Structural Investigation of the Molecular Chaperonin Tf55 From

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Open Access Theses & Dissertations

2014-01-01 Structural Investigation Of The olecM ular Chaperonin Tf55 From Thermophilic Archaeon Sulfolobus Solfataricus Sanjay Kumar Molugu University of Texas at El Paso, [email protected]

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Recommended Citation Molugu, Sanjay Kumar, "Structural Investigation Of The oM lecular Chaperonin Tf55 From Thermophilic Archaeon Sulfolobus Solfataricus" (2014). Open Access Theses & Dissertations. 1302. https://digitalcommons.utep.edu/open_etd/1302

This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. STRUCTURAL INVESTIGATION OF THE MOLECULAR CHAPERONIN TF55 FROM

THE THERMOPHILIC ARCHEON SULFOLOBUS SOLFATARICUS

SANJAY KUMAR MOLUGU

Department of Chemistry

APPROVED

______Ricardo Bernal Ph.D., Chair

______Mahesh Narayan Ph.D.

______Siddhartha Das Ph.D.

______Francia Giulio Ph.D.

______Charles Ambler, Ph.D. Dean of the Graduate School

Sanjay Kumar Molugu

2014

For Sulochana and Krishna Moorthy

STRUCTURAL INVESTIGATION OF THE MOLECULAR CHAPERONIN TF55 FROM

THE THERMOPHILIC ARCHEON SULFOLOBUS SOLFATARICUS

SANJAY KUMAR MOLUGU

THESIS

Presented to the Faculty of the Graduate School of

The University of Texas at El Paso

in partial fulfillment

of the requirements

for the Degree of

MASTER OF SCIENCE

Department of Chemistry

THE UNIVERSITY OF TEXAS AT EL PASO

December 2014 Acknowledgements

Foremost, I would like to express my sincere gratitude to my advisor

Dr. Ricardo Bernal for giving me a chance to work in his lab and for the continuous support throughout my Master’s study. Dr. Bernal’s motivation, enthusiasm and immense knowledge has helped me in all the time of research and made my research productive. I would like to thank my committee members Dr. Mahesh Narayan,

Dr. Siddhartha Das, Dr. Francia Giulio for their valuable comments and suggestions.

Furthermore I would also like to thank Dr. Sudheer Molugu for the cryo-EM data collection. I like to thank my fellow lab members for their help and valuable discussions.

I would also like to acknowledge UTEP and Welch Foundation for the financial support for my research.

v Abstract

Chaperonins are ubiquitous multi-subunit macromolecules that mediate the folding and assembly of certain proteins that cannot fold properly in vivo . The Archaea bacterium Sulfolobus solfataricus encodes for three chaperonin complexes formed from three different, but closely related subunits named α, β and γ. These subunits form a homo-18mer composed of all β subunits (TF55 β) at temperatures ranging between

80 oC-85 oC, a hetero-16mer composed of α and β subunits (TF55 αβ ) at temperatures ranging between 70 oC-75 oC and a hetero 18mer composed of α, β and γ subunits

(TF55 αβγ ) at temperatures ≤ 60 oC. Structures exist for the chaperonin TF55 all-β complex and for the TF55 αβ complex but not for the TF55 αβγ complex. Here we present the structural investigation of the chaperonin TF55 αβγ in the presence of ATP.

The cryo-EM reconstruction of TF55 αβγ chaperonin complex revealed a double-ring of

18 subunits with nine subunits in each ring. Each of the α, β, γ subunits are arranged in an alternating fashion where three hetero-trimers reside in each ring and the intact 18- meric complex is formed by two of these rings stacked back to back, similar to other chaperonin complexes.

vi Table of Contents

Acknowledgements ...... v

Abstract ...... vi

Table of Contents ...... vii

List of Figures ...... ix

List of Abbreviations and Notations...... x

Chapter 1 ...... 1

1.1 Introduction ...... 1

1.2 Protein folding in-vitro versus in-vivo ...... 1

1.3 Molecular Chaperones and Chaperonins ...... 2

1.4 The Chaperonin Architecture ...... 3

1.5 Group I chaperonins ...... 4

1.6 Group II Chaperonins from Eukarya and Archaea...... 8

1.7 TF55 chaperonin from Sulfolobus solfataricus overview ...... 13

Chapter 2 ...... 15

Biochemical Methods ...... 15

2.1 Bacterial cell Transformation ...... 15

2.2 Bacterial cell culture and protein expression ...... 16

2.3 Protein purification by column chromatography ...... 17

2.4 Electrophoresis and SDS PAGE ...... 20

vii Chapter 3 ...... 21

Cryo-EM overview and Single particle reconstruction technique ...... 21

3.1 Cryo-Electron Microscope (Cryo-EM) overview...... 21

3.2 Negative Staining ...... 21

3.3 Cryo-Freezing...... 22

3.4 Single particle cryo-EM reconstruction technique...... 23

3.5 Three Dimensional reconstruction using EMAN ...... 24

3.6 Contrast Transfer function (CTF) Correction ...... 24

Chapter4 ...... 27

Structure Determination of TF55-ATP Chaperonin Complex ...... 27

4.1 Introduction ...... 27

4.2 Materials and Methods ...... 28

4.2.3 TF55 αβγ Complex formation ...... 30

4.2.4 Cryo-electron microscopy ...... 30

4.3 Results and Discussion ...... 31

Conclusions ...... 38

References ...... 39

Curriculum Vitae ...... 46

viii List of Figures

Figure 1.1 Architecture of group I and II chaperonins ...... 4

Figure1.2 Structure of the GroEL–GroES...... 5

Figure 1.3 The folding cycle of the bacterial chaperonin system GroEL-ES ...... 6

Figure1.4: Proposed One-Stroke Mechanism of Mt-cpn60 ...... 8

Figure1.5 Current understanding of the group II chaperonin mechanism...... 10

Figure1.6 The eukaryotic TriC/CCT chaperonin...... 11

Figure1.7 Thermosome from archaebacteria...... 12

Figure1.8 mm-cpn from Methanococcus maripaludis...... 13

Figure1.9 Temperature dependent regulation of all the three subunits ...... 14

Figure 2.1 General steps involved in Transformation of bacterial cells ...... 16

Figure 2.2 General Mechanism involved in Ion Exchange Chromatography ...... 17

Figure 2.3 General Mechanism involved in Gel Filtration ...... 18

Figure 2.4: General Mechanism involved in Affinity Chromatography ...... 19

Figure 2.5: General Steps involved in SDS-PAGE ...... 20

Figure 3.1: Schematic representation of steps involved in Cryo-EM grid preparation .. 24

Figure 3.2 Contrast Transfer Function for the Philips CM200 FEG microscope...... 26

