CONTENTS

Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

List of figures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

List of Tables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . vi

Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . ix

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

Part I. Architecture and Characterization of a thermostable MoxR family of

AAA+ ATPase from Thermococcus kodakarensis

KOD1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

i
List of figures

Figure 1 Inferred evolutionary history of AAA+ATPases. ............................................

Figure 2. Phylogenetic tree of the MoxR AAA+ family. ..................................................

Figure 3 Thermococcus kodakaraensis KOD1 .................................................................

Figure 4 Assignment of highly conserved residues in TkMoxR protein .......................

Figure 5 Expression and purification of recombinant TkMoxR ...................................

Figure 6 Analysis TkMoxR by size exclusive chromatography....................................

Figure 7 Oligomerization of TkMoxR at different temperature ....................................

Figure 8 Electron micrograph and image processing of TkMoxR. ...............................

Figure 9 ATPase activity of purified TkMoxR protein at various pH value. ................

Figure 10 Analysis of TkMoxR with and without ATP by size exclusive chromatograph

Figure 11 The observation of TkMoxR in the presence of ATP on TEM......................

Figure 12 The binding of TkMoxR with circle and linear DNA. ..................................

Figure 13 Electrophoretic mobility shift assay of double stranded linear DNA in the prese
nce of TkMoxR at different condition...........................................................................

Figure 14 Observation of mixtures of 600 linear DNA and TkMoxR on TEM. ...........

Figure 15 A proposed assembly and functional mechanism of TkMoxR based on structura

l analysis........................................................................................................................

ii
List of tables

Table 1 Recent functional characterization of MoxR proteins ..........................................

Part I

Part II

Abbreviations

TEM Transmission electroron microscopy

2D 2-dimensional electrophoresis

SEC Size exclusion chromatography

iii
BCP Bacterioferritin comigratory protein

H2O2 Hydrogen peroxide

MDH Maleic dehydrogenase

MCO Metal-catalyzed oxidation

DNase I Deoxiribonuclease I

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

DTT Dithiothreitol

PCR Polymerase chain reaction

t-BOOH tert-Butly hydroperoxide

CHP Cumen hydroperoxide

iv
 Abstract

This study researched on correlations between the structure and function of

protein related to protective systems of organisms, and described by dividing it into 2

subjects largely.

First, The MoxR AAA+ ATPase, a member of AAA+ ATPase super family is

widespread but until now limited works were reported. The previously reported some

evidence suggest that MoxR AAA+ATPase have chaperone-like activities. However, the

structure as well as function of MoxR AAA+ ATPase are poorly understood. In this report,

the moxR gene from the hyperthermophile archaea Thermococcus kodakaraensis KOD1

was cloned. In the recently report, MoxR family of AAA+ ATPase from E.coli showed

hexameric structure, but the recombinant TkMoxR showed two different structures,

dodecamer and hexamer. TkMoxR in higher temperature (physiological condition)

showed dodecameric form, but in cold stress (room temperature) showed mostly

hexameric form and a few of dodecameric form. Also, in the presence of ATP, TkMoxR

form the higher oligomer like dodecamer and microtuble of hexamer. Finally, TkMoxR

can bound to broad ranges of dsDNA and releases dsDNA at the high temperature

suggesting that TkMoxR has DNA chaperone-like activity and decrease the gene

expression at transcription level to protect cell from the low temperature stress and

release DNA at the normal temperature. Based on these results, we suggested that

TkMoxR protein had dodecameric and hexameric form and it functioned as DNA

v
chaperones.

Second, BCP

vi
Part I. Architecture and Characterization of a thermostable

MoxR family of AAA+ ATPase from Thermococcus

kodakarensis KOD1

1. INTRODUCTION

The AAA+ATPase proteins (ATPases Associated with diverse cellular Activities)

are highly ubiquitous, found in all kingdoms of life and function as molecular chaperones,

helicase, ATPase subunits of protease or nucleic-acid-stimulated ATPase [1]. These

proteins typically assemble into hexameric ring structure and using energy from ATP

hydrolysis to change the conformation in molecular remodeling events. Members of

AAA+ ATPase superfamily contain a highly conserved AAA+ ATPase domain involved

Walker A (GxxGxGK[ST]}), Walker B (hhhhDE, h is an hydrophobic residue) and

Arginine finger that are responsible for ATP binding and hydrolysis [1-3].

Figure 1 Inferred evolutionary history of AAA+ATPases.

The figure shows several relative temporal epochs separated by the major evolutionary

transitions that mark their boundaries. The solid colored bars indicate the maximum depth

to which the AAA+ lineages can be traced with respect to these temporal epochs. The

dashed lines indicate uncertainty in terms of the exact point of origin of a lineage. The

ellipses bundle groups of lineages from which a new lineage with relatively limited

phyletic pattern could have potentially emerged via rapid divergence. Colored circles at

the terminal branches indicate broad functional categories: yellow, DNA replication and

repair; blue, transcription; pink, chaperone or protein unfolding/degradation; and white,

other specialized functions. (http://dx.doi.org/10.1016/j.jsb.2003.10.010 [1])

Among AAA+ATPase superfamily, MoxR family is belong to the helix-2 insert

clade and represented in all the major lineages of bacteria and archea [1] (Figure 1). This

family can divide into at least seven subfamilies base on phylogenetic analysis, including

MoxR Proper (MRP), TM0930, RavA, CGN, APE2220, PA2707, and YehL[2] (Figure 2).

