preclude real minimum be in existence. Additionally, the observed ceasing of protein function (like rebinding capabilities, or enzymatic activities) below a certain temperature (routinely called the glassy transition temperature) also precludes thermodynamically controlled folding and non thermodynamically controlled function.

Function is synonymous with mobility.

So after these initial remarks we can sketch out the initial fissure in this superficially accepted paradigm of protein folding.

1) People noticed that very similar sequences have different folding classes (homology modeling has natural limits)

2) People noticed that sometimes limited number of mutations changes the dynamic landscape and lead to change in folding class.

3) People discovered prions (different initial and final folding).

4) People discovered serpins (that change folding within the context of the interactions)..

5) People discovered transcription factors that fold only on the targets (nucleic acids or proteins).

6) The Paracelsus challenge was issued (Lynn) and in less than 50% mutations the protein was converted from one folding class to another.

7) People in the 1990's (R. Sauer) discovered that a single amino acid mutation change the folding from alpha to beta of a significant portion of the protein.

8) People discovered protein concerted motions (Caspar, Frauenfelder, Teeter) that encompass solvent and protein.

9) Kern at Brandeis showed that the motions control the enzymatic activity.

10) Teeter and Stec proposed that proteins are built of a composite of solid a liquid states components that proportional contribution controls the function (part of the protein is like solid and part is like liquid).

11) Stec published a crystal structure of a protein in which the identical sequence in two directly hydrogen bonded molecules attains the alpha and beta conformations at the same time at the same place proving existence of sequences that are completely ambivalent to the structure.

12) Wolynes published his "earth quake" model that stresses the notion of frustration or designed miss-optimization.

13) Hernandez published his musings on importance of solvent dynamics that followed the Frauenfelder suggestion of "solvent slaving".

As you see there are significant developments that no longer support a simplistic notion of sequence-folding-function direct relationship. The best proof is an entire career of Baker who is the most prominent protein modeler in the world now. He showed a complete failure of the energy based optimization schemes for protein modeling. Only addition of heuristic constraints allowed for a relative success of Rossetta based formalisms. As a good primer you should read is my paper in PNAS (Burra 2009)that shows why this was a failing notion. Another notion of a protein funnel is only a simplification and not necessarily reflect the reality. It was proven that sometimes the intermediate folding states are longer lived then final folding states. Additionally the protein kingdom has three general classes of proteins: relative well-stable, relatively unstable, never independently folding (fashionably called structurally-intrinsically disordered) that are almost equally populated in eukaryotes while they percentage contribution strongly depend on the kingdom and a class of an organism (mesophile, thermophile).

I hope it helps.

Bog

Dear Boguslaw,

Thank you for your comments.

I am not exactly sure how your points disagree with the notion that proteins fold to a sequence encoded free energy minima.

You are correct that Anfinsen stated the ‘thermodynamic hypothesis’ for protein folding. He also stated in his 1961 PNAS paper that ‘From chemical and physical studies of the reformed enzyme, it may be concluded that the information for the correct pairing of half-cystine residues in disulphide linkage, and for the assumption of the native secondary and tertiary structures, is contained in the amino acid sequence itself’. Therefore, he does specifically state that amino acid sequence encodes structure (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC223141/).

I’m not sure how reaching a thermodynamically stable state excludes life. I agree that proteins require motion, and the thermodynamic description accounts for this. Just because something is more stable does not mean it is fixed in that state - the very nature of thermodynamics means that the reaction is reversible and the protein can unfold. Proteins have motion in their thermodynamic minima and are therefore not static structures. Furthermore, differences in free energy change when the environmental conditions are altered e.g. temperature; ligand binding, which means that proteins can readily change structure in response to a stimulus.

In terms of hot and cold unfolding, these are both explained by thermodynamics. The equation for Gibbs free energy (G) is:

dG = dH - TdS

where H is enthalpy, T is the temperature and S is entropy. As you can see, the stability of protein (dG) will change when the temperature changes. Furthermore, proteins typically have different heat capacities in the unfolded and folded states, which means that dH is also temperature dependent. This explains why protein stability changes at high and low temperatures, and therefore why they unfold.