Figure 4.1: SDS PAGE gels showing the recombinant TF55 α, β and γ chaperonins purified separately and the purified TF55 αβγ complex ...... 33

Figure 4.2 Cryo-EM data collection and image processing of TF55 chaperonin ...... 34

Figure 4.3: The reconstructions showing TF55 single and double rings ...... 35

Figure 4.4: 18.5Angstroms resolution of TF55 chaperonin at 0.5 FSC cut off value ..... 35

Figure 4.4: X-ray coordinate fitting of β subunit into TF55 αβγ chaperonin complex...... 37

ix List of Abbreviations and Notations

% percent

α alpha

β beta

γ gamma

π 22/7 (3.14)

µm micro meter

0C degree centigrade

2D two-dimensional

3D three-dimensional

Å angstroms, unit of distance, 10 -10 M

ADP adenosine 5’-diphosphate

ATP adenosine 5’-triphosphate

BCA Bradford calorimetric assay

cis on the same side

CTF contrast transfer function

cryo-EM cryo-electron microscopy

DNA deoxy-ribonucleic acid

DNase deoxyribonucleic acid hydrolase

dsDNA double stranded deoxy-ribonucleic acid

e- Electron

Ea activation energy

EDTA ethylenediaminetetraacitic acid

x E. coli Escherichia coli

EM electron microscopy

Homo same

Hetero different

Hsp Heat shock proteins

FSC Fourier shell correlation

IPTG isopropyl β-D-1-thiogalactoside

In-vitro at laboratory conditions outside the living organism

In-vivo at physiological conditions within the living organism kDa kilo Dalton mM mill molar, 10 -3 Molar, unit of concentration

Mg/ml milligrams/milliliter, 10 -3g/10 -3L, unit of concentration

N2 nitrogen

Na + sodium ion nm nanometer

OD 600 optical density, 600 nanometer wavelength pH negative logarithm of H+ ion concentration of a solution

Pi inorganic phosphate

PAGE polyacrylamide gel electrophoresis

PDB protein databank bank

RNA ribonucleic acid

SDS sodium dodecyl sulphate

TEM transmission electron microscope

xi trans on the opposite side

Tris tris(hydroxymethyl)amino methane w/v weight/volume xg times the force of gravity

xii Chapter 1

1.1 Introduction

Proteins are the most versatile and structurally complex macromolecules in all living organisms where they perform many important biological functions. During protein synthesis, the ribosome catalyzes the translation of genetic information into a nascent polypeptide chain that folds into its functionally active structure co-translationally.

Defects in both protein folding and quality control are associated with variety of diseases such as cystic fibrosis, Huntington disease, Alzheimer’s disease, etc 1,2 . Protein folding is therefore an important field of study to better understand such diseases.

1.2 Protein folding in-vitro versus in-vivo

Christian Anfinsen in his experiments proved that under physiological conditions, the three dimensional structure of a protein is at the lowest Gibbs free energy state 3. To attain this, polypeptide chains follow folding pathways that bury hydrophobic surfaces inside the core of the structure 3-5. Under mild stress conditions such as low temperature or a high protein dilution, many proteins are able to fold spontaneously into their native state. However, under in-vivo conditions protein folding occurs in the presence of high concentrations of cellular protein (300-400 mg/ml) and other macromolecules that interfere with protein folding, leading to defective proteins and aggregation 5,6,7,8.

Furthermore, during protein translation, the nascent polypeptide chain is exposed to a complex cellular milieu contributing to the risk of misfolding and aggregation 9,10-12 .

Folding of such nascent polypeptide chains are therefore at risk. Such polypeptides are usually accompanied by a range of molecular chaperones and chaperonins that interact

1 co-translationally with nascent polypeptides to minimize misfolding until they attain a native conformation 9.

1.3 Molecular Chaperones and Chaperonins

A molecular chaperone is defined as any protein that interacts with, stabilizes or helps another protein to acquire its functionally active conformation, without being present in final structure 9,10 . Different types of structurally unrelated chaperones are present in cells, which are usually referred to as stress proteins or heat-shock proteins

(HSPs) as they are usually up regulated under the stress conditions where the probability of misfolding increases 13,14 . Chaperones are usually classified based on their molecular weight as HSP40, HSP60, HSP70, HSP90, HSP100 and small HSPs that are less than 40 kilo Daltons in size 15,16 . These proteins are involved in proteome- maintenance functions, including de novo folding, refolding of stress-denatured proteins, and assistance in proteolytic degradation. There are also a class of macromolecular complexes called chaperonins that are large double ring complexes of 800-900 kDa that function by encapsulating substrate protein for folding 17 . Chaperonins utilize the energy derived from ATP hydrolysis to undergo large conformational changes and provide a micro-environment protected from the unfavorable cytoplasmic conditions for folding 18 .

Chaperonins are classified into two groups. Group I chaperonins are seven membered rings found in bacteria (GroEL), mitochondria and chloroplasts (HSP60).

These chaperonins functionally cooperate with HSP10 proteins (GroES in bacteria) to form the lid of the protein-folding cage 9,19 . The Group II chaperonins in archaea bacteria

(thermosomes) and the eukaryotic cytosol (TriC) are usually eight or nine subunit rings

2 that do not require a co-chaperonin 9,20,21 . Chaperonins, by providing kinetic assistance, prevent or reverse protein misfolding that can lead to irreversible protein aggregation 19 .

1.4 The Chaperonin Architecture

All chaperonin subunits share a similar overall architecture consisting of three distinct domains: an equatorial domain connected via a hinge-like intermediate domain to the distal apical domain (see figure 1.1B) 22-24 . The equatorial domain provides the contact surface between the two rings and harbors the major sites of ATP binding 22,23 .

The intermediate domain consists of a hinge region that connects equatorial domain to the apical domain. The most flexible region of chaperonin is the apical domain that interacts with the substrate protein as well as co-chaperonin through hydrophobic interactions 25 . The flexibility of apical domains is responsible for large conformational changes that allow opening and closing of the ring cavity 21,25,26 .

The binding and hydrolysis of ATP induces multiple conformational changes within the subunits that opens and closes the apical domains where the substrate binding and encapsulation occur 22,27,28 . Two groups of chaperonins have been identified based on the necessity for a co-chaperonin22,27,28 . Group I chaperonins such as groEL from E. coli, need a co-chaperonin for closing and opening of the chaperonin ring.

Group II chaperonins such as the thermosome do not need a co-chaperonin. Instead the apical domains contain protrusions that extend to form an iris like structure that closes the ring at the expense of ATP hydrolysis 22,24 .