These proteins were wide distribution but remain poorly characterized. Recently studies

proposed MoxR ATPase have chaperone-like function in multiple stress response

pathways (Table 1) [4]. For example, RL3499 from Rhizobium leguminosarum is

important for stress tolerance in the cell envelope to help cell survival under both free-

living and symbiotic conditions between the bacterium and the host pea plant [5].

FTL_0200 contributes to stress resistance, intracellular multiplication, and virulence of

Francisella tularensiss [6]. RavA prevents inhibition of the inducible lysine

decarboxylase LdcI by ppGpp and is required for the cells to respond to acid stress at the

risk of depleting lysine amounts [7]. The Acidianus two-tailed virus protein p618, a

member of RavA supfamily, functions as a molecular chaperone within virion and that it

catalyzes and facilitates assembly of protein complexes involved in extracellular tail

development [8]. And CoxD from Oligotropha carboxidovorans OM5 functions as partial

unfolding of apo-CO dehydrogenase to assist in the stepwise introduction of sulfur and

copper in the [MoO3] center of enzyme [9].

 Figure 2. Phylogenetic tree of the MoxR AAA+ family.

The tree was constructed from 596 MoxR AAA+ (COG0714) sequences as

described in the Materials and Methods. Each subfamily is represented by a distinct color.

Small subgroups within the CGN branch are labeled. (Jamie Snider , Walid A. Houry,

Journal of Structural Biology Volume 156, Issue 1 2006 200 209,

http://dx.doi.org/10.1016/j.jsb.2006.02.009)

Table 1 Recent functional characterization of MoxR proteins
Protein Organism MoxR subfamily classification Cellular/molecular functions
Cell envelope development
Cell morphology
Rhizobium leguminosarum Biov Stress tolerance
RL3499 ar viciae MRP Bacterium-host symbiosis
Acid & oxidative stress resistance
FTL_0200 Francisella tularensis MRP Bacterial pathogenesis
Prevents inhibition of the inducible lysi
ne decarboxylase LdcI
Role in acid stress & stringent response
RavA Escherichia coli K-12 RavA s
DNA binding
Possible role in extracellular viral tail d
p618 Acidianus two-tailed virus RavA evelopment
Partial unfolding of apo-CO dehydrogen
Oligotropha carboxidovorans O ase
CoxD M5 APE2220 CO dehydrogenase maturation

Thermococcus kodakaraensis KOD1 was isolated from a solfatara (a vent

which emits sulphorous gases) on Kodakara Island, Japan [10] (Figure 3). It had

previously been reported as Pyrococcus sp. KOD1, however a detailed phylogenetic tree,

made possible by the recent accumulation of 16S rRNA sequences of various species in

the order Thermococcales, indicated that strain KOD1 is a member of the genus

Thermococcus and that strain KOD1 represents a new species of Thermococcus, which

was designated as Thermococcus kodakaraensis KOD1 sp. Nov [11].

Figure 3 Thermococcus kodakaraensis KOD1

T. kodakaraensis, is one of the organisms closest to the last common ancestor of

all life, whose whole genome sequence has been reported [11, 12]. They are of scientific

interest to understand the origin of life and its early evolution.

T. kodakarensis cells are irregular cocci 12 m in diameter, often occurring in

pairs, and are highly motile by means of lophotrichous flagella (Figure 3a). The cell wall

consists of a layer of di-ether and tetra-ether lipids, and an outer glycoprotein coat.[10, 11]

T. kodakarensis is an obligate anaerobe, and a heterotroph, growing rapidly on a variety

of organic substrates in the presence of elemental sulfur, producing hydrogen sulfide gas.

The generation time is estimated to be 40 minutes under optimum conditions.[10] The

requirement for elemental sulfur is relieved when pyruvate or starch is used for growth. In

the absence of sulfur, hydrogen is produced instead of hydrogen sulfide.[11] Growth is

possible at temperature ranging from 60100 C, with an optimum at 85 C.[11] Like

other marine organisms, high salt concentrations are required for optimal growth, and cell

lysis may occur in dilute solutions.

In the present study, we purify and characterize structure, biochemical of

hypothetical protein - MoxR-related ATPase from T. kodakaraensis KOD1. Using size

exclusion chromatography and transmission electron microscope (TEM), we reveal

the architecture and characterization of TkMoxR.

2. MATERIAL AND METHODS

2.1. Microoganisms strainstrain and media

Thermococcus kodakaraensis KOD1, donated by Japan Collection of

Microoganisms, RIKEN BioResource Center, Japan. T. kodakaraensis KOD1 was

cultured in the 280 Thermococcus medium [12] before cell extraction and genomic DNA

isolation.

