Molecular Biophysics I
2005
Session #4

Folding and Unfolding ...
in the Cellular Environment

February 16, 2005
BioMed 205/207
1:00 p.m. - 3:50 p.m.

"Anfinsen's work was of seminal importance in establishing the central principle of self-assembly of proteins, but it probably delayed by a decade or two the initiation of investigations of protein folding in vivo--- the trouble was we believed that the polypeptide sequence was all that there was to it." ¹

In this session we consider some of the challenges presented to folding proteins when they are removed from an artificial, controlled environment in vitro, and relocated to the cellular environment, in particular, within cells. (A later session will be devoted to protein unfolding in cells, so in this session we concentrate on protein folding.)

As we discussed last session, protein refolding in vitro, as characterized by the typical Anfinsen experiment, occurs as follows: 1) start with purified proteins in their native conformation, at low concentration; 2) 'denature' using high concentrations of urea or guanidine hydrochloride; 3) rapidly dilute the denaturant, and follow protein refolding experimentally (complete refolding is measured by restitution of original enzyme activity, for example).

At least three major differences serve to distinguish protein folding in cells from protein refolding in vitro:

• Proteins are initially synthesized by the ribosome in a vectorial manner, with nascent chain growth occurring  from the N-terminus to the C-terminus. We have seen that protein folding can be very fast, in fact it is much faster than the rate of synthesis. But we also know that some proteins show high contact order, i.e. residues contacting each order in the fully folded state are far-removed from each other in sequence. How does the cell handle both the synthesis and the folding of these different nascent polypeptides?

• The interior of a typical cell represents a very crowded environment; this condition leads to a phenomenon termed 'volume exclusion'. How does this 'macromolecular crowding' influence protein folding? What about protein aggregation?

• The cell interior is compartmentalized, and nascent proteins must be routed to many different destinations, e.g., to the cytoplasm; to other organelles, such as mitochondria; for export out of the cell; for integration into the cell's membrane network. How does the cell recognize where a particular protein is to be routed? When and where does the protein fold?

Exciting new data have forced investigators to reevaluate earlier answers to these questions, and have once again revealed how nature makes use of protein conformation to accomplish its purposes. Two of our course general review papers (Frydman, 2001; Young et al, 2004) contain some discussion relative to these issues, so you might wish to consult them in addition to the specific papers listed below.

The references below are arranged to cover three major topics:

1. Folding events at (and within) the ribosome (Refs. 1 to 5). Since we shall not have time to consider the original data in detail, we'll rely on on one review (Ref. #1) and several 'comment' papers (Refs. 2-5). Note: These papers cover work with BOTH prokaryotic and eukaryotic systems, where the processes and components are similar, but not identical.  The 'trigger factor'  (Ref. #4) is found in E. coli; in higher organisms its function is carried out by different factors (the NAC).  In eukaryotes, proteins destined for the ER first encounter the SRP (signal recognition particle), and then the translocon channel into the ER.

2. Cotranslational folding (Refs. #6 & 7)

3. Macromolecular crowding (Refs. #8 to 11). Note: Refs. # 9 & 10 pretty much say the same thing, but in slightly different ways, using different figures.

Student Assignments: (Student roster is also available via the Library ERes site)

Students #1, 2, 3 & 4: You should all read the review paper by Arthur Johnson (Ref. #1); thereafter, each student can be responsible for one of the next four papers (Student 1: Ref. 2; Student 2: Ref. 3; Student 3: Ref. 4; & Student 4: Ref. 5)

Student #5: Ref. 6; Student 6: Ref. 7

Student #7: Ref. 8; Student #8: Ref. 9; Student #9: Ref. 10; & Student #10: Ref. 11 !

Reference List
(PDF copies of Refs #7 & #8 are available on the Library ERes site)

1.    Johnson, A.E. Functional ramifications of FRET-detected nascent chain folding far inside the membrane-bound ribosome.
             Biochemical Society  Transactions, 32: 668-672, 200

2.    Etchells, S.A. and Hartl, F.U. The dynamic tunnel.  Nature Structural & Molecular Biology, 11: 391-392, 2004.

3.    Rospert, S. Ribosome function: Governing the dispatch fate of a nascent polypeptide.  Current Biology, 14: R386-R388,2004.

4.    Horwich, A. Cell biology - Sight at the end of the tunnel.  Nature, 431: 520-522, 2004.

5.    Bowie, J.U. Cell biology: Border crossing.  Nature, 433: 367-369, 2005.

6.    Fedorov, A.N. and Baldwin, T.O. Cotranslational protein folding.  J.Biol.Chem., 272: 32715-32718, 1997. (check ERes site)

7.    Basharov, M.A. The posttranslational concept of protein folding: How valid is it?  Biochemistry (Moscow), 65: 1184-1191, 2000. (check ERes site)

8.    Goodsell, D.S. Inside a living cell.  Trends Biochem.Sci., 16: 203-206, 1991.

9.    Minton, A.P. The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media.  J.Biol.Chem., 276: 10577-10580, 2001.

10.   Ellis, R.J. Macromolecular crowding: obvious but underappreciated.  Trends Biochem.Sci., 26: 597-604, 2001.

11.   Shtilerman, M.D., Ding, T.T., and Lansbury, P.T. Molecular crowding accelerates fibrillization of alpha-synuclein: Could an increase in the cytoplasmic protein concentration induce Parkinson's disease?  Biochem., 41: 3855-3860, 2002.

¹ Mary-Jane Gething, Nature 388: 329-330, 1997