Figure 1.1 Architecture of group I and II chaperonins (A) Structures of the GroEL/ES complex (left) and the thermosome (right), respectively. Group I chaperonins like GroEL-GroES require a co-chaperonin (red).The image on the right side corresponds to the thermosome utilize a built in lid structure 22,23 . ( B) Crystal structures of a single subunit of GroEL(left) and the thermosome (right), respectively 22 . The equatorial domain (red) is linked via the hinge-like intermediate domain (yellow) to the apical domain (green). In contrast to the group I chaperonin, group II chaperonins contain apical protrusions extending from the tip of the apical domain.

1.5 Group I chaperonins

1.5.1 groEL/ES

The groEL/ES chaperonin complex from E. coli assists in folding a large number of cytosolic proteins 29,30 . Approximately 250 different proteins interact with groEL upon synthesis, and of these, 85 proteins are predicted to be obligate chaperonin substrates.

4 The general mechanism of action of groEL /ES involves encapsulation of single nonnative substrate protein molecule in a cage -like structure, thereby allowing folding to occur 31,32 . The groEL chaperonin is about 800 kDa c ylindrical complex with ATPase activity, consisting of two heptameric rings stacked back to back to form a central cavity in each ring for encapsulating substrate protein (see figure 1.2 a). The co -chaperonin groES is a dome shaped heptameric ring of about 70 k Da total that contacts the apical groEL domains via flexible loop sequences thereby capping the groEL cylinder 29,33

(See figure 1.2 b).

Figure1.2Structure of the GroEL –GroES. (a).The double ring groEL chaperonin (blue). ( b)The binding of the heptameric GroES lid(red) to the cis ring of GroEL 34 . 1.5.2 The groEL /ES machinery

The ATPase cycle of the group I chaperonin system has been studied extens ively. Each individual ring in groEL represents a functional unit, whose individual subunits have to work in a fully synchronize d fashion 17,23 . Subunits within each ring are coupled via positive co-operativity in ATP binding which allows th em to act in a concerted fashion to create the closed folding chamber 23,35,36 . In addition, n egative cooperativity between the rings causes ATP binding to one ring while inhibiting ATP

5 binding to the adjacent ring 35,36 . This feature ensures that only one ring, the so called cis-ring, is folding-active at a given time, allowing groEL/ES chaperonin to function as a

"two-stroke" motor where the two rings alternate during the reaction cycle.

Binding of ATP to the cis ring induces conformational changes which triggers the binding of substrate protein 37 . This scenario initiates the attachment of the heptameric groES lid, resulting in encapsulation of the substrate within the cavity 37,38 . The ATP hydrolysis creates an active folding environment for substrate protein to fold itself by hiding the hydrophobic residues inside the core of the substrate protein 37,38 . ATP hydrolysis in the cis-ring facilitates ATP binding to the trans -ring, which in turn results in release of ADP, groES, and the native substrate protein from the cis -ring 17,39 (see figure

1.3). The co-chaperonin groES then binds to the “new” cis -ring, and a new round of folding starts 40 .

Figure 1.3 The folding cycle of the bacterial chaperonin system GroEL-ES Binding of ATP to GroEL cis-ring induces slight conformational changes that result in increased affinity for the substrate and GroES. GroES binding displaces protein into the cis-cavity. The substrate protein folds during hydrolysis of ATP. Binding of ATP to the trans-ring finally discharges ADP, GroES and the encapsulated substrate from the cis-

6 ring and the next folding cycle starts in the new cis-ring. Adapted from Zachariah L. Hilden brand and Ricardo A. Bernal et al. (2006) 29 . 1.5.3 Hsp60/10

Hsp60 and its co-chaperonin Hsp10 are structurally and functionally similar to groEL/ES, but differ in their protein folding mechanisms 41 . The chaperonin Hsp60 is abundant in mitochondria where it catalyze the folding of different proteins destined for the mitochondrial matrix 42 . It was observed that Hsp60 along with its co-chaperonin

Hsp10 in mitochondria facilitates the folding of RuBisCO (ribulose-1,5 bisphosphate carboxylase oxygenase) in an ATP-dependent one-stroke mechanism 43 (see figure

1.4). It was also observed that Hsp60/10 chaperonin has the ability to fully function as single ring and can substitute groEL/ES complex in E. coli and fold proteins with same efficiency 43,44 .

The chaperonin Hsp60/10 complex performs protein folding as single ring species which is in contrast to two stroke mechanism of groEL/ES complex that utilizes the negatively cooperative allosteric interactions between its two rings in order to facilitate protein folding 45,46 . However, the driving force for the single ring protein folding mechanism is not completely understood.

Figure1.4: Proposed One -Stroke Mechanism of Mt-cpn60 1. Misfolded protein binds to apical domains of the Mt -cpn60 followed by 7ATP molecules and GroES . 2. ATP hydrolysis creates a productive protein folding environment. 3. The ADP molecule is released along with the substra te protein. 4. The empty single ring now accepts a substrate protein. Modified from Hayer -Hartl et al. (2006) 29 .

1.6 Group II Chaperonins from Eukarya and Archaea

Although, both the group I chaperonin s and the group II chaperonin s are double- ring structures and share sequence similarities, they differ from each other in two major aspects. First, the group I chaperonin s are composed of identical subunits and ha ve seven subunits per ring whereas group II chaperonins are composed of 2 -8 paralogous subunits with 30-40% identity to one another and each ring has 8-9 subunits 46-51 .

Second, the group II chaperonins do not require any groES-like co-chaperonin. Instead these chaperonins possess a helical protrusion that acts like “built in lid”. These two major differences are the result of the evolution of group II chaperonins for assisting the folding of different archaeal and eukaryotic proteins 51 . It was observed that structural

8 and mechanistic differences between the two groups have a significant functional impact on substrate specificity 50,51

1.6.1 Substrate protein folding mechanism

Despite intensive studies on structure and function of bacterial groEL/ES chaperonin, little is known about the Group II chaperonin mechanism. Owing to the absence of sufficient structural information, the current understanding of the Group II chaperonin mechanism is derived from limited cryo-EM and X-ray crystal structures.

The cryo-EM structures include the TriC chaperonin from eukaryotic bovine tissue, the mm-cpn60 from mesophilic methanogenic Archaean Methanococcus maripaludis and the X-ray crystal structure includes the thermosome from Thermoplasma acidophilum 22,47,53 .

In the absence of nucleotide, all group II chaperonins adopt a symmetrically open conformation that can bind unfolded substrate protein 54-57 . The binding of ATP induces conformational changes that close the ring (see figure 1.5). In TriC and the thermosome, ATP hydrolysis is required to induce a conformational change during which the apical protrusions of neighboring subunits assemble into an iris-like β-sheet and that leads to a conformational state that supports substrate folding 58,59 . The current understanding of the ATPase cycle in Group II chaperonins is based on a collection of isolated conformational states that still need to be interconnected.

