The Folding Energy Landscape of MerP AnnAnn----ChristinChristin Brorsson ______________________________________________ Department of Biochemistry Umeå University Umeå, Sweden 2004 Copyright by Ann-Christin Brorsson Front cover: The 3-dimensional structure of MerP Printed in Sweden by Solfjädern Offset AB, Umeå 2004 ISBN 91-7305-710-X The Folding Energy Landscape of MerP ByByBy AnnAnn----ChristinChristin Brorsson Department of Biochemistry Umeå University Umeå, Sweden 2004 Akademisk avhandling Som med vederbörligt tillstånd av Rektorsämbetet vid Umeå universitet för avläggande av filosofie doktorsexamen vid teknisk-naturvetenskapliga fakulteten kommer att offentligen försvaras på Kemiska institutionen, Umeå universitet, hörsal KB3A9, KBC, fredagen den 1 oktober 2004, kl. 10.00. Fakultetsopponent: Prof. Daniel Otzen Institute of Life Sciences Section of Biotechnology Aalborg University Denmark Abstract This thesis is based on studies, described in four papers, in which the folding energy landscape of MerP was investigated by various techniques. MerP is a water-soluble 72 amino acid protein with a secondary structure consisting of four anti-parallel β-strands and two α-helices on one side of the sheet in the order β1α1β2β3α2β4. The first paper describes the use of CD and fluorescence analysis to examine the folding/unfolding process of MerP. From these experiments it was found that the protein folds according to a two-state model in which only the native and unfolded forms are populated without any visible intermediates. With a rate constant of 1.2 s -1, the folding rate was found to be unusually slow for a protein of this size. The studies presented in the second and third papers were based on measurements of native-state amide proton exchange at different temperatures (Paper II) and GuHCl concentrations (Paper III) in the pre- transitional region. In these studies partially unfolded forms were found for MerP which are essentially unrelated to each other. Thus, in the folding energy landscape of MerP, several intermediates seem to occur on different folding trajectories that are parallel to each other. The slow folding rate of MerP might be coupled to extensive visitation of these conformations. Hydrogen exchange in MerP did also reveal structure-dependent differences in compactness between the denatured states in GuHCl and H 2O. In the last paper multivariate data analysis was applied to 2-dimensional NMR data to detect conformational changes in the structure of MerP induced by GuHCl. From this analysis it was suggested that regions involved in the most flexible part of the protein structure are disrupted at rather low denaturant concentrations (< 2.1 M GuHCl) while the native structures of the most stable parts are still not completely ruptured at 2.9 M GuHCl. Finally, the stability, kinetics, contact order and folding nuclei of six proteins with similar topology (MerP, U1A, S6, ADA2h, AcP and HPr) were compared. In this analysis it was found that their folding properties are quite diverse, despite their topological similarities, and no general rules that have been formulated yet can adequately predict their folding behaviour. Keywords: protein folding and stability; hydrogen exchange; intermediate; partial unfolding ISBN 91-7305-710-X Papers in this thesis This thesis is based on the following papers which are included as supplements after the thesis. In the text, they will be referred to by their roman numerals. I. Aronsson, G., Brorsson, A-C., Sahlman, L. and Jonsson, B-H. (1997) Remarkably slow folding of a small protein, MerP. FEBS Lett. 411 , 359-364. II. Brorsson, A-C., Kjellson, A., Aronsson, G., Sethson, I., Hambraeus, C. and Jonsson, B-H. (2004) The “Two-State folder” MerP forms partially unfolded structures that show temperature dependent hydrogen exchange. J. Mol. Biol. 340 , 333-344. III. Brorsson, A-C., Sethson, I. and Jonsson, B-H. (2004) Hydrogen exchange in MerP reveals structure-dependent differences in compactness between the denatured states in GuHCl and H 2O. Submitted to Journal of Molecular Biology. IV. Berglund, A., Brorsson, A-C., Jonsson, B-H. and Sethson, I. (2004) The equilibrium unfolding of MerP characterized by multivariate analysis of 2D NMR data. Submitted to Journal of Magnetic Resonance. Abbreviations MerP Mercury binding Protein CD Circular Dichroism GuHCl Guanidine HydroChloride MVDA Multi Variate Data Analysis NMR Nuclear Magnetic Resonance PCA Principal Component Analysis PLS Partial Least Squares U1A RNA binding domain of the U1 small nuclear ribonucleoprotein A S6 Ribosomal protein S6 ADA2h Activation Domain of procarboxypeptidase A2 AcP Muscle AcylPhosphatase HPr Histidine-containing Phosphocarrier protein CspB Cold shock protein B DNA DeoxyriboNucleinAcid UV UltraViolet