Binding and enzymatic activities will also have free energy profiles which change with temperature, explaining why protein function is temperature dependent.

I agree that designing a protein structure de novo is currently very difficult. However, protein design researchers, including David Baker, have had great success by starting from a known protein structure and sequence. Perhaps the best example of this is sequence consensus design, where a protein produced from a collection of the most conserved residues across protein homologues has the same fold and greater stability (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917058/).

Therefore, I don’t see how your points disagree with the notion that the amino acid sequence encodes structure(s) that are the free energy minima.

Dear Michael,

I am sorry, I cannot help you. You have to find this out on your own, either by understanding what I am writing about or by your own experience. In every science in a paradigm change the element of faith (belief) is the key and some wrong paradigms obviously persisted for more than 1000 years (recall Ptolomean earth centered system).

I am sorry, but if you do not see any contradiction with Anfinsen in fact that the same sequence can fold to two completely opposed structures you can only blame your teachers for not explaining mathematical logic. If you do not see that liquids do not have preferred shape (fold) as determined by thermodynamics you can only blame your physics teachers. If you do not see that function has a lot of different meanings in biology but most of them is associated with mobility (that by definition is opposed to defined structure/fold) you can only blame your biology teachers.

(From your answer, I surmise, that all these behaviors are covered by simple thermodynamics, therefore everything is fine).

Thermodynamics does not explain what is the optimal angle of the sand pile. This is a self-organized system. There is not even approximate theory. The new paradigm that I am trying to show you a glimpse of is that proteins are self-organized entities. Some properties are obviously partially explained by thermodynamics but many of their features are not. Some proteins are stabilized by more than 50kcal/mol barriers some of them do not have any stabilization energy. Some proteins live for millenia in a folded state some stop existing in microsecond scales. Some are designed for performing a single motion some are completely like liquid or gel. These do not retain any structure so how you apply Anfinsen principle to these cases I do not clearly see. Moreover a lot of proteins behave in entropy defying ways.

This is probably not an appropriate place to criticize and what Baker is doing because he is very successful in selling his stuff. I can only direct you to his own admissions (if I correctly remember in discussion in Science around 2008) when he admitted that he is producing mostly dead proteins. He obviously made significant progress since that time and he even recently claims he can produce enzymes (catalysts) but for the people like me who tried to use his stuff in many different combinations is like going to the doctor sick, counting on a quick remedy and getting answer in a year that is 90% incorrect.

This is mostly in line with a sobering recent realization of NIH in the US that around 90% all biology science results are NOT repeatable. Scientist publish what worked not a majority of experiments that do not, even if this is the same experiment.

I am sorry again, but I grew a bit skeptical over the years.

Do what you do and enjoy it. Your beliefs are your beliefs, you change them when you change them.I am here only to signal the change.

Bog

Dear Boguslaw,

Thank you for the extensive explanation. I downloaded your paper and will surely read it. You mentioned in point

10) Teeter and Stec proposed that proteins are built of a composite of solid a liquid states components that proportional contribution controls the function (part of the protein is like solid and part is like liquid).

Is it possible to determine which amino acids are responsible for the solid and which for the liquid part?

Could you also give me the reference for the following point?

11) Stec published a crystal structure of a protein in which the identical sequence in two directly hydrogen bonded molecules attains the alpha and beta conformations at the same time at the same place proving existence of sequences that are completely ambivalent to the structure.

Are the structural variations here related to the speed of folding or rather the environment of the protein? Or something totally different?

Renata

Dear Michael,

Thank you for your reply and references. I have a follow-up question regarding what you said about aggregation:

In the cell, some proteins require chaperones to fold correctly, but this is often to avoid aggregation with the high concentrations of intracellular proteins. In the test tube, these proteins can often fold on their own, and therefore follow Anfinsen behaviour (there are some exceptions).