Figure1.5 Current understanding of the group II chaperonin function The open complex in the absence of a nucleotide binds to an unfolded substrate through the apical domains and the binding sites in the central cavity (red lines). ATP binding induces conformational changes in the ring followed by the closure of the ring. ATP is hydrolyzed. Folding probably occurs at this stage of the cycle The inorganic phosphate (P i) dissociation is likely to trigger reopening of the lid and the release of folded substrate The cycle is probably asymmetric but the mechanism that keeps both rings in different stages of the hydrolytic cycle is unclear . Adopted from Frydman et al. 2004 53 .

1.6.2 TriC /CCT from Eukaryotic cytosol

The chaperonin TriC/ CCT ( TCP-1-Ring Complex) plays an important role in foldi ng of various proteins including cytoskeletal components, cell cycle regulators and tumor suppressor proteins 59,60 . Furthermore, it was recently found that TriC plays an important role in protecting cells against formation of cytotoxic conformations of proteins with extended polyglutamine repeats that underlie Huntington’s disease and other neurodegenerative disorders61 -63 . Substrates like tubulin can only be folded by TriC suggesting that TriC has its own mechanistic features that are abs ent in other 10 chaperonin systems 64,65 . Furthermore, it is believed that because of its unique subunit composition, TriC has more substrate -specificity in protein folding 66 . It i s believed that specialized substrate binding occurs through hydrophobic interactions but the extent of these interactions in a stru ctural context remains unclear 66 . Of the Group II chaperonins,

TriC is most structurally complex because each ring is composed of eight different individual subuni ts and is related by Dihedral symmetry 67,68 (see figure 1.6) .

Figure1.6 The eukaryotic TriC/CCT chaperonin. (a – c) End, side, and center -slabbed views of the hetero-oligomericdouble - ringed TriC/CCT complex in the closed conformation (PDB 3IYG). The eight different subunits within each ring are colored accordingly to illustrate the intra -ring subunit heterogeneity 25 .

1.6.3 Thermosome from Archaea

Archaea bacteria are evolutionarily distinct from prokaryotes and eukaryotes and form their own kingdom 68 . However they are more related to the eukaryotic cells than to bacteria 69-71 . The group II thermosome, found in thermophilic archaea is structurally related to the Temperature Factor55 (TF55) chaperonin from Sulfolobus solfataricus and is a functional homolog of the eukaryotic TriC 72,73 (see figure 1.7a, b ). The

11 thermosome structure consists of two eight -me mbered rings that are stacked back to back with two fold symmetry, each ring formed by an alternating α and β subunit 22 . The primary sequence identity between α and β subunits is about 60% and are virtually identical pert aining to their fold and domain arrangements 22 . Moreover, the α subunit of the thermosome exhibits 46% sequence similarity with that o f bacterial groEL monomer where both have the characteristics of limited domain movements 22,23 . All of the subunits of a thermosome consists of β-sheet protrusions at the distal ends of the apical domains that form a lid like structure that functions in an ATP-dependent manner 47,74 .

Figure1.7 Thermosome from archaebacteria. (a – b) End and side vie ws of the thermosome chaperonin from Ther moplasma acidophilum (PDB 1A6D) 25 .

1.6.4 The Methanococcus maripaludis chaperonin (mm-cpn)

The Archaeal chaperonin mm -cpn from Methanococcus maripaludis has an eight fold double barrel structure similar to other group II chaperonins 75 . However , the subunits of mm-cpn are homogenous (see figure 1.8a), similar to the group I chaperonin groEL, rather than heterogeneous as seen in TriC/CCT 14,75,76 . Although, the subunit

12 homogeneity of mm-cpn may suggest functional similarity to groEL, the sequence identity and structure showed that mm-cpn is more closely related to TriC/CCT 21 .

Mm-cpn also contains built-in lid structure (see figure 1.8b), which is the characteristic feature of group II chaperonins and exhibits allosteric regulation similar to that of TriC/CCT 3,77 . Like the other group II chaperonins, mm-cpn has a natural affinity towards substrates containing β-sheet folds that are generally prone to thermodynamically unfavorable folding and aggregation 78,79 . It is thought that affinity of group II chaperonins towards β-sheet structures is due to its subunit heterogeneity 80 .

Such a hypothesis is not applicable to mm-cpn due to its subunit homogeneity.

Therefore, further work is needed to determine the cause of this phenomenon 78,79 .

Figure1.8 mm-cpn from Methanococcus maripaludis. (a-b)End and side views of Homo-oligomeric double-ringed mm-cpn in closed conformation(PDB3IZI)25 .

1.7 TF55 chaperonin from Sulfolobus solfataricus overview

Sulfolobus species grow at extreme temperatures between 75 oC-80 oC and at low pH in the range of 2.5-3.0, making them thermophiles and acidophiles. The TF55

13 chaperonin complex from Sulfolobus solfataricus is encoded by three different, but closely related genes, named α, β, and γ. This is consistent with the 9 fold symmetry of the purified comp lex seen by electron microscop y22 . The crystallization of TF 55 purified from Sulfolobus at normal growth tempe ratures resulted in crystals with tetragonal symmetry but only α and β subunits were present, similar to the crys tal structure of the

16-meric T. acidophilum thermosome 22 . The electron micrographs obtained from preparations from heat-shocked (85 oC) Sulfolobus cells and the crystal structure from

Sulfolobus cells grown at optimal temperatures (75 o C) showed that the

γ subunit is not heat resistant and denatures at a normal Sulfolobus growth temperatures of 75 oC81 (see f igure 1.8 b and c). Hence, TF55 appears to have three different chaperonin complexes for different temperatures. There is an 18 -mer α-β-γ complex under cold-shock conditions (60 oC) (see figure 1.8 a), a 16-mer α-β complex at normal growth temperatures (75 o C) and an all-β 18-mer forms under heat shock conditions (85 oC) 82 (see figure 1.8 b and c).

