The N-terminal region of HtrA heat shock protease from Escherichia coli is essential for...

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Biochimica et Biophysica Acta 1649 (2003) 171–182

The N-terminal region of HtrA heat shock protease from

Escherichia coli is essential for stabilization of HtrA primary

structure and maintaining of its oligomeric structure

Joanna Skorko-Gloneka, Dorota Zurawaa, Fabio Tanfanib, Andrea Scireb,Alicja Wawrzynowc, Joanna Narkiewicza, Enrico Bertolid, Barbara Lipinskaa,*

aDepartment of Biochemistry, University of Gdansk, ul. Kladki 24, 80-822 Gdansk, Polandb Institute of Biochemistry, Universitr Politechnica delle Marche, Via Ranieri, Ancona, Italy

cFaculty of Sciences, Universitr Politechnica delle Marche, Via Ranieri, Ancona, Italyd International Instutute of Molecular Biology, ul. Trojdena 4, Warsaw, Poland

Received 17 January 2003; received in revised form 22 April 2003; accepted 25 April 2003

Abstract

HtrA heat shock protease is highly conserved in evolution, and in Escherichia coli, it protects the cell by degradation of proteins

denatured by heat and oxidative stress, and also degrades misfolded proteins with reduced disulfide bonds. The mature, 48-kDa HtrA

undergoes partial autocleavage with formation of two approximately 43 kDa truncated polypeptides. We showed that under reducing

conditions, the HtrA level in cells was increased and efficient autocleavage occurred, while heat shock and oxidative shock caused the

increase of HtrA level, but not the autocleavage. Purified HtrA cleaved itself during proteolysis of substrates but only under reducing

conditions. These results indicate that the autocleavage is triggered specifically by proteolysis under reducing conditions, and is a

physiological process occurring in cells. Conformations of reduced and oxidized forms of HtrA differed as judged by SDS-PAGE,

indicating presence of a disulfide bridge in native protein. HtrA mutant protein lacking Cys57 and Cys69 was autocleaved even without the

reducing agents, which indicates that the cysteines present in the N-terminal region are necessary for stabilization of HtrA peptide.

Autocleavage caused the native, hexameric HtrA molecules dissociate into monomers that were still proteolytically active. This shows that

the N-terminal part of HtrA is essential for maintaining quaternary structure of HtrA.

D 2003 Elsevier B.V. All rights reserved.

Keywords: HtrA protease; HtrA structure; Heat shock; Autodegradation

1. Introduction and Wren [3] and by Clausen et al. [4]). HtrA degrades

In response to stress such as heat shock, cells react by

elevated expression of chaperone proteins and proteases.

These proteins protect cellular proteins against denatura-

tion, facilitate proper folding and degrade irreversibly

denatured proteins, whose accumulation in cell might be

toxic [1,2].

HtrA (DegP) protein of Escherichia coli is a heat-

shock-induced serine protease, localized on the periplasmic

side of the inner membrane and indispensable for bacterial

survival at temperatures above 42 jC (reviewed by Pallen

1570-9639/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S1570-9639(03)00170-5

* Corresponding author. Tel.: +48-58-3059278; fax: +48-58-3010072.

E-mail address: [email protected] (B. Lipinska).

abnormally folded proteins formed in periplasmic space at

elevated temperatures [5,6] and at lower temperatures, it

has a chaperone function [7]. It has been shown that HtrA

participates in cellular defense against oxidative stress, by

degradation of oxidatively damaged proteins localized in

the cell envelope, especially those associated with the

membranes [8]. HtrA is also involved in the removal of

proteins lacking proper disulfide bonds [5,6,9]. Bacterial

HtrA, apart from protection against thermal and oxidative

stress, has been implicated in virulence of pathogenic

bacteria (reviewed by Pallen and Wren [3] and Clausen

et al. [4]).

HtrA is part of a large family of related serine proteases,

members of which are found in most organisms, including

humans. To date, at least four human HtrA homologs have

J. Skorko-Glonek et al. / Biochimica et Biophysica Acta 1649 (2003) 171–182172

been identified [4] and there is a strong evidence that they

are involved in a cellular stress response, including heat

shock or inflammation processes [10–12]. Recently, evi-

dence emerged suggesting that hHtrA1 may function as a

tumor suppressor protein [13] and there is an increasing set

of reports indicating that hHtrA2 plays a regulatory role in

apoptosis (reviewed by Clausen et al. [4]). The HtrA gene is

highly conserved among mammalian species: the amino

acid sequences encoded by HtrA cDNA clones from cow,

rabbit, and guinea pig are 98% identical to human HtrA

[10]. Since there is a high homology between the E. coli and

human HtrA proteases (more than 40% identity with the

major domain of humHtrA1) [10] and both function in stress

response, research concerning the E. coli version of HtrA

may help to understand structure, function and physiological

role of the human protein(s).

