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Folding of nascent protein chains / amyloid fibrils

Research project at the Oxford Centre for Molecular Sciences funded by a BBSRC "David Phillips Research Fellowship" 1996--1999. Sponsor: Prof. C. M. Dobson, FRS. The project somehow deviated from the objectives shown below and developed into a project on the formation of amyloid fibrils by the model peptides derived from CspB.

To investigate the mechanisms by which newly synthesized proteins fold to their native three- dimensional structures in the cell.

Description of the programme:
Background. Despite much effort, the "second half of the genetic code" has not yet been deciphered. Although the instructions for the formation of three-dimensional protein structures are encoded in the primary sequences, we don't know how to read them or to use them to create new proteins. In vitro studies of protein folding usually start from the fully unfolded polypeptide chain in free solution. In contrast, folding in the cell never occurs in this way. Unfolded polypeptide chains in the cell are always attached to high molecular weight complexes, be it the ribosomal translation machinery, if the chain is still in statu nascendi, or the chaperone machinery, if the chain is folded post-translationally, transported, or refolded after accidental unfolding. As yet, very few researchers have addressed the issue of nascent chain folding.

Previous work leading to the proposed project: During my post-doctoral stay with Dr S.E. Radford at the OCMS (May 1993-April 1996), I have helped to develop a new methodology to study the conformational properties of protein folding intermediates bound to the molecular chaperone GroEL by a combination of hydrogen exchange kinetics with electrospray ionization mass spectrometry (ESI-MS). In collaboration with Dr C.V. Robinson of the OCMS mass spectrometry facility, I have analysed the conformational properties of a partly folded, GroEL-bound state of bovine alpha-lactalbumin, which were found to resemble those of a molten globule state in free solution (Robinson, 1994). In recent work, I have studied the structures of two different GroEL-bound productive folding intermediates of dihydrofolate reductase (DHFR) (M. Groß, C. V. Robinson, S. E. Radford, 1996). Building on this work as well as on experience gained during my doctoral research about the interaction and assembly of ribosomes and functional ribosomal complexes, I now propose to investigate the conformation of nascent protein chains still bound to the ribosomes. The technique of hydrogen exchange analysed by electrospray- ionization mass spectrometry, which we successfully applied to the similarly complex problem of substrate recognition by the molecular chaperone GroEL (Robinson, 1994), will be developed further to address the nascent chain/ribosome system.Rationale of the proposed project: While investigations of molecular chaperones have proven highly useful in bringing in vitro studies of protein folding closer to the biological conditions, it has been found that folding of freshly synthesized proteins already occurs co-translationally in a high- molecular weight complex of ribosomes, nascent chain and chaperones (Frydman, 1994). A truly biological approach to protein folding will therefore have to address the ribosome- bound protein as well as the role of molecular chaperones in folding. The size and complexity of these systems preclude analysis by NMR, while their dynamic nature rules out investigation by X-ray crystallography. Immunological detection of protein structures in a nascent chain have yielded ambiguous results (Friguet, 1994). Hence I suggest to use the approach of hydrogen exchange protection analysed by ESI-MS, and to combine it with more site-specific investigations of simple peptidess derived from N-terminal sequences.
Experimental strategy: In order to apply the hydrogen exchange/ESI-MS methodology to this very complex system, methodological investigations with ribosomes alone will be needed. Preliminary investigations are under way. Given that ribosomal proteins tend to have isoelectric points in the basic range, it is anticipated that the selection of a rather acidic model protein will enable us to detect the mass of the latter without too much interference from the former. The messenger-RNA of a suitable model protein will be used in a synchronized in vitro translation system, which can be stopped after a well-defined number of amino acids, thus producing an incompletely synthesized polypeptide chain bound to the ribosome as a peptidyl-tRNA (Frydman, 1994; Kudlicki, 1995). These complex but well- defined assemblies will be subjected to hydrogen-exchange mass spectrometry. As in my previous chaperone work, the fact that non-covalently linked assembly systems tend to dissociate during the evaporation in the mass spectrometer can be used advantageously to detect a single small component, such as the nascent chain, from a complicated high molecular weight system. While our current methodology detects the bulk hydrogen exchange kinetics of populations of molecules, the newly developed Fourier transform ion cyclotron resonance mass spectrometry can potentially provide us with site-specific information. Additional help in interpreting the results in a site-specific way will be obtained from parallel in vitro investigations of N-terminal peptides of the model protein, corresponding in length to the nascent polypeptides produced on the ribosomes. Peptides will be synthesized in-house by the OCMS peptide synthesis facility. Methodologies well established in this laboratory, including circular dichroism (CD) and high-field NMR spectroscopies, will be used to study the conformational properties of these peptides in detail. As yet, such a study has only been carried out for one protein, the serine protease inhibitor CI-2 (Prat Gay, 1995).In addition to conformational studies of the peptides alone, these will be coupled with their C-termini to inert nanoscopic (i.e. ribosome-sized) carriers, to mimic the partial immobilization in the cell. Dendrimers of up to x molecular weight are now available on a kg scale and can be made in a way as to be chemically inert and invisible to the spectroscopic method used to investigate the attached peptide. For instance, 1H- NMR studies could be carried out with deuterated dendrimers. By combining the analysis of the full biological machinery with the more detailed but less authentic information which will be obtained from peptide models, I hope to achieve substantial progress in our understanding of how proteins fold in the living cell.


  1. Groß M (1996): FEBS Lett 390, 249-252 -- Linguistic analysis of protein folding
  2. Groß M & Dobson CM (1997): Chimia 51, 443
    Folding of nascent protein chains (Poster abstract)
  3. Groß M, Wilkins DK, Pitkeathly MC, Chung EW, Higham C, Clark A & Dobson CM * (1999): Protein Science 8, 1350-1357
    Formation of amyloid fibrils by peptides derived from the bacterial cold-shock protein CspB
  4. Higham CE, Jaikaran ETAS, Fraser PE, Groß M & Clark A* (2000): FEBS Letters 470, 55-60
    Preparation of synthetic human islet amyloid polypeptide (IAPP) in a stable conformation to enable study of conversion to amyloid-like fibrils FULL TEXT
  5. Wilkins DK, Dobson CM & Groß M * (2000): European Journal of Biochemistry, 267, 2609-2616
    Biophysical studies of the development of amyloid fibrils from a peptide fragment of cold shock protein B
  6. Groß M (2000): Current Protein and Peptide Science 1, 339-347
    Proteins that convert from alpha helix to beta sheet: Implications for folding and disease
  7. Jaikaran ETAS *, Higham CE, Serpell LC, Zurdo J, Gross M, Clark A & Fraser PE (2001): Journal of Molecular Biology 308, 515-525
    Identification of a novel human islet amyloid polypeptide beta-sheet domain and factors influencing fibrillogenesis


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Dr Michael Groß

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