Figure1.9 Temperature dependent regulation of all the three subunits (a) Formation of αβγ complex at 60 oC (cold shock) (b) Formation of αβ complex at normal growth temperatures (c) Formation of all β complex at 85 oC (heat shock)

14 Chapter 2

Biochemical Methods

2.1 Bacterial cell Transformation

Transformation is a process by which foreign DNA is introduced into bacterial cells. Since DNA is very hydrophilic molecule, it won’t easily pass through the bacterial cell membrane. Furthermore, the size of the DNA is much larger than any bacterial porin. In order to make bacteria uptake the plasmid, the cells are made competent by suspending the cells in solution of calcium chloride. The plasmid DNA containing an antibiotic resistant gene can then be forced into the competent cells by incubating the cells and DNA together on ice for an hour followed by placing them briefly at 42 oC (heat shock) and then transferring them back onto the ice for additional 2 minutes. This heat shock procedure successfully transfers foreign DNA into the bacterium. The cells are then grown for an hour in a nutrient rich medium to allow the growth of antibiotic resistant proteins and then plated on antibiotic containing agar plates. The plates are left at 37 oC overnight and the colonies are expected to be seen by naked eye the next day (see figure 2.1). Only bacteria containing the resistant gene towards the respective antibiotic grows and other cells die. A single colony is picked up and cell culture is cultivated.

Figure 2.1 General steps involved in Transformation of bacterial cells Adopted from Introduction to Gen etic Analysis by Griffiths AJF.

2.2 Bacterial cell culture and protein expression

A bacterial cell culture is grown by picking up a single isolated colony from the streaked plate and transferring it with a loop into 20ml of bacterial growth media with antibiotic in a baffled Erlenmeyer flask and incubated in a shaker at 37 oC overnight . 1 ml of this culture is then transferred into a fresh 1 liter of LB media and allowed to grow in the presence of antibiotic. When the OD 600 reaches 0.6-0.8, protein production is induced by adding Isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells are incubated 4 more hours a nd then harvested by centrifugation at 10 ,000xg. The cells are lysed with lysozyme and the cell extract is treated with DNase and streptomycin sulf ate to free the lysate from genetic material. The lysate free of DNA and cellular debris is now ready for col umn chromatography .

16 2.3 Protein purification by column chromatography

The crude lysate contain ing the protein of interest is subjected to a series of columns packed with chromatographic matrix material that helps in purifying the protein to homogeneity. Three types of column chromatography are used for this project including Ion exchange, Gel Filtration and Affinity Chromatography (His-tagged protein purification).

2.3.1 Ion Exchange Chromatography

A method for the purification of proteins and other charged molecules is Ion exchange chromatography. In cation exchange chro matography, positively ch arged molecules are attracted towards a negatively charged solid support. I n anion exchange chromatography, negatively charged molecules are attracted to a positively charged solid support (see figure 2.2).

Figure 2.2 General Mechanism involved in Ion Exchange Chromatography Adopted from The Dynamic Chemistry E -textbook by Michael Blaber

To optimize binding of all charged molecules, the mobile phase is generally a low to medium conductivity (i.e., low to medium salt concentration) solution. The positively charged ionic groups of the sample are adsorbed onto the negatively c harged solid

17 functional groups of the column. By increasing the salt concentration (generally by using a linear salt gradient) the molecules with the weakest ionic interactions start to elute from the column first. Molecules that have a stronger ionic inte raction require a higher salt concentration and elute later in the gradient.

2.3.2 Gel Filtration ( Size Exclusion Chromatography)

Figure 2.3 General Mechanism involved in Gel Filtration Adopted from The Dynamic Chemistry E -textbook by Michael Blaber

Gel filtration does not depend on any chemical interaction with the protein; rather it is based on the physical property of the protein , the effective molecular radius (relates to mass for most typical globular proteins). Gel filtration resin can be though t of as beads which contain pores of a defined size range (see figure 2.3). Large proteins which cannot enter these pores pass around the outside of the beads. Smaller proteins which can enter the pores of the beads have a longer, complex path before they exit the bead. Thus, a sample of proteins passing through a gel filtration column will separate based on molecular size: the large ones will elute first and the smaller ones will elute last.

18 2.3.3 Affinity Chromatography (His-tagged Proteins Purification)

Figure 2.4: General Mechanism involved in Affinity Chromatography Adopted from BIO-RAD Applications & Technologies.

The DNA sequence specifying a string of six to nine histidine residues is frequently used in vectors for production of recombinant proteins. The result is expression of a recombinant protein with a 6xHis or poly -His tag fused to its N - or C- terminus. Expressed His-tagged proteins can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions including nickel, cobalt and copper. Nickel is the most widely available m etal ion for purifying His - tagg ed proteins. Elution and recovery of captured His -tagged protein from the column is accomplished with a high concentration of imidazole in the buffer. However, the imidazole from the sample has to be removed as it can denature the purified protein.

Therefo re, the sample has to be purified from Imidazole immediately by running through a desalting column (see figure 2. 4).

19 2.4 Electrophoresis and SDS PAGE

Figure2.5: General Steps involved in SDS -PAGE. Adopted from The Dynamic Chemistry E -textbook by Thomas Chasteen

The separation of macromolecules in an electric field is called electrophoresis. A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. The SDS (also called lauryl sulfate) is an anionic detergent and acquires an overall negative charge within a wide pH range (see figure 2.3a) . A polypeptide chain binds SDS in proportion to its relative mole cular mass. The hydrophobic moiety on SDS destroys most of the complex structure s of proteins, and the negative charge is strongly attracted toward an anode (positively -charged electrode) in an electric field ( see figure

2.3b). Because the charge-to -mass ratio is nearly the same among SDS -denatured polypeptides, the final separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides. In order to analyze the proteins of our interest, Coomassie Blue Dye is used to stain the gel (see figure 2.3c), followed by destaining with a mixture of acetic acid, ethanol and water.

20 Chapter 3

Cryo-EM overview and Single particle reconstruction technique

3.1 Cryo-Electron Microscope (Cryo-EM) overview

The cryo-electron microscopy (cryo-EM) is a form of transmission electron microscopy where a small amount of sample is flash frozen to cryogenic temperatures usually by plunging the grid holding the sample in liquid ethane 83 . The frozen grid is then transferred to the microscope and kept at liquid nitrogen temperature. Images are collected by using low electron dose (1-10 electrons per square angstrom) 83,84 . The cryogenic temperature is maintained so as to minimize the damage caused by radiation and to preserve the sample in a native state 85 . Whenever a protein cannot be crystallized due to instability or its inability to crystallize, Cryo-EM is used as alternative to crystallography 86 . Cryo-EM along with single particle reconstructions has now become an important tool to visualize large, macromolecular assemblies at sub- nanometer and atomic resolutions 87-91107-111 .

