Molecular Biophysics I
2005
Session #3

How do proteins fold...really?
February 09, 2005
BioMed 205/207
1:00 p.m. - 3:50 p.m.

"Protein folding is arguably the single most important process in biology." 1


For today's class, we focus on the more conventional conformation of proteins: the folded or 'native' conformation required for the protein's major biological activity. (Note: Most conformational diseases, characterized by the polymerized, 'primordial' conformation, exhibit a gain-of-function phenotype.)

As we have seen previously, the phenomenon by which a linear polypeptide folds into (generally) one unique, three-dimensional conformation has been referred to as the 'protein folding problem'. But, in actuality, it consists of at least two separate problems: 1) How does the amino acid sequence dictate the final three dimensional structure? and 2) How is that final structure achieved within the allowable time scale? Today's session will deal primarily with the second of these questions.

To set the stage, we first consider several significant contributions from the past, namely Levinthal's paradox, Anfinsen's work with ribonuclease, and Ptitsyn's identification of the 'molten globule'.

Much of what is known today about protein folding was derived from in vitro experiments carried out under controlled, but not necessarily physiologic, conditions, i.e., the so-called 'refolding', or 'Anfinsen'  experiments. This is where purified proteins at low concentration are first denatured, generally using a chaotropic agent such as guanidine hydrochloride, and then re-folding is allowed to occur as a result of rapid dilution of the denaturant. Under appropriate conditions, the 'denatured' protein then rapidly refolds to it's 'native' conformation.

As aptly demonstrated by Levinthal, denatured proteins cannot possibly sample all available conformational space during the refolding process, so other explanations must prevail: we'll consider a few of these, namely:

We also look at a review paper by Dill & Chan that describes, in readable fashion, the 'new view' of protein folding, i.e., a process involving concepts such as ensembles, energy landscapes and folding funnels.

Theory and experiments together are beginning to present a unified picture of folding, as reported by Daggett & Fersht.

Finally, to establish that progress is indeed being made, we briefly consider recent work by David Baker in which an 'artificial' protein structure (not yet found in nature) was designed, and its predicted structure confirmed by actual synthesis and high resolution x-ray crystallography.

STUDENT ASSIGNMENTS: All students should read reference #9 (Daggett & Fersht), because we'll spend some time on it in class. For the remainder of the papers, see below (and the class roster for student numbers). Where two students have the same paper, you may collaborate, and apportion between yourselves, each discuss half, etc.: your choice. All figures will be available for projection. Concentrate on the main message...and its significance.

Reference List

1.    Anfinsen, C.B. Principles that govern the folding of protein chains.  Science, 181: 223-230, 1973.
 Student #5: Note: This paper presents Anfinsen's Nobel lecture, and also may be accessed through the Nobel Foundation
[ http://nobelprize.org/chemistry/laureates/1972/anfinsen-lecture.html ]. A PDF version from the Nobel site has been placed on the course web page in the Library ERes site. Please limit your discussion to the work with ribonuclease ONLY (the first ≈ five pages; Figs. 1, 2, & 3 only).

2.    Ptitsyn, O.B. How the molten globule became.  Trends Biochem.Sci., 20: 376-379, 1995.
 Student #6: Note: Don't miss the student perspective: Who were Ptitsyn's main colleagues? Did they receive due credit? This paper conveys more than the molten globule story itself.

3.    Denton, M. and Marshall, C. Laws of form revisited.  Nature, 410: 417-417, 2001.
 Student #7

4.    Baker, D. A surprising simplicity to protein folding.  Nature, 405: 39-42, 2000.
Simplicity is not the word to describe this paper, however! Again, try to get the gist of the message without getting bogged down in the details. Student #8

5.    Baldwin, R.L. and Zimm, B.H. Are denatured proteins ever random coils?  Proc.Natl.Acad.Sci.USA, 97: 12391-12392, 2000.

6.    Plaxco, K.W. and Gross, M. Unfolded, yes, but random? Never!  Nature Structural Biology, 8: 659-660, 2001.
 Students #9 & 10: Papers #5 & #6 are related; each is a comment paper on some recent work. What we need here is the take-home message form these two studies.

7.    Tompa, P. Intrinsically unstructured proteins.  Trends in Biochemical Sciences, 27: 527-533, 2002.
 Student #1: Note: This is a review paper, filled with details. Try to summarize...what are IUPs, give a few examples, and some thoughts as to their biological advantage (reason for being).

8.    Dill, K.A. and Chan, H.S. From Levinthal to pathways to funnels.  Nature Structural Biology, 4: 10-19, 1997.
 Students #2 & 3: Note: A PDF version of this paper is available from the course page in the Library ERes site. Discuss Figs. 1 through 6 with accompanying text; skip the last 3 Figs & their text material.

9.    Daggett, V. and Fersht, A. The present view of the mechanism of protein folding.  Nature Reviews Molecular Cell Biology, 4: 497-502, 2003.
 All Students

10.   Kuhlman, B., Dantas, G., Ireton, G.C., Varani, G., Stoddard, B.L., and Baker, D. Design of a novel globular protein fold with atomic-level accuracy.  Science, 302: 1364-1368, 2003.
 Student #4: Go lightly with this paper; try to get past the experimental details, and convey their overall objective & the results obtained. As a first start, look at the accompanying 'comment' paper (Jones, D.T. Learning to speak the language of proteins.  Science, 302: 1347-1348, 2003) and the press clipping from HHMI
[ http://www.hhmi.org/news/baker3.html ].

1 R. John Ellis
   Curr. Opin. Struct. Biol. 4: 117, 1994