Are chaperones needed to prevent the aggregation of extracellular proteins? Could it be that the structure of a protein obtained in vitro is different from the structure of the same protein after chaperones acted on it in vivo?

Renata

Dear Renata,

There is much background material to explain before I can answer your direct question. The human knowledge acquisition process was shaped by millenia. One of the most productive rules of knowledge acquisition is reductionism. By this principle if you subdivide the problem on smaller pieces and find answers to them you will be available to assemble to answer that will cover the larger problem. This is a very productive principle but as any human activity it has tremendous weaknesses. The best example is provided by a history of ribosome and its structure obtained in early 2000's. People for a long time new that this is a large nucleoprotein complex. So they tried to isolate individual subcomponents and try to establish folding principles for individual one (RNA as well as protein) Peter White had a program for a long time to "divide and concur". So for a long time Ada Yonath was ridiculed as a visionary who tried to crystallized and obtain the structure of the entire thing. When finally structure emerged in 2001 there were many surprises. One of them was that structures of the individual components were only approximate because proteins have extensive tails that were never folded in absence of the RNA component and stabilized the entire structure. The catalytic center turned out to be completely RNA driven. The recognition of mRNA and the protein channel had a very mixed signature. The final result was possible only thanks to a talented postdoc Nenad Ban and a long standing project for DNA and RNA processing proteins granted to Thomas Steitz. Finally it was possible to assemble the team who was able to tackle the phasing of this large complex by using an innovative methodology of the metal clusters. The moral of this story is that reductionism is powerful but sometimes only the holistic approach to science provides real progress.

In my opinion protein folding problem is an ill-posed (not solvable because is not well defined) problem that only approximate solutions are possible with extreme imposed conditions. Computational methods are welcomed by they have only very limited use. When I was studying physics in late 1970's there was a fashion for "self avoiding" walks. The progress was made and some people like Shakhnovich (protege of Karplus) made careers out of it but really not that much useful science came out of it. It is because what is important is understanding the changeable paradigm. It looks like life is tinkering on the edge between stable and unstable world. What it practically means is that proteins are self organized systems that do not have any uniform organizing principle. The only universal principle is a utilitarian need for life (function). So understanding the composition and function of protein is important. In my opinion the most substantial contributions on both issues were provided by Dorothy Kern from Brandeis and Peter Wolynes. Dorothy provided a direct evidence that the catalytic properties are directly associated with amount of mobility that is imposed (designed) into the protein structure. So consideration of the protein structure is pointless unless you consider the dynamic stability. Wolynes with collaboration with bunch of people provided a lot of facts and basis for the thermodynamic understanding of protein structure and proved the so called "frustration" is at the basis of protein design. So he basically proved that real proteins are designed to NOT be at their thermodynamic minima in terms of relation to closely spaced sequences. In order to general the dynamic signature they have purposely misplaced residues. So asking about the thermodynamic profile is becoming almost an academic exercise. However some very smart people like Freire and Hilser utilized the heuristic knowledge to produce a semi-thermodynamic scheme to predict how variants differ in their thermodynamic stability using the partition function development approximation by transferring 5-10 AA sequences between solvated and folded environments. All these facts are setting an interesting question what is answerable what is not.

My contribution is rathe modest. Two papers with Martha Teeter provided a basis for a notion that proteins are designed to dynamically couple with solvent Full‐matrix refinement of the protein crambin at 0.83 Å and 130 K

B Stec, R Zhou, MM Teeter Acta Crystallographica Section D 51 (1995), 663-681, and Global distribution of conformational states derived from redundant models in the PDB points to non-uniqueness of the protein structure PV Burra, Y Zhang, A Godzik, B Stec

Proceedings of the National Academy of Sciences 106 (2009), 10505-10510. Paper with George Phillips provided an evidence that crystallography is able to capture the NMR accessible native assemble Sampling of the native conformational ensemble of myoglobin via structures in different crystalline environments