3.2 Negative Staining

Biological samples are made of atoms including nitrogen, carbon, oxygen which have almost the same scattering power as water. This creates very low intrinsic contrast. The biological sample is also radiation sensitive, because electrons produce free radicals that causes oxidative damage 86-92 . The sample is often negatively stained, to avoid such problems 92,93 . Negative staining is easy to perform and this technique not only provides high contrast but also low sensitivity to electron beam 94-96 . In this technique, the sample is embedded in a low concentration of salt solution of a heavy

21 atom like methyl amine tungstate or uranyl acetate and dried to a thin layer which is then introduced into the microscope 93,97,98 . The benefit of this technique is that there is no radiation damage to the sample because the image is generated by the heavy atom stain. The protein is vaporized instantaneously as the electron beam hits the sample but as long as the cast of the sample stays intact, images can be collected 98 . However, the drawback of this technique is that the images collected are at low resolution of up to 20

Angstroms 94,95,99 .

3.3 Cryo-Freezing

Cryo-freezing is a technique for fixation or stabilization of biological samples as the first step in specimen preparation of cryo-electron microscopy. This method involves ultra-rapid cooling of samples embedded in aqueous solutions to the temperature of liquid nitrogen (-196 oC) or below so that they do not have time to reorganize into crystal and remain amorphous. This ice is called vitreous ice 100-102 . The sample is introduced into microscope without any evaporation of water and the sample can withstand the high vacuum of the microscope because of the low temperature. Radiation damage is also minimized in this frozen hydrated state because the free radicals generated by inelastic scattering of the electrons with the sample cannot move quickly in the solid ice at liquid nitrogen temperature. Low electron dose is still required because the radiation damage is minimized but not completely eliminated. The images produced will be of low contrast since low electron dose is used 102 . The basic steps involved in preparing Cryo

EM grid are outlined in Figure 3.1.

Figure 3.1: Schematic representation of the steps involved in Cryo -EM grid preparation 31 Adopted from Molugu et al.

3.4 Single particle cryo-EM reconstruction technique

Single particle cryo–EM technique has become one of the most popular tool s to get the three-dimensional structure of macromolecules using two-dimensional projections of the molecule. In this method a large number of m icrographs are collected using cryo-EM . The micrographs are then processed by using different types of

23 statistical and image processing techniques to get final three-dimensional reconstruction 103-105 .

Many software packages are available now to process cryo-EM data using single particle reconstruction and other reconstruction techniques 125-129 . Some of the software packages include EMAN, IMAGIC, SPIDER, Relion and others 105-108 . EMAN (Electron

Micrograph Analysis) is used in this project to generate three-dimensional reconstruction.

3.5 Three Dimensional reconstruction using EMAN

There are three stages in single particle reconstruction procedure. First, all the individual particles are extracted from the digital micrographs. A series of alignment programs are then used to process the selected particles images and based on their orientations, particles are grouped into individual classes. The particles with in each class are mutually aligned and averaged into a class average. By using a number of class averages, a rough 3D reconstruction map is calculated. The preliminary model is iterated through many cycles of refinement until the data has converged. Additionally, the contrast transfer function (CTF) correction and amplitude correction of the microscope need to be performed so as to achieve high resolution of the reconstruction 31 .

3.6 Contrast Transfer function (CTF) Correction

The goal of single particle reconstruction is to regenerate the correct three- dimensional structure of a molecule based on two dimensional projections of the molecule. However, the images generated by the electron microscope are not true

24 projections of the specimen 125 . They suffer from a set of artifacts including the CTF and the envel ope functions of the microscope . The CTF can cause serious artifacts in a 3D reconstruction if not corrected properly 109,130 . The contrast transfer function defines the transfer of contrast from the sample on the image. The biological samples produce little contrast in vitreous ice at perfect focus. So, images are collected at under focus, which produces phase contrast but leads to systemic alteration of the i mage data. This alteration is described as the contrast transfer function (CTF).

Figure 3.2 Contrast Transfer Function for the Philips CM200 FEG microscope. The CTF defines the transfer of contrast from the sample on to the image 110 Contrast changes from positive to negative and back repeatedly passing through spatial resolutions that contain no contrast at all.

When the contrast of the CTF is negative, positive phase contrast occurs, which means that atoms will appear dark on a bright back ground. When the contrast of the

CTF is positive, negative phase contrast occurs which means that atoms will appear

25 bright on dark background 109 . CTF correction is performed by inverting phases by 180 o

(inverting contrast) 111 .

26 Chapter4

Structure Determination of TF55-ATP Chaperonin Complex

4.1 Introduction

The chaperonin TF55 from Sulfolobus solfataricus is encoded by three different, but closely related α, β, γ subunits. The subunit α was cloned into the PET-Duet vector system and is resistant to the antibiotic ampicillin. The Subunits β and γ were cloned into the PET24 vector systems and are resistant to kanamycin. The gene carrying vectors were transformed into BL21 and DH5 ( α) cells and the protein expression was induced with 1mM IPTG and purified to homogeneity. The three subunits were purified as monomers and later mixed together in equi-molar ratios and in the presence of a

5mM ATP at 60 oC in an attempt to procedure TF55 αβγ complex. The now formed complex was purified from monomers and impurities by running the assembled complex through a size exclusion column. The sample was flash frozen at -196 oC using the aforementioned cryo-plunge freezing technique. The cryo-EM data was collected using the JEOL 3200 FS at The University of Texas at El Paso. The cryo-EM data was processed using EMAN 1.9 and EMAN2.0 software suites and the reconstruction of

TF55 αβγ complex was solved to 18.5 Angstroms resolution. The resolution was calculated using EOTEST algorithm of EMAN 1.9 105 . The reconstruction revealed a double ringed TF55 αβγ chaperonin complex with a two 3-fold hetero-oligomeric rings that are stacked back to back into an 18 subunit complex.

27 4.2 Materials and Methods

4.2.1 Transformation of α, β, γ plasmids into BL21 cells

The chaperonin from Sulfolobus solfataricus , TF55, is encoded by three different but closely related genes α, β and γ. The plasmids carrying α, β and γ genes were received from our collaborator Dr. Daniela Stock (VICTOR CHANG CARDIAC

RESEARCH LAB, AUSTRALIA). The plasmids are transformed into the BL21 cells using the aforementioned heat shock transformation protocol. The transformed bacterial growth was screened using respective antibiotic.

4.2.2a Purification of TF55 α subunit

1L of Luria Bertani broth in a baffled flask was inoculated with 1ml of overnight culture and induced with 1M IPTG upon reaching OD 600 =0.6, and then left shaking at

30 oC for 4 hours. The cells were harvested by centrifugation at 5000xg for 30 minutes and the cell pellet was lysed in lysis buffer (50mM Tris and 10mM EDTA) in conjunction with a treatment of hen egg white lysozyme (Sigma) and multiple freeze/thaw cycles.

