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The Birth and Growth of the Protein-Folding Nucleus: Studies of Protein Folding Focused on Critical Contacts, Topology and Ionic Interactions

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The Birth and Growth of the Protein-Folding Nucleus: Studies of Protein Folding Focused on Critical Contacts, Topology and Ionic Interactions The birth and growth of the protein-folding nucleus: Studies of protein folding focused on critical contacts, topology and ionic interactions Linda Hedberg ©Linda Hedberg, Stockholm 2008 ISBN 978-91-7155-704-9 pages 1-73 Printed in Sweden by US-AB, Stockholm 2008 Distributor: Department of Biochemistry and Biophysics Till mamma och pappa Abstract Proteins are among the most complex molecules in the cell and they play a major role in life itself. The complexity is not restricted to just structure and function, but also embraces the protein folding reaction. Within the field of protein folding, the focus of this thesis is on the features of the folding transition state in terms of growing contacts, common nucleation motifs and the contribution of charged residues to stability and folding kinetics. During the resent decade, the importance of a certain residue in struc- ture formation has been deduced from φ-value analysis. As a comple- ment to φ-value analysis, I present how scatter in a Hammond plot is related to site-specific information of contact formation, φ´(βTS), and this new formalism was experimentally tested on the protein L23. The results show that the contacts with highest φ growth at the barrier top were distributed like a second layer outside the folding nucleus. This contact layer is the critical interactions needed to be formed to over- come the entropic barrier. Furthermore, the nature of the folding nucleus has been shown to be very similar among proteins with homologous structures and, in the split β−α−β family the proteins favour a two-strand-helix motif. Here I show that the two-strand-helix motif is also present in the ribosomal protein S6 from A. aeolicus even though the nucleation and core com- position of this protein differ from other related structure-homologues. In contrast to nucleation and contact growth, which are events driven by the hydrophobic effect, my most recent work is focused on electro- static effects. By pH titration and protein engineering the charge con- tent of S6 from T. thermophilus was altered and the results show that the charged groups at the protein surface might not be crucial for pro- tein stability but, indeed, have impact on folding kinetics. Further- more, by site-specific removal of all acidic groups the entire pH de- pendence of protein stability was depleted. List of publications This thesis is based on the following publications, referred to by their Roman numerals: I Maxwell, K. L., Wildes, D., Zarrine-Afsar, A.,De Los Rios, M. A., Brown, A. G., Friel, C. T. Hedberg, L., Horng, J. C., Bona, D., Miller, E. J., Vallee-Belisle, A., Main, E. R., Bemporad, F., Qiu, L., Teilum, K., Vu, N. D., Edwards, A. M., Ruczinski, I., Poulsen, F. M., Kragelund, B. B., Michnick, S. W., Chiti, F., Bai, Y., Hagen, S. J., Serrano, L., Oliveberg, M., Raleigh, D. P., Wittung Stafshede, P., Radford, S. E., Jackson, S. E., Sosnick, T. R., Marqusee, S., Davidson, A. R., Plaxco, K. W. “Protein folding: Defining a “standard” set of ex- perimental conditions and a preliminary kinetic data set of two-state proteins.” Protein Science (2005), 14:602- 616 II Hedberg, L. and Oliveberg, M. “Scattered Hammond plots reveal second level of site-specific information in protein folding: φ’(β‡)” Proc. Natl. Acad. Sci. USA (2004), 101 (20):7606-11 III Olofsson, M., Hansson, S., Hedberg, L., Logan, D. T., Oliveberg, M. “Folding of S6 structures with divergent amino acid composition: pathway flexibility within partly overlapping foldons” J Mol Biol (2007) 365, 237- 248 IV Hedberg, L., Kurnik, M., Oliveberg, M. “Depletion of pH dependence upon removal of amino acids” Manu- script Contents Introduction ..................................................................................................9 Protein folding: random or not? ...............................................................................9 Proteins: folding and stability .................................................................................11 Two state folding..................................................................................................11 The kinetics of protein folding ...........................................................................13 How can we measure the kinetics of protein folding?..................................15 Stopped flow..........................................................................................................17 Kinetic parameters: what do they tell?.................................................................20 φ-value analysis ....................................................................................................20 φ values and their reliability...............................................................................22 m-value analysis...................................................................................................23 Transition-state movement and Hammond behaviour ................................24 Scattered Hammond plots..................................................................................26 The nature of proteins ..............................................................................................28 The denatured state ............................................................................................28 The native state....................................................................................................30 The role of charged groups and electrostatic interactions upon protein folding ..........................................................................................................................31 Electrostatic interactions ....................................................................................31 Water and hydration............................................................................................33 Chemical denaturants..........................................................................................34 Linear free energy relation; to be or not to be…...........................................35 The model systems ...................................................................................37 L23 ................................................................................................................................37 S6A ................................................................................................................................38 S6T.................................................................................................................................39 Charge variants of S6T..............................................................................................40 S6wt..........................................................................................................................41 S6half charged ..............................................................................................................42 S6half basic .................................................................................................................42 S6half carb ..................................................................................................................43 S6no carb....................................................................................................................43 S6no basic and S6no charges ........................................................................................43 Summary of papers ..................................................................................44 PAPER I ........................................................................................................................44 Experimental conditions......................................................................................44 Curve-fitting and models for interpreting kinetic data ................................45 Conclusions............................................................................................................45 PAPER II.......................................................................................................................46 Stabilisation of L23 by Na2SO4 ..........................................................................46 The folding event: φ -value analysis ................................................................47 Calculation of φ .....................................................................................................51 Scattered Hammond plots..................................................................................51 Conclusions............................................................................................................53 PAPER III .....................................................................................................................54 Folding kinetics .....................................................................................................56 φ -value analysis...................................................................................................56 Minimal folding motif...........................................................................................58 Conclusions............................................................................................................58 PAPER IV......................................................................................................................59 pH dependence and acidic residues .................................................................59 Effects in the protein folding reaction..............................................................61
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