2.2. Cloning, expression and purification of the TkMoxR protein

DNA manipulation was performed by standard procedures, as described by

Sambrook and Russell [13]. The full-length of TkMoxR gene was amplified from T.

kodakaraensis KOD1 genomic DNA by PCR, and cloned into the pET-28a expression

vector (Novagen). The primerss (Forward primerprimer: 5- TAC CAT ATG AAG ATT

GAG GAA GTA C -3, Reverse primer: 5- TAC CTC GAG TCA ATC GAA TTT TGG

AAC CGG -3) were designed base on the T. kodakaraensis KOD1 genome sequence. The

PCR product and pET28 (a) plasmid were digested by NdeI and XhoI, and the digested

product were then ligated by DNA ligase. The ligation products were transformed into

Escherichia coli BL21 (DE3) plys heat shock transformation method. Finally, the

recombinant vector (TkMoxR-pET28a) was confrimed by DNA sequencing.

The expression of TkMoxR was induced in Escherichia coli BL21 (DE3) Plys

(Stratagene) by the addition of 1 mM isopropyl--galactopyranoside (IPTG), once

cultures reached an O.D. about 0.6 at 600 nm. After 4 h, the cells were harvested and

resuspended with lysis buffer (50 mM KH2PO4, 0.3 M KCl, 5 mM imidazole, pH 8.0).

After disruption of cells by sonication, the sample was heated at 70C for 20 min. The

heat-stable supernatant was separated by centrifugation and loaded onto a Profinity

IMAC Ni-charged resin (Bio-Rad, Hercules, CA) which was pre-equilibrated with lysis

buffer. TkMoxR eluted at 250 mM imidazole. TkMoxR was further purified by Size-

exclusion Chromatography Assay (Bio-Rad; Biologic DuoflowTM Chromatography

system) on a SuperdexTM 200 (10/300) column (GE healthcare) equilibrated with 50 mM

HEPES, 5 mM MgCl2 (pH 7.0) containing 300 mM NaCl. The purification of TkMoxR

protein was examined by SDS-PAGE according to standard procedure [14].

2.3. Size exclusion chromatography assay

The size-exclusion chromatography assays were performed using DuoflowTM

Chromatography system with SuperdexTM 200 (10/300) column (GE healthcare). The

protein sample and molecular mass standards were applied to SuperdexTM 200 (10/300)

column and eluted in HEPES buffer [50 mM HEPES, 5 mM MgCl2, 0.3M NaCl (pH 7.0)].

Thyroglobumin (699 kDa), ferritin (440 kDa), catalase (232 kDa), and ovalbumin (43

kDa) (GE healthcare) were used as standard proteins.

2.4. ATPase activity assay

ATPase activity was measured by determining free phosphate released after ATP

hydrolysis using a colorimetric assay as reported previously [15, 16]. TkMoxR was added

in buffer with 50 mM HEPES, 5 mM MgCl2, 300 mM NaCl (pH 7.0) containing various

concentrations of ATP substrate. Solutions with different pH for TkMoxR were generated

using 10 mM concentrations of citrate, acetate, MES, HEPES, or TAPS buffers. Assay

was performed in 200 L volumes at 45oC in Eppendorf tubes at different time and added

400 L of developed solution (1 mM malachite green, 8.5 mM ammonium molybdate and

1 M HCl). Color development was allowed to proceed for 1 min and then stopped by the

addition of 100 L 37% citric acid. Absorbance was measured at 660 nm and converted to

moles of phosphate produced using a KH2PO4 standard curve.

2.5. DNA-protein binding assay

A series of linear dsDNA (from 100 bp to 1500 bp) were amplified follow

Forever ladder premix personnalizer kit of Seegene. pET19b, pET28a from Novagen

were used as circle dsDNA. The DNA-protein binding assay was performed by

incubating dsDNA or ssDNA with TkMoxR in a 25 mM Tris-HCl (pH 7.4) buffer

containing 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 mM DTT at room

temperature. DNA-protein complexes were applied to glow-discharged carbon coated

copper grids. Grids were imaged in a Tecnai Transmission Electron Microscope by

dark-field or bright-field. Also, the ability of TkMoxR to bind and protect DNA was

evaluated on a 1% (w/v) agarose gel after staining with ethidium bromide.

 2.6. Electron microscopy and image processing

The purified TkMoxR was applied to glow-discharged carbon coated copper

grids. After 3 min for protein absorption, the grids were rinsed with droplets of deionized

water and stained with 2% (w/v) uranyl acetate. Electron micrographs were recorded on

CCD camera at a nominal magnification of 42000. For image processing, the SEMPER

[17] and EM [18] software packages were used. From digitized micrographs, smaller

frames of 64 64 pixels containing individual particles were extracted interactively.

These images were aligned translationally and rotationally, using standard correlation

methods [19].

3. RESULTS

3.1. TkMoxR is one member of MoxR-Proper subfamily of MoxR AAA+

family

To analysis the characterization of this protein, firstly we compared TkMoxR

amino acid sequence with proteins belong to MoxR AAA+ ATPase family. Figure 4

showed that TkMoxR has highly conserved with MoxR proper subfamily (more than 80%

sequence identity). Detail on amino acid sequence of TkMoxR, we found that TkMoxR

contains COG0714 conserved domain predicted with MoxR-like ATPase function and

locate from amino acid 1st to 315th . This region contains AAA+ domain (ATPase

Associated with a wide variety of cellular Activity), a group of ATPase belongs to the

ASCE (for additional strand, catalytic E) division of the P-loop NTPase fold. The domain

contains: Walker A motif, located from amino acid postion fron 45 to 52; Walker B motif,

located from amino acid postion from 104 to 109; and arginine finger. When compare

with the common Walker A with consensus GxxGxGK[S/T] sequence, we found that

Walker A on TkMoxR is EDLPGLAKT similar with ExxPGixKT, consensus sequence of

MRP subfamily)[2].

