Effects of Macromolecular Crowding on Protein Folding - in-vitro equilibrium and kinetic studies on selected model systems Alexander Christiansen Kemiska Institutionen Umeå 2013-11-20 Responsible publisher under swedish law: the Dean of the Faculty of Science and Technology This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7459-764-6 Elektronisk version tillgänglig på http://umu.diva-portal.org/ Tryck/Printed by: Service Center KBC Umeå Sweden 2013 众鸟高飞去 孤云独去闲 相看两不厌 只有敬停山 —李白 The birds have vanished down the sky. Now the last cloud drains away. We sit together, the mountain and I, Until only the mountain remains. -Li Bai (Translated by Xiaowei Song) Table of Contents ABSTRACT ............................................................................................. III LIST OF ABBREVIATIONS ...................................................................... VIII ENKEL SAMMANFATTNING PÅ SVENSKA ................................................ VI LIST OF PUBLICATIONS ........................................................................ VIII 1. INTRODUCTION .................................................................................. 2 1.1 PROTEIN FOLDING ................................................................................. 3 1.2 MACROMOLECULAR CROWDING ............................................................... 6 1.2.1 CELL AND CELLULAR ORGANIZATION ............................................................. 6 1.2.2 THEORETICAL MODELS OF EXCLUDED VOLUME EFFECTS ON PROTEINS ................ 8 1.2.3 EXPERIMENTAL STUDIES OF MACROMOLECULAR CROWDING EFFECTS .............. 11 1.2.4 COMPUTER SIMULATIONS OF CROWDING EFFECTS ........................................ 15 1.3 AIM OF THE PROJECT ....................................................................... 17 2. MATERIALS AND METHODS ............................................................... 18 2.1 PROTEIN EQUILIBRIUM STABILITY ............................................................. 18 2.2 PROTEIN FOLDING KINETICS .................................................................... 20 2.2.1 NON-LINEARITIES AND ADDITIONAL PHASES .................................................. 22 2.3 COMPARING KINETIC AND EQUILIBRIUM MEASUREMENTS ............................. 22 2.3.1 PHI-VALUE ANALYSIS ................................................................................. 23 2.3 SPECTROSCOPY .................................................................................... 24 2.3.1 CD SPECTROSCOPY ................................................................................... 24 2.3.2 FLUORESCENCE SPECTROSCOPY .................................................................. 25 2.4 CROWDER PREPARATION ....................................................................... 25 2.5 DIFFERENTIAL SCANNING CALORIMETRY (DSC) ............................................ 25 2.6 MODEL PROTEINS ................................................................................ 26 2.6.1 APOAZURIN ............................................................................................. 27 2.6.2 CYTOCHROME C ....................................................................................... 27 2.6.3 APOFLAVODOXIN ..................................................................................... 28 2.7 THEORETICAL MODELS OF EXCLUDED VOLUME EFFECTS ON PROTEIN STABILITY ... 29 i 3. RESULTS ............................................................................................ 33 3.1 EFFECT OF CROWDING ON EQUILIBRIUM .................................................... 33 3.1.1 CYTOCHROME C ....................................................................................... 34 3.1.2 APOAZURIN ............................................................................................. 38 3.1.3 SUMMARY OF THE EQUILIBRIUM DATA ........................................................ 43 3.2 EFFECTS OF CROWDING ON FOLDING KINETICS ............................................ 44 3.2.1 APOAZURIN FOLDING KINETICS ................................................................... 44 3.2.2 APOFLAVODOXIN FOLDING KINETICS ........................................................... 45 3.2.3 FAST FOLDING KINETICS OF CYTOCHROME C ................................................. 48 3.2.4 EFFECT OF VISCOSITY ON FOLDING KINETICS .................................................. 49 3.2.5 SUMMARY OF THE EFFECTS OF CROWDING ON FOLDING KINETICS .................... 50 4. DISCUSSION....................................................................................... 52 4.1 ATTRACTIVE INTERACTIONS? ................................................................... 55 4.2 CROWDING EFFECTS ON KINETICS ............................................................ 58 4.3 IN-VITRO VS IN-VIVO CONDITIONS ............................................................ 59 5. CONCLUSION AND SUMMARY ............................................................ 62 6. OUTLOOK .......................................................................................... 63 ACKNOWLEDGEMENTS .......................................................................... 65 REFERENCES .......................................................................................... 67 ii Abstract Protein folding is the process whereby an extended and unstructured polypeptide is converted into a compact folded structure that typically con- stitutes its functional form. The process has been characterized extensively in-vitro in dilute buffer solutions over the last few decades. However, in- vivo, it occurs inside living cells whose cytoplasm is filled with a plethora of different macromolecules that together occupy up to 40% of its total volume. This large number of macromolecules restricts the space available to each individual molecule, which has been termed macromolecular crowding. Macromolecular crowding generates excluded volume effects and also in- creases the importance of non-specific interactions between molecules. It should favor reactions that reduce the total volume occupied by all molecules within the cytoplasm, such as folding reactions. Theoretical models have predicted that the stability of proteins’ folded states should be increased by macromolecular crowding due to unfavorable effects on the extended un- folded state. To understand protein folding and function in living systems, we need to have a defined quantitative link between in-vitro dilute condi- tions (under which most biophysical experiments are conducted) and in-vivo crowded conditions. It is therefore important to determine how macromo- lecular crowding modifies the biophysical properties of proteins. The work underlying this thesis focused on how macromolecular crowd- ing tunes proteins’ equilibrium stability and kinetic folding processes. To mimic the crowded cellular environment, synthetic sugar-based polymers (dextrans of different sizes and Ficoll 70) were used as crowding agents (crowders) in controlled in-vitro experiments. In contrast to previous studies which often have focused on one protein and one crowder at a time, the goal here was to perform systematic analyses of the relationships between the size, shape and concentration of the crowders and the equilibrium and kinet- ic properties of structurally-different proteins. Three model proteins (cyto- chrome c, apoazurin and apoflavodoxin) were investigated under crowding by Ficoll 70 and dextrans of various sizes, using a range of spectroscopic techniques such as far-UV circular dichroism and intrinsic tryptophan fluo- rescence. Thermodynamic models were used to explain the experimental results. It was discovered that the equilibrium stability of all three proteins in- creased in the presence of crowding agents in a crowder concentration- dependent manner. The stabilization effect was around 2-3 kJ/mol and was greater for the various Dextrans than for Ficoll 70 at the same mass concen- iii tration but independent of dextran size (for dextrans ranging from 20 to 70 kDa). A theoretical crowding model was used to investigate the origins of this stabilization. In this model, Dextran and Ficoll were modeled as elongated rods and the protein was represented as a sphere, with the folded sphere representation being smaller than the unfolded sphere representation. Nota- bly, this model was able to reproduce the observed stability changes while only accounting for steric interactions. This correlation showed that when using sugar-based crowding agents, excluded volume effects can be studied in isolation with no contributions from nonspecific interactions. Time-resolved experiments using apoazurin and apoflavodoxin revealed an increase in the folding rate constants while the unfolding rates were un- changed by the presence of crowding agents. For apoflavodoxin and cyto- chrome c, the presence of crowding agents also altered the folding pathway such that it became more homogeneous (cytochrome c) and gave less mis- folding (apoflavodoxin). These results showed that macromolecular crowd- ing restricts the conformational space of the unfolded polypeptide chain, making its conformations more compact. This in turn eliminates access to certain folding/misfolding pathways. The results of the kinetic and equilibrium measurements on three model proteins, together with available data from the literature, demonstrate that macromolecular crowding effects due to volume exclusion are on the order of a few kJ/mol. Considering the numerous