The lysate was treated with porcine liver DNase (Sigma) and MgCl 2 to a final concentration of 25mM and incubated on a shaker for 30 minutes to remove all the nucleic acid. Then the sample was heated on a heat block at 70 oC for 30 minutes to denature contaminating proteins. The sample was spun down and the supernatant was collected and applied to a nickel column, followed by elution with an imidazole gradient

(20mM Tris-Cl pH7.5, 2mM MgCl 2, 1mM EDTA, 50mM NaCl and 1M Imidazole). The fractions containing purified α protein were collected and the protein was concentrated and applied to S75 size exclusion column that is pre equilibrated with buffer containing

20mM Tris-Cl pH7.5, 2mM MgCl 2, 1mM EDTA, and 150mM NaCl. This step removes

28 the Imidazole from sample and further purifies the protein from other contaminants. The sample homogeneity was determined by SDS PAGE and concentration was determined with BCA assay. The samples were stored at the final concentration of 1mg/ml in 50 uL aliquots at -80°C.

4.2.2b Purification of TF55 β subunit

The β protein expression, cell lysis and DNAse treatment was identical to α protein purification protocol. However, after DNAse treatment, the lysate was spun down and the supernatant was collected and incubated with streptomycin sulfate to a final concentration of 3% at 4 oC for 30minutes to remove endogenous DNA. Then sample was then spun down and the supernatant was then incubated with ammonium sulfate to a final concentration of 40% at 4 oC for 30minutes .The precipitated protein was then re-suspended in a buffer containing 20mM Tris-Cl pH7.5, 2mM MgCl 2, 1mM

EDTA, and 150mM NaCl (Buffer A) and heated in a heat block at 80 oC for 30minutes to denature contaminating proteins. The sample was spun down and the supernatant was applied to Superdex75 size exclusion column that was equilibrated with buffer A. The fractions containing β protein were pooled and applied onto MONO-Q anion exchange column. The β protein was cleared of impurities through MONO-Q and the fractions corresponding to β protein were concentrated. The sample homogeneity was determined by SDS PAGE and the concentration was determined using BCA assay.

The samples were stored at the final concentration of 0.5mg/ml in 50 uL aliquots at -

80°C

29 4.2.2c Purification of TF55 γ subunit

After ammonium sulfate precipitation, the sample was heated on a heat block at

60 oC for 30 minutes to denature contaminating proteins. The sample was then spun down and the supernatant was applied to a Superdex75 size exclusion column that was equilibrated with buffer A. The fractions containing protein of interest were pooled and applied onto MONO-Q anion exchange column. The γ protein was further cleared of impurities through the MONO-Q and the fractions corresponding to desired protein were collected and concentrated. The homogeneity and the concentration of γ protein were determined by SDS-PAGE (10%) and BCA respectively. The samples were stored at the final concentration of 0.8mg/ml in 50uL aliquots at -80 oC.

4.2.3 TF55 αβγ Complex formation

For formation of TF55 αβγ complex, an equimolar concentrations of the γ protein first, then subunit α, followed by subunit β, were added in an attempt to avoid α/β complex forming preferentially. To make the complex, 25mM of MgCl 2 and 5mM ATP were sequentially added to the solution containing the protein. The complex is formed only in the presence of ATP. The formed complex was separated from monomers using the superose6 size exclusion column. The fraction containing the complex was analyzed for homogeneity by SDS-PAGE and concentration was determined by using

BCA assay. The sample concentration was found to be 0.5mg/ml.

4.2.4 Cryo-electron microscopy

The data set of the TF55 αβγ complex was collected using the JEM 3200 FS microscope (JEOL, Japan) operating at an accelerating voltage of 300kV by

30 Dr. Ricardo Bernal & Dr. Sudheer Molugu. Images were collected using US4000 4kx4k camera at 65364x magnification. Individual TF55 chaperonin particles were interactively extracted as 160×160 pixel fields from the digital micrographs using the Interactive

Boxing algorithm of the EMAN2 software package 112 . Full CTF correction was done using the ctfit algorithm of the EMAN software package 105 . A preliminary set of 32000 particles were picked from 160 micrographs taken at a defocus range between -2.0 to -

4.0 µm under focus.The particles were then grouped into reference free 2D class averages using the refine2d.py program of EMAN software package 105 .The program refine2d.py applies multivariate statistical analysis follwed by K-means classification of the raw particles 105 . The process was repeated for 9 iterations and a total of 200 class averages were generated ,each containing upto 50 similar particle images. The class averages with top views and circular views are handpicked separately and used for making two different 3-Dimensional initial models by using the program make3d . By creating two sub-directories, one with each initial model several cycles of refinements were performed. The final resolution of the TF55 chaperonin was estimated using

Fourier shell correlation (FSC) curve and criterion of 0.5 105 .

4.3 Results and Discussion

4.3.1 Chaperonin purification

All the three subunits α, β and γ subunits were expressed individually and purified to homogeneity (see figures 4.1a, b, and c) according to the procedures explained under materials& methods. The TF55 chaperonin complex was formed by mixing equimolar amounts of α, β and γ in the presence of ATP and MgCl 2 and was

31 further purified to separate the complex from monomers using size exclusion chromatography(see figure 4.1d).

Figure 4.1: SDS PAGE gels showing the recombinant TF55 α, β and γ chaperonins purified separately and the purified TF55 αβγ complex . a.Lane 1 showing TF55 α chaperonin with a molecular weight of 59.6kilo Daltons Lane 2 showing the ladder with different molecular weights in kilo Daltons. b.Lane 1 showing TF55 β chaperonin with a molecular weight of 59.9 kilo Daltons Lane 2 showing the ladder with different molecular weights in kilo Daltons. c.Lane 1 showing TF55 γ chaperonin with a molecular weight of 59.3kilo Daltons Lane 2 showing the ladder with different molecular weights in kilo Daltons. d.Lane 1 showing the ladder with different molecular weights in kilo Daltons Lane 2 showing TF55 αβγ chaperonins. 4.3.2 Cryo-EM reconstruction TF55 chaperonin complex

The data collection and image processing was performed according to the procedures explained under materials and methods. Initially cryo-EM images showed filaments of chaperonin; we observed end-to-end chains of chaperonins

(See figure 4.2a). It was previously shown that presence of Mg +2 the chaperonin forms chains. The reason for aggregation of protein by Mg+2 is that the divalent ion is able to

32 crosslink proteins via ionic interactions. When an equimolar amount of EDTA was added to the sample, it chelated excess Mg 2+ ions and broke the chains into individual chaperonin molecules (See figure 4.2b).

The CTF corrected particles were grouped into 200 reference-free class averages (see figure 4.2c). Out of 32000 particles, 8000 particles were used in the

18.5Å resolution final reconstruction (see figure 4.3a).