Figure 4 Assignment of highly conserved residues in TkMoxR protein

Sites of residues conserved in >80% of MoxR proper subfamily members are black box.

The Walker A, Walker B motif, and Argine finger are indicated with the bar above. The

origins sequences as follows: TkMoxR Thermococcus Kodakarensis KOD1 (TkMoxR);

Pyrococcus horikoshii OT3 (P.horikosh); Pyrococcus furiosus DSM 3638 (P.furiosus);

Pseudomonas aeruginosa PAO1 (P.aerugino) Pelobacter carbinolicus DSM 2380

(P.carbinol); Mycobacterium bovis AF2122/97 (M.bovis AF) from NCBI database. The

numbers at the first of each amino acid seqences line on the right-hand side refer to the

numbers of amino acid residues.

3.2. Cloning, expression and purification of the recombinant TkMoxR

protein

To clone TkMoxR gene, we utilized a PCR to amplify TkMoxR gene from T.

kodakaraensis KOD1 as described in Materials and Methods, the resulting DNA

sequence comprised an open reading frame of 954 bp, predicting a protein composed of

317 amino acid residues with a molecular mass of 35384 Da. The TkMoxR gene was

cloned into pET28a and expression in E. coli BL21 (DE3) Plys (Figure 5a). The

purification of recombinant TkMoxR was performed by Ni-NTA chromatography as

described in Materials and Methods. SDS-PAGE analysis of recombinant TkMoxR

revealed a molecular weight of approximately 37 kDa (Figure 5b).

Figure 5 Expression and purification of recombinant TkMoxR

(a) The expression of TkMoxR in E. coli BL21 (DE3) Plys. Lane M: protein

marker; Lane 1: Total proteins from non-induced cells; Lane 2-8: total proteins from

IPTG-induced cells. (b) TkMoxR was purified by Ni-NTA column: Lane M, marker; 1,

Extraction protein was heat at 70oC for 20 minutes; 2, 3, Unbound protein; 4, Elute with

30mM Imidazol; 5, Elute with 30mM Imidazol; 6,7: Elute with 250mM Imidazol.

3.3. TkMoxR showed two different forms such as dodecameric and

hexameric structure.

In order to determine structure of TkMoxR protein, the purified protein was

analyzed by size exclusive chromatography. The size exclusive chromatography showed

two peaks (Figure 6), first peak with molecular weight about 440 kDa, and the second

peak about 232 kDa. To check the homogenety of TkMoxR protein, we loaded each

fraction on native PAGE and SDS-PAGE. The result on SDS-PAGE gel showed that these

peaks were homogeneous of TkMoxR with molecular weight about 37 kDa, and the

native-PAGE gel showed the fractions from fraction at 26 to 28 appear band with

molecular weight about 440 kDa beside the same band in fraction from 29 to 31 with

molecular weight about 232 kDa (Panel in Figure 6).

In order to separate two bands, the fresh protein was analyzed immediately on

size exclusive chromatography and each fractions were loaded on native PAGE and SDS-

PAGE gel. The result showed that TkMoxR form two peaks with molecular weight about

440 kDa and 232 kDa. When analyze on native PAGE gel, the first peak also appeared

band with molecular weight about 232 kDa. These experiment was repeat at several time,

we found that the TkMoxR protein corresponding with 440 kDa was not stabled, and at

the room temperature, it usually change to form with size about 232 kDa. Base on the

analysis and theoretical calculation from TkMoxR sequence, we suggest the 232 kDa

band is hexameric oligomer and 440 kDa is dodecarmer of TkMoxR. Here we also try to

recover dodecameric structure of TkMoxR by heating TkMoxR at 85oC and checked on

native PAGE. The native PAGE gel showed the band about 440 kDa, corresponded with

dodecameric structure of TkMoxR (Figure 7).

 Figure 6 Analysis TkMoxR by size exclusive chromatography

Analysis of fresh TkMoxR (black line) and after incubate at room temperature

for 2 hour (dash line) by size exclusive chromatography. The arrows indicated the

molecular mass standard proteins. Above panel: Eluted proteins from size exclusive

chromatography on 7.5% native PAGE and 12.5% SDS PAGE

 Figure 7 Oligomerization of TkMoxR at different temperature

TkMoxR purified was incubated at 25oC and 80oC for 48 hours and subjected to

7.5% native PAGE.

In order to clasify the oligomeric structure of TkMoxR, we performed electron

microscopy (EM) analysis of TkMoxR from first peak and second peak. The electron

micrographs of the negatively stained TkMoxR from second peak showed only one kind

of TkMoxR form (Figure 8A1) while TkMoxR from first peak showed two form clearly:

the top-view and the side-view of TkMoxR (Figure 8B1). These results were the same

when we analysis TkMoxR by native PAGE. To improve the oligomer structure,

determine size and characteristic structure, we using TEM image of TkMoxR for image-

processing using EM and SEMPER program. The averages of hexameric TkMoxR

revealed a flower-shaped structure with a 6-fold symmetry and heavy stain accumulation

at its center (Figure 8 B2). The dimension of it and diameter of the center hole were

approximately 15 nm and 3 nm respectively. The averages of top-view TkMoxR from

first peak showed similar structure with hexameric form but more compact with

dimension and diameter of the center hole were about 15 nm and 3 nm. The side-view of

TkMoxR from first peak suggested it was maded from stack of 2 hexamer to form

dodecamer with the gap between two stack about 2 nm (Figure 8 A3). These images as

well as the gelfiltration analyses indicated that TkMoxR proteins showed two different

oligomer forms such as dodecamer and hexamer and hexamer assembly was main form at

the room temperature.