Table of Contents Introduction 1 Historical background of proteins 1 Protein structure and function 1 The protein folding problem 4 Conformational properties of proteins 5 Protein stability 5 Protein folding 7 Protein unfolding 14 Protein misfolding 17 Techniques used in this study 18 Nuclear Magnetic Resonance 18 Hydrogen Exchange 21 Fluorescence Spectroscopy 27 Circular Dichroism Spectroscopy 27 Multivariate Data Analysis 28 The protein MerP 29 Summary of papers 30 I Fluorescence and CD analysis of the folding properties of MerP 30 II Native-state HX of MerP at increasing temperatures 33 III Native-state HX of MerP at increasing concentrations of GuHCl 38 IV Multivariate analysis of 2D NMR data 42 Comparison of proteins with similar topology to MerP 44 Conclusions 46 Acknowledgements 47 References 48 Papers I-IV Introduction Historical background of proteins Four classes of molecules constitute the main components in living systems: proteins, carbohydrates, lipids, and nucleic acids. Of these components proteins have the widest diversity in structure and function. Proteins were first discovered in 1838 by the Swedish scientist Jöns Berzelius. The original meaning of proteins is first thing , derived from the Greek word for “pre-eminent”. Over the following century it was found that the building blocks in proteins are made of 20 different amino acids linked together in an unbranched biopolymer known as a polypeptide chain. In the early 1950s, Linus Pauling and Robert Corey proposed two possible hydrogen-bonded configurations for the polypeptide chain: a helical formation,1 and a pleated-sheet configuration 2 today known as an α-helix and a β-strand, respectively. At almost the same time Fred Sanger, a Cambridge scientist, managed to develop new methods for amino acid sequencing and was able to determine the complete sequence of insulin (a hormone protein). 3 The first detailed protein structures were solved, in 1957, of myoglobin (a protein that stores oxygen) by John Kendrew and colleagues 4 and in 1959 of haemoglobin (an oxygen transport protein) by Perutz and his collaborators.5 Protein structure and function An amino acid contains both amino and carboxylic functional groups. In the polypeptide chain the amino acids (also called residues) are linked together by a peptide bond, and one molecule of water is released with the formation of each peptide link. Figure 1 shows a schematic diagram of two amino acids linked together by a peptide bond. The only part that differs between the various amino acids is their side chains (labelled R 1 and R 2 for the respective amino acids in Figure 1) except for proline, which has a side chain covalently linked to the amide nitrogen and therefore lacks an amino group. According to the chemical properties of the side chain the amino acids are classified into four different groups: acidic, basic, hydrophilic (polar) and hydrophobic (non-polar). 1 Peptide group Peptide bond Amin o group Carboxyl group Amide proton Figure 1. Schematic drawing of two amino acids linked together by a peptide bond. Protons are coloured white, carbon atoms grey, oxygen atoms black and nitrogen atoms light grey. The side chains of the amino acid are labelled R 1 and R 2 for the amino acids to the left and right, respectively. Before a protein can function it has to fold into a unique 3-dimensional structure called its native state. The protein structure can be described at four levels: • The primary structure is the order of the amino acids in the polypeptide chain. • The secondary structure is composed of repetitive 3-dimensional conformational units. The key to this structure is hydrogen bonding between the amide protons and carbonyl groups of specific pairs of residues. Most frequently the secondary structure constitutes either an α-helix or a β-sheet structure. In an α-helix the structure is stabilized by a series of hydrogen bonds from C=O of residue n to N-H of residue n + 4 (see Figure 2a), while in the β-sheet the structure is stabilized by hydrogen bonds between the C=O and N-H groups of two adjacent β-strands (see Figure 2b). Very often the structural units between elements of secondary structure constitute well-defined (non-repeating) turn structures. 2 a b Figure 2. Schematic drawings of secondary structural units. (a) α-helix, (b) β-sheet. The colour scheme is the same as described in Figure 1. Hydrogen bonds are shown by dotted lines. For clarity only the backbone atoms are shown. • The tertiary structure is the overall 3-dimensional configuration of the polypeptide chain, which is stabilised by interactions between the side chains of the residues. The tertiary structure can be divided into one or more subdomains that constitute local parts with specific tertiary structure elements.