Figure 4.2 Cryo-EM data collection and image processing of TF55 chaperonin complex. a. An example of cryo-EM image of TF55 chaperonin complex in the absence of EDTA. The image is taken at 5000x magnification. The micrograph displays chains of chaperonin complexes. b. An example of cryo-EM image of TF55 chaperonin complex in the presence of EDTA. The image is taken at 5000x magnification. The micrograph displays different orientations of individual chaperonin particles. c. Top and side class average views of TF55 chaperonin obtained by averaging 100 similar oriented particles for each view .Left column represents the projections and right column represents the class averages. The box size of each class average is 160×160 Å.

Figure 4.3: The TF55 chaperonin complex reconstructions showing single and double rings a. The end and side view of double ring TF55 chaperonin showing D3 symmetry and is open on both the ends. The chaperonin is about 175 Å in height and 160 Å in diameter. Each subunit is directly interacting with its symmetry related subunit across the ring. b. The apical end, side view and equatorial end of single ring TF55 chaperonin showing C3 symmetry. The chaperonin single ring is open on apical end and closed on equatorial end.

Figure 4.4: 18.5Angstroms resolution of TF55 chaperonin at 0.5 FSC cut off value The TF55 chaperonin in the ATP bound state is at 18.5 Å resolution at 0.5 FSC cutoff value

34 4.3.3 Structural Features observed in TF55 chaperonin complex

From the reconstruction, it was observed that TF55 chaperonin forms a complex of 18 subunits consisting of two rings stacked back to back and exhibiting three fold symmetry. Each ring consists of three hetero-trimers of subunits α, β and γ alternating in the ring. The complete chaperonin complex is about 175 Å in height and 160 Å in diameter.

The cryo-EM images showed many end-views, but few side views, perhaps because of the hydrophobic properties of the apical domain extensions which favor the air-solvent interface. An initial model was developed from a set of class averages and was used for refining the data. After several cycles of refinement, the reconstruction did not reveal any structural features and did not improve. Therefore, we used a multirefine algorithm that computationally separated the chaperonin particles into two different data sets resulting in two different reconstructions; one with C3 and one with D3 symmetry.

The two structures were refined for several cycles and the resolution improved substantially.

In the double ring conformation, the TF55 chaperonin complex looks like a cylindrical, “barrel-like” structure with D3 symmetry, consisting of two back to back identical rings (see figure 4.3a) and appears to be closed on both the ends.

Furthermore, each subunit of each ring interacts directly with its symmetry related subunit directly across and on the opposite ring. In the single ring reconstruction of the

TF55 chaperonin with C3 symmetry, the apical end is closed (see figure 4.3b).

However, we do not know if the single ring conformation is an off-pathway intermediate

(artifact) and further investigation is required to know its physiological importance.

35 4.3.4 Atomic Structure Fitting

A single β subunit of the crystal structure of TF55 all β complex was fit into the final reconstruction of TF55 αβγ complex using the program chimera 113 . Docking the crystal structure of single β subunit into the cryo-EM density of TF55 αβγ complex exhibited a probable β subunit location in the trimer. As shown in the figure 4.4, the apical domain of the β subunit fits fairly well into the cryo-EM density. However, the β coordinates could not fit into the alpha and gamma subunits of the cryo-EM density.

Figure 4.4: X-ray coordinate fitting of β subunit into TF55 αβγ chaperonin complex. A single β subunit of TF55 all β crystal structure fitted into the probable β subunit of cryo-EM density of TF55 αβγ chaperonin complex. 4.3.5 Physiological importance of TF55 chaperonins in Sulfolobus solfataricus

36 The TF55 chaperonin from Sulfolobus solfataricus has the ability for form different complexes at different temperatures. The TF55 all β complex formed at the heat shock temperatures of 85 oC, the αβ complex at normal growth temperature of 75 oC and αβγ complex at cold shock temperatures ≤ 60 oC. We propose a novel protein folding mechanism that highlights the necessity of these chaperonins for the survival of the bacterium at different stress conditions. Formation of different complexes at different temperatures indicates the specificity for the substrates that are vulnerable to particular temperature fluctuations in the environment. The temperature dependent subunit interactions between α, β and γ subunits highlights its physiological importance in folding a broad spectrum of proteins. This scenario could possibly help the bacterium to overcome the substrate specificity of group II chaperonins that limits the number of proteins or the type of proteins it could fold.

It was previously shown that the TF55 chaperonin all β was able to rescue several enzymes like ME (malic enzyme), GDH (glutamate dehydrogenase), ADH

(alcohol dehydrogenase) against denaturation when incubated under high temperature conditions (90 oC). These substrate proteins were folded in-vitro by the TF55 chaperonin back into native enzymes with catalytic activity 114 . Furthermore, the chaperonin TF55 αβ was also able to prevent the aggregation of lysozyme at 70 oC and refolded it into a functionally active state114 . However, further investigation is required in order to know the functional importance of TF55 αβγ complex.

37 Conclusions

The present work contributes to the structural investigation of the chaperonin complex TF55 αβγ from archaea bacterium Sulfolobus solfataricus . The TF55 chaperonin is the only group II chaperonin that is catalytically active when swapping subunits α, β and γ depending on temperature. Our structural studies with cryo-EM confirmed the complex formation of α, β and γ into a double ringed 18mer with three fold dihedral symmetry.

Our conclusions include:

A. The ATP bound state of TF55 is a double ringed structure with D3 symmetry.

However, we found a single ring in-vivo assembled structure of TF55 with C3

symmetry. We do not know if the single ring conformation of TF55 is an off-

pathway intermediate (artifact). Further investigation would be necessary to

conclude the physiological importance of single ring conformation of TF55.

B. The chaperonin filaments are formed because of the presence of Mg +2 . We

conclude that the addition of equimolar concentration of EDTA would chelate

excess Mg +2 thus breaking the TF55 filaments into individual chaperonin

molecules.

C. Docking the crystal structure of single β subunit into the cryo-EM density of

TF55 αβγ complex exhibited a probable beta subunit location in the trimer.

However, the location of alpha and gamma subunit is yet to be determined.

D. We conclude that TF55 chaperonin forms different complexes at different

temperatures so as to fold a broad spectrum of proteins under different

temperature extremes.

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45 Vita

Sanjay Kumar Molugu earned his degree in Pharmacy from Jawaharlal Nehru

Technological University in 2011 with First class and Distinction. He then joined Master of Science degree in Chemistry at U.T.E.P in 2013.While pursuing his Master’s degree,

Sanjay Kumar Molugu worked as teaching Assistant for one and half years and then as

Research Assistant for half year funded by Welch Foundation

Permanent Address: 20 Headley Drive Princeton New Jersey 08540 Contact Information: 915-242-3100