 Figure 8 Electron micrograph and image processing of TkMoxR.

(A) Electron micrograph and image processing of dodecameric TkMoxR. (1)

Electron micrograph of TkMoxR from first peak stained by 2% UAC. White arrow

showed top-view and black arrow showed side-view of TkMoxR. Image-processing of

TkMoxR using EM and SEMPER software showed average of top-view (2) and average

of side-view (3) of dodecameric TkMoxR. (B) Electron micrograph and image processing

of hexameric TkMoxR. (1) Electron micrograph of TkMoxR from second peak. White

arrow showed hexameric oligomer. (2) Averages of top-view of hexamer TkMoxR and (3)

Average of bottom-view hexameric TkMoxR.

3.4. TkMoxR form higher oligomer like chain in the presence of ATP

Proteins belong to AAA+ ATPase usually employ the energy obtained from ATP

hydrolysis to remodel protein [3]. To analysis these characterization, we measured the

ATPase activity and checked the oligomerization of TkMoxR at different concentration of

ATP. When measured ATPase activity, we found that TkMoxR have highest activity at the

optimal pH 7.0 and it hydolased ATP to release 19.5 mM inorganic phosphate per mg

protein per min (Figure 9). In order, TkMoxR was incubated with different concentration

of ATP from 0.1 mM to 10 mM for 10 minutes then was analyzed on 7.5% native PAGE

(Figure 10B). The result showed that TkMoxR changed the oligomer structure to form

dodecamer and higher oligomer when increasing the concentration of ATP. To improved

this result, we analyzed mixture of TkMoxR with 1mM ATP using size exclusive

chromatograph. The gelfiltration graph (Figure 10A) showed the TkMoxR form

dodecamer structure (correspond to 440 kDa) and higher oligomer structure. When

compared TkMoxR in the presence and absence of ATP using 7.5% native PAGE, the

fraction contained most of TkMoxR shift from about 232 kDa molecular weight to 440

kDa, corresponded to dodecameric structure, but when analyzed these fractions on 7.5%

native PAGE, we found that the main band about 232 kDa corresponded to hexamer, and

the other is oligomer (Figure 10C). Because the dodecameric structure was not stable and

then changed to the main form, hexamer. To observed the oligomer structure of TkMoxR

in the presence of ATP, we stained mixture diluted of TkMoxR with 1mM ATP with

2%UAC and checked on TEM. The electron micrograph showed that in the presence of

ATP, some TkMoxR stack binded together to make chain form (Figure 11).

Figure 9 ATPase activity of purified TkMoxR protein at various pH value.

(A) ATPase activity of TkMoxR at different concentration of ATP. ATPase

activity was measured by determining the free phosphate release after ATP hydrolysis

using a colorimetric assay. (B) ATPase activity of TkMoxR at different pH. TkMoxR was

dialysis with different buffer and measure ATPase activity.


 Figure 10 Analysis of TkMoxR with and without ATP by size exclusive
chromatograph

(A) Size exclusive chromatograph of TkMoxR protein after incubated with and without 1

mM ATP for 10 min at room temperature using superdexTM 200. (B) The

oligomerization of TkMoxR at different concentration of ATP. TkMoxR was incubated

with different concentration of ATP for 10 minute at room temperature then was analyzed

on native PAGE 7.5% (C) The analysis of TkMoxR with and without ATP after

gelfitration on Native and SDS-PAGE

Figure 11 The observation of TkMoxR in the presence of ATP on TEM

TkMoxR was incubate 10 min with ATP and was prepared for TEM. The yellow boxs

show the binding of TkMoxR stack together to form a chain, the red circles show the

hexamer TkMoxR.

 3.5. TkMoxR binds to double strand DNA and exposes DNA-chaperone

activity

The previous works reported that AAA+proteins are involved in a wide variety

of different functions in which the energy extracted from ATP hydorlysis is used in

molecular remodeling events. They include the helicase involved in DNA repilcation,

metal chelatases [1]. We therefore investigated whether TkMoxR interacts with DNA

through electrophoretic mobility shift assay (Figure 12, Figure 13) and TEM (Figure 14).

