On 2016 Apr 28, Jorg Rosgen commented:

The processes in question are associated with such small energetic changes, that everything happens within the range of normal thermal fluctuations. That's why protein folding can be both spontaneous and reversible.

On 2016 Apr 14, Ariel Fernandez commented:

Walter:

In so far as we agree that protein folding in vitro is spontaneous under suitable renaturation conditions, the process is thermodynamically irreversible. Hence, the intermediate states of the system, comprised of protein chain and solvent statistical bath, are irretrievable [1]. In thermodynamic terms there is no such thing as a spontaneous reversible process. Ariel Fernandez

[1] Ariel Fernandez Stigliano. Biomolecular Interfaces, Chapter 3 (Springer, Heidelberg, 2015) http://link.springer.com/book/10.1007/978-3-319-16850-0

On 2016 Apr 13, S Walter Englander commented:

I disagree with several issues in this comment.

1. The protein folding question is not a sterile one. It is centrally important to understand how proteins fold and why they fold in that way. The protein folding reaction is essential to all living things, arguably the most important reaction in all of biology. It determines many currently forefront biological behaviors and diseases.

2. Folding is not irreversible. At the molecular level, free energy downhill processes are spontaneous but certainly not irreversible. Proteins spontaneously fold energetically downhill to their lowest free energy native state, as per Anfinsen, but even under fully native conditions they continue to unfold and refold repeatedly over time, cycling through all possible higher energy states. One result is that all higher energy states, including those that carry the major U to N folding flux, are populated at equilibrium, each one according to its Boltzmann factor [K = e^-G/RT].

3. The effort is far from futile. Hydrogen exchange (HX) methods can observe that cycling and characterize the major intermediates (structure, G, ASA). For cytochrome c we found four major partially folded intermediates. Each differs from the next by one native-like foldon unit. In the present paper we used advanced HX MS methods to define the major partially folded cyt c states DURING kinetic folding. They are the same as the high free energy states we found before at equilibrium native conditions. One conclusion is that cyt c and, we suspect, proteins in general fold through defined N-like intermediates, adding one foldon unit at each step.

4. Summary: Fifty plus years after Anfinsen there is still not general consensus about how protein folding works and why it works that way. The reason is that it has been so hard to define folding intermediates and pathways, although not impossible as Fernandez asserts. Our HX experiments indicate that proteins are made of cooperative foldon units that provide built-instructions for the folding process. Stepwise folding puts a sequence of cooperative foldon units into place, one at a time in an ordered foldon-dependent pathway (the HOW question), just as they are evolutionarily tailored to fit together in the native protein (the WHY question).

All of this is true for cyt c (the present paper) and for some other proteins as well (see paper for refs).

On 2016 Apr 05, Ariel Fernandez commented:

In a recent paper, Hu et al. [1] reported a careful and detailed characterization of the folding pathway for a soluble protein. I am always confused by this kind of results. Under the appropriate enabling conditions, protein folding in vitro is known to be spontaneous [2] and therefore thermodynamically irreversible [3]. The endpoints of the protein folding process, i. e. the denatured random coil ensemble and the native state, are of course recoverable by restoring respectively denaturing or renaturing conditions [2]. Yet, at a variance with thermodynamics, protein scientists incorrectly regard this restoration of folding endpoints as meant to imply that protein folding is a reversible process [2]. Be as it may, according to the tenets of thermodynamics, the folding and unfolding pathways are untraceable and irreproducible as it would be the case for any spontaneous process [3]. In fact, the very notion of “pathway” for a spontaneous process is thermodynamically meaningless because dissipative forces intervene in such processes causing a net increase in the entropy of the universe, the hallmark of irreversibility. Thus, as with any spontaneous process, the actual intermediate states associated with the protein folding process are irretrievable. Therefore I think that the sterile controversy on whether protein folding in vitro is actually a two-state process or proceeds through intermediates is not even an issue: The two–state model is simply a realization that intermediates are irretrievable in a thermodynamically spontaneous process [4].

Notwithstanding such thermodynamic considerations, an active quest for folding intermediates continues to this day [1, 5]. In my view, this search remains futile from a thermodynamic perspective, unless some sort of paradox holds (thermodynamics is full of paradoxes) that, at the very least, needs to be properly dispelled before the saga of the quest for folding intermediates continues. To the best of my knowledge this has not been done. Real folding intermediates not only remain elusive: I am afraid they do not exist, and claims to the contrary violate the second law of thermodynamics. Ariel Fernandez

References

1. Hu W., Kan Z.-Y., Mayne L. & Englander, S. W. Proc. Natl. Acad. Sci. USA 113, 3809-3814 (2016).

2. Anfinsen, C.B. Science 181, 223-230 (1973).

3. Planck, M. Treatise on Thermodynamics, 3rd edition, Dover, New York (2010).

4. Fernandez, A. Biomolecular Interfaces (ISBN 978-3-319-16849-4), Springer, Berlin (2015).

5. Vendruscolo M. & Dobson, C.M. Nature Chem. Biol. 9, 216-217 (2013).

On 2016 Apr 05, Ariel Fernandez commented:

In a recent paper, Hu et al. [1] reported a careful and detailed characterization of the folding pathway for a soluble protein. I am always confused by this kind of results. Under the appropriate enabling conditions, protein folding in vitro is known to be spontaneous [2] and therefore thermodynamically irreversible [3]. The endpoints of the protein folding process, i. e. the denatured random coil ensemble and the native state, are of course recoverable by restoring respectively denaturing or renaturing conditions [2]. Yet, at a variance with thermodynamics, protein scientists incorrectly regard this restoration of folding endpoints as meant to imply that protein folding is a reversible process [2]. Be as it may, according to the tenets of thermodynamics, the folding and unfolding pathways are untraceable and irreproducible as it would be the case for any spontaneous process [3]. In fact, the very notion of “pathway” for a spontaneous process is thermodynamically meaningless because dissipative forces intervene in such processes causing a net increase in the entropy of the universe, the hallmark of irreversibility. Thus, as with any spontaneous process, the actual intermediate states associated with the protein folding process are irretrievable. Therefore I think that the sterile controversy on whether protein folding in vitro is actually a two-state process or proceeds through intermediates is not even an issue: The two–state model is simply a realization that intermediates are irretrievable in a thermodynamically spontaneous process [4].

Notwithstanding such thermodynamic considerations, an active quest for folding intermediates continues to this day [1, 5]. In my view, this search remains futile from a thermodynamic perspective, unless some sort of paradox holds (thermodynamics is full of paradoxes) that, at the very least, needs to be properly dispelled before the saga of the quest for folding intermediates continues. To the best of my knowledge this has not been done. Real folding intermediates not only remain elusive: I am afraid they do not exist, and claims to the contrary violate the second law of thermodynamics. Ariel Fernandez

References

1. Hu W., Kan Z.-Y., Mayne L. & Englander, S. W. Proc. Natl. Acad. Sci. USA 113, 3809-3814 (2016).

2. Anfinsen, C.B. Science 181, 223-230 (1973).

3. Planck, M. Treatise on Thermodynamics, 3rd edition, Dover, New York (2010).

4. Fernandez, A. Biomolecular Interfaces (ISBN 978-3-319-16849-4), Springer, Berlin (2015).

5. Vendruscolo M. & Dobson, C.M. Nature Chem. Biol. 9, 216-217 (2013).

On 2016 Apr 13, S Walter Englander commented:

I disagree with several issues in this comment.

1. The protein folding question is not a sterile one. It is centrally important to understand how proteins fold and why they fold in that way. The protein folding reaction is essential to all living things, arguably the most important reaction in all of biology. It determines many currently forefront biological behaviors and diseases.

2. Folding is not irreversible. At the molecular level, free energy downhill processes are spontaneous but certainly not irreversible. Proteins spontaneously fold energetically downhill to their lowest free energy native state, as per Anfinsen, but even under fully native conditions they continue to unfold and refold repeatedly over time, cycling through all possible higher energy states. One result is that all higher energy states, including those that carry the major U to N folding flux, are populated at equilibrium, each one according to its Boltzmann factor [K = e^-G/RT].

3. The effort is far from futile. Hydrogen exchange (HX) methods can observe that cycling and characterize the major intermediates (structure, G, ASA). For cytochrome c we found four major partially folded intermediates. Each differs from the next by one native-like foldon unit. In the present paper we used advanced HX MS methods to define the major partially folded cyt c states DURING kinetic folding. They are the same as the high free energy states we found before at equilibrium native conditions. One conclusion is that cyt c and, we suspect, proteins in general fold through defined N-like intermediates, adding one foldon unit at each step.

4. Summary: Fifty plus years after Anfinsen there is still not general consensus about how protein folding works and why it works that way. The reason is that it has been so hard to define folding intermediates and pathways, although not impossible as Fernandez asserts. Our HX experiments indicate that proteins are made of cooperative foldon units that provide built-instructions for the folding process. Stepwise folding puts a sequence of cooperative foldon units into place, one at a time in an ordered foldon-dependent pathway (the HOW question), just as they are evolutionarily tailored to fit together in the native protein (the WHY question).

All of this is true for cyt c (the present paper) and for some other proteins as well (see paper for refs).

On 2016 Apr 14, Ariel Fernandez commented:

Walter:

In so far as we agree that protein folding in vitro is spontaneous under suitable renaturation conditions, the process is thermodynamically irreversible. Hence, the intermediate states of the system, comprised of protein chain and solvent statistical bath, are irretrievable [1]. In thermodynamic terms there is no such thing as a spontaneous reversible process. Ariel Fernandez

[1] Ariel Fernandez Stigliano. Biomolecular Interfaces, Chapter 3 (Springer, Heidelberg, 2015) http://link.springer.com/book/10.1007/978-3-319-16850-0

On 2016 Apr 28, Jorg Rosgen commented:

The processes in question are associated with such small energetic changes, that everything happens within the range of normal thermal fluctuations. That's why protein folding can be both spontaneous and reversible.