DA Kondrashov, W Zhang, R Aranda IV, B Stec, GN Phillips Jr

Proteins: Structure, Function, and Bioinformatics 70 (2008), 353-362. With Adam Godzik we had a stab at isolating the sequence components that are related to liquid-like or solid-like components Between order and disorder in protein structures: analysis of “dual personality” fragments in proteins

Y Zhang, B Stec, A Godzik Structure 15 (2007), 1141-1147. The experimental work about a ambivalent sequence was published in Crystal structure of the tetrameric inositol 1‐phosphate phosphatase (TM1415) from the hyperthermophile, Thermotoga maritima KA Stieglitz, MF Roberts, W Li, B Stec The FEBS journal 274 (2007), 2461-2469.

The final reflection has to be made about chaperones and foldability in general. A lot of info you can get from global analysis presented in many papers of Adam Godzik. In one of them he analyzed several genomes and concluded that the foldable proteins contribution to different genomes changes and varies. The many proteins are not simply foldable. If it comes to chaperones it appears that their need was strictly associated with lowering of proteins stability in higher organisms like eukaryotes. Specialization of function dissects the proteins into clades that are less and less stable. Chaperones generally slow down folding by number of hydrophobic interactions therefore preventing misfolding therefore reaching a local thermodynamic minima in non-native structures. Functions of chaperones are many but specialization of cells provided a larger need to chaperones. So defects in Chaperones like HSP70 and 90 are associate in human cells with a lot of cancers. Your question about the extracellular proteins and intracellular proteins have nothing to do with a folding problem. All protein must be produced in a similar manner (ribosomes) and transported or by a cell death they must be exposed. Proteins are proteins and they are carriers of life therefore they cannot be "solid state" like crystals of sugars or cholesterol but must couple dynamically with solvents to carry the function. Desiccation is one of natural and unnatural method of protein preservation. I know that this post might be a bit of a informational overload but I do not have a lot of occasions (time) to come back to explain things in detail.

Good Luck in your studies.

Bog

Dear Renata,

That's a great question!

(Are chaperones needed to prevent the aggregation of extracellular proteins? Could it be that the structure of a protein obtained in vitro is different from the structure of the same protein after chaperones acted on it in vivo?)

Changes in environmental conditions can alter free energy differences (which explains why proteins can fold to different structure in different conditions). There are a couple of papers that have directly tried to address whether the altered intracellular environment affects protein stability and folding rates vs in vitro. These two examples showed that there was little difference for a variety of proteins. Article Single-molecule spectroscopy of protein conformational dynam...

Article Structural disorder of monomeric α-synuclein persists in mam...

I am not an expert on chaperones, but from my understanding, they usually act to alter folding rates, rather than significantly altering the fold of the monomer. The final structure with and without the chaperone is therefore usually the same. See this for more details:

Article Molecular chaperones in protein folding and proteostasis

In terms of extracellular proteins, this review may answer your question.

Article Extracellular Chaperones and Proteostasis

Dear David,

thank you. I am not an expert in the field. Could you please describe briefly the advantages of your model?

Renata

Thank you, everybody, for your contributions and references.

And thanks to David for the clarification. I will take a look at your paper.

My purpose in asking the question was to update myself in the current situation in Protein Structure Prediction as I am hoping to develop a quantum algorithm for this problem.

Renata

My idea is that only long-range interaction can solve this paradox.

Thank you, Alexey. Could you please elaborate a bit on your idea?

In my paper (Congenital programs of the behavior and nontrivial...2014) and in our paper with professor Meijer (On a generalized Levinthal’s paradox...2018) it was proposed a quantum model that takes into account the long-range interactions between atoms. According to this model, the interaction Hamiltonian contains a term that is responsible for the contribution of the distant neighbors of a region surrounding a given particle.

Thank you for the references. I got both papers and will take a look at them once I am done with my present projects. Upfront though, if you could just advise me briefly what kind of medium between atoms, if any, is usually assumed for the long-range interactions?

I thnik that this is non-local quantum field for biologically important molecules....

Thank you. Appreciate your help.