The TkMoxR was incubated with different kinds of DNA at different condition and the

mixtures of protein and DNA were analyzed by agarose gel. The results showed that

TkMoxR only can bind to dsDNA, both of circle dsDNA and linear DNA (Figure 12). To

check whether the length of DNA affect to TkMoxR binding ability, we synthesized a

series (10-80 bp, 100 1500 bp) of polynucleotide and incubated them with TkMoxR for

check the DNA-protein interaction. We found that TkMoxR only can bind to larger DNA

(longer than about 100 bp). The mixtures of TkMoxR and DNA also were incubated at

different temperature in the absence or presence of ATP and at different time and then

analyzed on EMSA (Figure 13a). These results showed that TkMoxR bind to DNA at

lower temperature (4oC and 25oC) and but at the optimal growth temperature (85oC)

TkMoxR bound to DNA and after certainly time, the DNA from DNA-protein complex

was released and appear the DNA band similar with DNA control on agarose gel (Figure

13b). In improve it more clearly, we incubated the mixture of DNA and TkMoxR at 85oC

in the presence of ATP and at the interval time, aliquot was checked on agarose gel. The

results showed that TkMoxR bound to DNA and then released after 10 minutes with DNA

band was observed to be higher than the control suggested DNA were released.

Figure 12 The binding of TkMoxR with circle and linear DNA.

TkMoxR bind to plasmid DNA (a) and dsDNA 600 bp (b) at different ratio DNA:

TkMoxR. DNA were incubated with 30 minutes at different mole ratio [DNA]/[Protein]

(Lane 1 to 8: 1:0; 1:0.0625; 1:0.125; 1:0.25; 1:0.5; 1:1; 1:2, 1:4; 1:8) in the presence of 5

mM MgCl2, 1 mM ATP at 25oC

Figure 13 Electrophoretic mobility shift assay of double stranded linear DNA in the
presence of TkMoxR at different condition.

(a) TkMoxR bind to dsDNA 600 bp at different temperature. DNA was incubate with

TkMoxR at diffrent temperature in 10 minutes in the presence or absence of 1 mM ATP.

(b) at 85oC with interver time.

To observation the complex of DNA and TkMoxR by TEM, the mixtures of

DNA-TkMoxR were incubated at 85oC for 1, 10, 15, and 20 minutes in the presence of 1

mM ATP and stained by 2%UAC and observed on TEM. The electron micrograph

showed TkMoxR bound to DNA and made cluster of mixture protein-DNA at the early

time (Figure 14). We also used single strand DNA (10 80 bp), and single strand plasmid

DNA (M13mp18) for agarose electrophoresis to investigate the interaction of TkMoxR

with these DNA but TkMoxR did not binding (data not show).

Figure 14 Observation of mixtures of 600 linear DNA and TkMoxR on TEM.

The mixture of DNA and protein were incubated at 85oC at different time and were

negative-stained by 2% UAC

4. DISCUSSION

In this study, we showed that TK1846 gene from T. kodakarensis KOD1

encoded for TkMoxR protein, MoxR-like AAA family ATPase belong to MoxR proper

subfamily of MoxR AAA+ ATPase family. We also described the structure and function

of TkMoxR of hyperthermophilic origin.

TkMoxR showed dodecameric structure in the phishiological condition and

almost hexameric structure in low temperature. Our analysis of TkMoxR by size

exclusive chromatography reveals that it have dodecameric and hexameric structure and

mostly TkMoxR become to hexamer form in the low temperature stress. Electron

micrograph also showed that almost TkMoxR from second peak were hexamer (Figure 6)

and TkMoxR from first peak appear top-view and side-view corresponded with

dodecameric structure. Using EM and SEMPER software for image-processing, we found

that TkMoxR hexamer had different structure corresponded with top-view and bottom-

view; and have a litter different when compare with top-view of dodecamer.

TkMoxR bind to double strand DNA at the low temperature stress. DNA-protein

binding data showed (Figure 12) that TkMoxR can bind both linear DNA and circle DNA;

and bind stronger at the room temperature. It also showed that TkMoxR bind to DNA at

the early time and then release DNA as DNA control in the physiological temperature.

When compare the interaction of TkMoxR with DNA, the data showed that TkMoxR

binding DNA stronger in the absence of ATP. In the presence of ATP, TkMoxR exposed

ATPase activity and conformation change to higher oligomer (Figure 9, Figure 10).

With these data, we proposed a model of TkMoxR (Figure 15), its assemble and

functional mechanism during the procedure. At the physiological condition, TkMoxR

appears as dodecamer form in the cytoplasm. When environment temperature decrease,

dodecarmer TkMoxR change to hexamer form and bind to DNA compactly, so the gene

expression at transcription level is decrease. In the presence of ATP at the optimal

temperature growth, TkMoxR hydrolysis ATP and change structure to form dodecamer

structure. In the in-vitro condition, at the high concenctration of ATP, some TkMoxR

protein change structure and form chain of protein.

Figure 15 A proposed assembly and functional mechanism of TkMoxR based on

structural analysis


5. REFERENCES

[1] J. Snider, G. Thibault, W.A. Houry, The AAA+ superfamily of functionally diverse

proteins, Genome biology, 9 (2008) 216.

[2] T. Fukui, H. Atomi, T. Kanai, R. Matsumi, S. Fujiwara, T. Imanaka, Complete

genome sequence of the hyperthermophilic archaeon Thermococcus

kodakaraensis KOD1 and comparison with Pyrococcus genomes, Genome Res, 15

(2005) 352-363.

[3] J.S.a.D.W. Russell, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor

Laboratory, Cold Spring Harbor, N.Y, New York, 2001.

[4] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head

of bacteriophage T4, Nature, 227 (1970) 680-685.

