Mechanochemical Methods for Single Molecule Biochemistry and Studies of Thiol-Disulfide Exchange in Proteins Pallav Kosuri Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2012 ©2012 Pallav Kosuri All rights reserved ABSTRACT Mechanochemical Methods for Single Molecule Biochemistry and Studies of Thiol-Disulfide Exchange in Proteins Pallav Kosuri In this thesis, I develop single molecule methods to detect the formation of covalent bonds in proteins. With the use of mechanical force, I investigated the mechanism of disulfide formation in the protein synthesis pathway, and how enzymes catalyze this process. Disulfide bonds between cysteine residues serve as essential structural and functional components in many proteins. In humans, formation of these covalent bonds takes place during oxidative protein folding, a process that is catalyzed by the enzyme Protein Disulfide Isomerase (PDI). Formation of incorrect disulfides can be pathogenic. It is therefore of utmost importance to understand how PDI effectively recognizes which atoms to join, even in environments crowded with cysteines. However, this and other fundamental aspects of oxidative folding remain unexplored due to our current lack of precise experimental methods. I used a custom built Atomic Force Microscope (AFM) to simultaneously detect protein folding and disulfide formation in single protein molecules. This powerful assay allowed me to determine the sequence of reactions during PDI catalysis of oxidative folding. I discovered that PDI actually does not recognize which cysteines to join together in its protein substrate. Instead, the enzyme relies on the natural folding pathway of its substrate to guide the pairing of cysteines. This remarkable finding can explain how one enzyme can catalyze the oxidative folding of a wide variety of proteins. While disulfide bonds are known to stabilize proteins, it has remained unknown how other cysteine modifications affect the mechanical properties of proteins. I show in a series of experiments that protein mechanics are strongly modulated by S-glutathionylation, a common post-translational modification involved in cellular redox signaling. These modulatory effects are specific to cryptic cysteines that only become exposed under strain. Relevant to the many proteins that function under mechanical force, this mechanism provides a way to dynamically adjust protein and tissue elasticity. Strain-dependent cysteine modification may represent a new paradigm in mechanochemical regulation. In a final set of experiments, I show that substrate conformation alters the chemical properties of thioredoxin, an enzyme that catalyzes the reduction of disulfide bonds. The ability of a substrate to influence the p Ka of a catalytic residue may play an important role in determining substrate specificity. The studies I present in this thesis provide fundamental new insights into the protein synthesis pathway, mechanochemical regulation and enzyme catalysis. Together with the methods provided here, my work sets the stage for the emerging field of mechanical biochemistry. Table of Contents 1 Background and Significance .................................................................................................... 2 1.1 The art of making an extracellular protein ........................................................................ 2 1.2 Single molecule biology .................................................................................................... 4 1.3 Force as a probe of biochemistry ....................................................................................... 5 1.4 Disulfide bonds .................................................................................................................. 7 1.4.1 Disulfide chemistry ................................................................................................. 7 1.4.2 Disulfides in biology ............................................................................................... 9 1.4.3 Disulfide formation in cells ................................................................................... 10 1.5 Thiol-disulfide oxidoreductases ....................................................................................... 11 1.5.1 Thioredoxin ........................................................................................................... 12 1.5.2 Protein Disulfide Isomerase .................................................................................. 16 1.5.3 Glutaredoxin .......................................................................................................... 20 1.6 Glutathione ...................................................................................................................... 21 1.7 Studies of oxidative protein folding in vitro .................................................................... 22 1.7.1 Conventional in vitro assays of oxidative folding ................................................. 23 1.7.2 Small molecule catalysis of oxidative folding in vitro .......................................... 24 1.7.3 PDI catalysis of oxidative folding in vitro ............................................................ 27 2 Experimental Techniques ......................................................................................................... 30 2.1 The Atomic Force Microscope ........................................................................................ 30 2.1.1 Basic principle ....................................................................................................... 31 2.1.2 AFM head .............................................................................................................. 34 2.1.3 Laser ...................................................................................................................... 34 i 2.1.4 Fluid cell ................................................................................................................ 34 2.1.5 Piezoelectric actuator ............................................................................................. 35 2.1.6 Sample slide ........................................................................................................... 36 2.1.7 Cantilever .............................................................................................................. 36 2.1.8 Data acquisition ..................................................................................................... 37 2.1.9 Supporting structures ............................................................................................. 37 2.1.10 Force-clamp ......................................................................................................... 38 2.1.11 Calibration ........................................................................................................... 41 2.2 Protein engineering .......................................................................................................... 45 2.2.1 Protein expression and purification ....................................................................... 46 2.3 Single Molecule Force Spectroscopy .............................................................................. 47 2.3.1 Protein mechanics under force .............................................................................. 48 2.3.2 Mechanical protein unfolding ................................................................................ 50 2.3.3 Force based assays of protein folding ................................................................... 55 2.3.4 Disulfide detection using force .............................................................................. 56 2.3.5 Disulfide cleavage under force .............................................................................. 58 2.3.6 Setup of an experiment .......................................................................................... 60 2.3.7 Pulse protocols ....................................................................................................... 61 2.3.8 Data Analysis ......................................................................................................... 62 3 Single Molecule Study of Oxidative Protein Folding .............................................................. 65 3.1 Physiological significance of PDI activity ...................................................................... 66 3.2 Oxidative folding pathways in cells ................................................................................ 67 3.3 Spontaneous disulfide oxidation kinetics ........................................................................ 69 ii 3.4 PDI-mediated oxidation of folded proteins ..................................................................... 70 3.5 Creating a mixed disulfide complex between PDI and a single unfolded protein ........... 71 3.6 Mixed disulfide complexes with PDI enable oxidative folding ...................................... 73 3.7 Detection of spontaneous enzyme release ....................................................................... 74 3.8 Measurement of the PDI spontaneous release rate .......................................................... 78 3.9 Kinetics of oxidative protein folding from an extended state .......................................... 82 3.10 Interchangeability of oxidoreductase enzymes