[5] J. Snider, I. Gutsche, M. Lin, S. Baby, B. Cox, G. Butland, J. Greenblatt, A. Emili,

W.A. Houry, Formation of a distinctive complex between the inducible bacterial

lysine decarboxylase and a novel AAA+ ATPase, The Journal of biological

chemistry, 281 (2006) 1532-1546.

[6] P.A. Lanzetta, L.J. Alvarez, P.S. Reinach, O.A. Candia, An improved assay for

nanomole amounts of inorganic phosphate, Analytical Biochemistry, 100 (1979) 95-

97.

[7] W.O. Saxton, T.J. Pitt, M. Horner, Digital image processing: The Semper system,

Ultramicroscopy, 4 (1979) 343-353.

[8] W. Baumeister, B. Dahlmann, R. Hegerl, F. Kopp, L. Kuehn, G. Pfeifer, Electron

microscopy and image analysis of the multicatalytic proteinase, FEBS Lett, 241

(1988) 239-245.

[9] M. van Heel, J. Frank, Use of multivariate statistics in analysing the images of

biological macromolecules, Ultramicroscopy, 6 (1981) 187-194.

[10] K.I. Kim, G.W. Cheong, S.C. Park, J.S. Ha, K.M. Woo, S.J. Choi, C.H. Chung,

Heptameric ring structure of the heat-shock protein ClpB, a protein-activated

ATPase in Escherichia coli, J Mol Biol, 303 (2000) 655-666.

[11] J. Snider, W.A. Houry, MoxR AAA+ ATPases: a novel family of molecular

chaperones?, Journal of structural biology, 156 (2006) 200-209.

Part II. Characterization of a thermostable peroxidase BCP from

Thermococcus kodakarensis KOD1

1. INTRODUCTION

2. MATERIAL AND METHODS

2.1. Microoganisms strainstrain and media

Thermococcus kodakaraensis KOD1, donated by Japan Collection of

Microoganisms, RIKEN BioResource Center, Japan. T. kodakaraensis KOD1 was

cultured in the 280 Thermococcus medium [12] before cell extraction and genomic DNA

isolation.

2.2. Cloning, expression and purification of the TkBCP protein

DNA manipulation was performed by standard procedures, as described by

Sambrook and Russell [13]. The full-length of TkMoxR gene was amplified from T.

kodakaraensis KOD1 genomic DNA by PCR, and cloned into the pET-28a expression

vector (Novagen). The expression of TkMoxR was induced in Escherichia coli BL21

(DE3) Plys (Stratagene) by the addition of 1 mM isopropyl--galactopyranoside (IPTG),

once cultures reached an O.D. about 0.6 at 600 nm. After 4 h, the cells were harvested

and resuspended with lysis buffer (50 mM KH2PO4, 0.3 M KCl, 5 mM imidazole, pH 8.0).

After disruption of cells by sonication, the sample was heated at 70C for 20 min. The

heat-stable supernatant was separated by centrifugation and loaded onto a Profinity

IMAC Ni-charged resin (Bio-Rad, Hercules, CA) which was pre-equilibrated with lysis

buffer. TkMoxR eluted at 250 mM imidazole. TkMoxR was further purified by Size-

exclusion Chromatography Assay (Bio-Rad; Biologic DuoflowTM Chromatography

system) on a SuperdexTM 200 (10/300) column (GE healthcare) equilibrated with 50 mM

HEPES, 5 mM MgCl2 (pH 7.0) containing 300 mM NaCl. The purification of TkMoxR

protein was examined by SDS-PAGE according to standard procedure[14].

2.3. Size exclusion chromatography assay

The size-exclusion chromatography assays were performed using DuoflowTM

Chromatography system with SuperdexTM 200 (10/300) column (GE healthcare). The

protein sample and molecular mass standards were applied to SuperdexTM 200 (10/300)

column and eluted in HEPES buffer (50 mM HEPES, 5 mM MgCl2, 0.3M NaCl (pH 7.0)).

Thyroglobumin (699 kDa), ferritin (440 kDa), catalase (232 kDa), and ovalbumin (43

kDa) (GE healthcare) were used as standard proteins

2.4. Assay of peroxidase (Prx) activity

Prx activity was measured by the ferrous oxidation xylenol (FOX)

method[20] and [21]. Incubation mixture (100 l of total volume) contained the following:

50 mM HEPES buffer (pH 7.4), 1 mM DTT, TkOsmC protein and various concentrations

of H2O2 and t-BOOH (tert-butyl hydroperoxide). After incubating samples at RT or 80

C, the reaction was stopped by the addition of ice-cold FOX reagent and sample

absorbance was measured at 560 nm. The amount of hydroperoxide was calculated from

the sample absorption using the molar extinction coefficient () of

2.24 105 M 1cm 1.

2.5. Aggregation assays

The aggregation of malate dehydrogenase (MDH) was monitored by measuring

the apparent absorption due to light scattering in a spectrophotometer. MDH was

incubated in 50 mM HEPES (pH 8.0) buffer at 45C or 95C with various concentrations

of TkAhpC. Thermal aggregation of the substrate was determined by monitoring the

turbidity increase at an absorbance of 340 nm.

2.6. Electron microscopy and image processing

The purified TkMoxR was applied to glow-discharged carbon coated copper

grids. After 3 min for protein absorption, the grids were rinsed with droplets of deionized

water and stained with 2% (w/v) uranyl acetate. Electron micrographs were recorded on

CCD camera at a nominal magnification of 42000. For image processing, the SEMPER

[17] and EM [18] software packages were used. From digitized micrographs, smaller

frames of 64 64 pixels containing individual particles were extracted interactively.

These images were aligned translationally and rotationally, using standard correlation

methods [19].

[1] L.M. Iyer, D.D. Leipe, E.V. Koonin, L. Aravind, Evolutionary history and higher order
classification of AAA+ ATPases, Journal of structural biology, 146 (2004) 11-31.

[2] J. Snider, W.A. Houry, MoxR AAA+ ATPases: a novel family of molecular chaperones?,
Journal of structural biology, 156 (2006) 200-209.

[3] J. Snider, G. Thibault, W.A. Houry, The AAA+ superfamily of functionally diverse proteins,
Genome biology, 9 (2008) 216.

[4] K.S. Wong, W.A. Houry, Novel structural and functional insights into the MoxR family of
AAA+ ATPases, Journal of structural biology, 179 (2012) 211-221.

[5] E.M. Vanderlinde, S.A. Magnus, D.D. Tambalo, S.F. Koval, C.K. Yost, Mutation of a
broadly conserved operon (RL3499-RL3502) from Rhizobium leguminosarum biovar viciae
causes defects in cell morphology and envelope integrity, Journal of bacteriology, 193
(2011) 2684-2694.

[6] J. Dieppedale, D. Sobral, M. Dupuis, I. Dubail, J. Klimentova, J. Stulik, G. Postic, E. Frapy,

K.L. Meibom, M. Barel, A. Charbit, Identification of a putative chaperone involved in stress
resistance and virulence in Francisella tularensis, Infection and immunity, 79 (2011) 1428-
1439.

[7] M. El Bakkouri, I. Gutsche, U. Kanjee, B. Zhao, M. Yu, G. Goret, G. Schoehn, W.P.

Burmeister, W.A. Houry, Structure of RavA MoxR AAA+ protein reveals the design
principles of a molecular cage modulating the inducible lysine decarboxylase activity,
Proceedings of the National Academy of Sciences of the United States of America, 107
(2010) 22499-22504.

[8] U. Scheele, S. Erdmann, E.J. Ungewickell, C. Felisberto-Rodrigues, M. Ortiz-Lombardia,

R.A. Garrett, Chaperone role for proteins p618 and p892 in the extracellular tail
development of Acidianus two-tailed virus, Journal of virology, 85 (2011) 4812-4821.

[9] A. Pelzmann, M. Ferner, M. Gnida, W. Meyer-Klaucke, T. Maisel, O. Meyer, The CoxD

protein of Oligotropha carboxidovorans is a predicted AAA+ ATPase chaperone involved
in the biogenesis of the CO dehydrogenase [CuSMoO2] cluster, The Journal of biological
chemistry, 284 (2009) 9578-9586.

[10] M. Morikawa, Y. Izawa, N. Rashid, T. Hoaki, T. Imanaka, Purification and

characterization of a thermostable thiol protease from a newly isolated hyperthermophilic
Pyrococcus sp, Applied and Environmental Microbiology, 60 (1994) 4559-4566.

[11] H. Atomi, T. Fukui, T. Kanai, M. Morikawa, T. Imanaka, Description of Thermococcus

kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as
Pyrococcus sp. KOD1, Archaea, 1 (2004) 263-267.

[12] T. Fukui, H. Atomi, T. Kanai, R. Matsumi, S. Fujiwara, T. Imanaka, Complete genome

sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and
comparison with Pyrococcus genomes, Genome Res, 15 (2005) 352-363.

[13] J.S.a.D.W. Russell, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y, New York, 2001.

[14] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of
bacteriophage T4, Nature, 227 (1970) 680-685.

[15] P.A. Lanzetta, L.J. Alvarez, P.S. Reinach, O.A. Candia, An improved assay for nanomole
amounts of inorganic phosphate, Analytical Biochemistry, 100 (1979) 95-97.

[16] J. Snider, I. Gutsche, M. Lin, S. Baby, B. Cox, G. Butland, J. Greenblatt, A. Emili, W.A.
Houry, Formation of a distinctive complex between the inducible bacterial lysine
decarboxylase and a novel AAA+ ATPase, The Journal of biological chemistry, 281 (2006)
1532-1546.

[17] W.O. Saxton, T.J. Pitt, M. Horner, Digital image processing: The Semper system,

Ultramicroscopy, 4 (1979) 343-353.

[18] R. Hegerl, The EM Program Package: A Platform for Image Processing in Biological
Electron Microscopy, Journal of structural biology, 116 (1996) 30-34.

[19] W. Baumeister, B. Dahlmann, R. Hegerl, F. Kopp, L. Kuehn, G. Pfeifer, Electron

microscopy and image analysis of the multicatalytic proteinase, FEBS letters, 241 (1988)
239-245.

[20] S.P. Wolff, Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for
measurement of hydroperoxides, Methods in Enzymology, 233 (1994) 182-189.

[21] J. Lesniak, W.A. Barton, D.B. Nikolov, Structural and functional characterization of the
Pseudomonas hydroperoxide resistance protein Ohr, EMBO Journal, 21 (2002) 6649-6659.