E. coli HtrA is synthesized as a 50-kDa preprotein from

which a signal peptide of 26 amino acids is removed, most

probably by signal peptidase [14,15]. The mature HtrA is a

48-kDa protein consisting of 448 residues of which His105,

Asp135 and Ser210 form the catalytic triad residues [3,16].

It has been shown previously in our laboratory [16] and

others [5] that the mature HtrA undergoes partial degra-

dation. Preparations of purified HtrA contained, in addition

to the mature 48-kDa protein, two major degradation

products of approximately 43 kDa, arising in consequence

of cutting occurring after Cys69 or after Gln82 of the

mature protein. Since the preparations of mutant, proteo-

lytically inactive HtrA proteins, did not contain the deg-

radation products, we assumed that the degradation was an

autocatalytic process. Moreover, the autocleavage was not

observed in purified preparations of the wild-type HtrA,

even upon prolonged incubation, which suggested that

some other cellular factor(s) may participate in this process

[16]. A similar phenomenon of self-degradation has been

reported in the case of the cloned human HtrA1 homolog.

hHtrA1 expressed in the in vitro transcription/translation

system and in heterologous systems exhibited autocatalytic

cleavage [10]. Gray et al. [11] demonstrated that the

cloned wild-type human HtrA2 expressed in baculovirus

system had molecular weight of 41 kDa, while HtrA2 with

the putative active site serine mutation gave rise to a 58-

kDa product. It has been shown recently that cleavage of

the first 133 amino acids of hHtrA2 occurred during

import of this protein to mitochondria, resulting in a

release of pro-apoptotic hHtrA2 into mitochondrial inter-

membrane space [4].

Recently, the crystal structure of HtrA has been resolved

[4,17]. According to the published model, HtrA is a hex-

americ molecule formed by staggered association of two

trimeric rings. This association results in formation of a

molecular cage with proteolytic sites located on the inner

wall of the central cavity. The two trimers are mainly

connected by the long LA loops, located in the N-terminal

parts of the HtrA polypeptides. The LA loops of opposing

trimeric rings are wound around each other, forming three

corner pillars of the central cavity of protein. Additionally,

the hexamer structure is stabilized by interaction of PDZ1

and PDZ2 domains, localized in C-terminal parts of poly-

peptides, with their symmetry mates. Thus, the HtrA hex-

amer might be considered as a relatively loose bound dimer

of two trimers.

Considering this model, the HtrA autocleavage cuts

occur close to the C-terminal end of the LA loop. Theoret-

ically, such cleavage should destabilize the quaternary

structure of HtrA. However, the 3D structure of the entire

C-terminal region of the LA loop has not been determined

by structural analysis.

The aim of this work was to study the mechanism of

HtrA autocleavage resulting in removal of the N-terminal

fragment and its impact on HtrA structure. We show here

that the autocleavage occurs during proteolysis under

reducing conditions and two cysteines (C57 and C69)

are involved in this process, most probably by reduction

of a disulfide bridge, and that the autocleavage leads to

dissociation of the HtrA oligomer.

2. Materials and methods

2.1. Bacteria and plasmids

The strains and plasmids used are listed in Table 1.

2.2. Chemicals

Anti-HtrA polyclonal rabbit antibodies were purified on

the affinity column (HtrA bound to Sepharose 4B, Phar-

macia). Goat anti-rabbit immunoglobulins conjugated with

alkaline phosphatase were purchased from Promega.

Nitrocellulose type BA 83 0.2 Am for immunoblotting

was obtained from Schleicher & Schuell. The Ni-NTA

agarose was from Qiagen. Carboxymethyl dextran (CMD)

dual-well cuvettes and the suitable coupling kit, consisting

of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)

and of N-hydroxysuccinimide (NHS), were from Affinity

Sensors.

The other chemicals were purchased from Sigma or

Fluka and were of the highest quality.

2.3. Protein purification

The HtrA full-length protein and the HtrAS210A were

purified basically as described previously [21]. The only

modification was the lack of DTT in the buffer for opening

the cells, which prevented the autodegradation of HtrA.

The truncated HtrA (S-HtrA) was obtained as follows.

Approximately 5 mg of purified HtrA was dialyzed over-

night against 50 mM Tris/HCl (pH = 8.0) buffer. Then,

HtrA was incubated with 50 mg of lysozyme in the

presence of 2 mM DTT for 60 min at 37 jC. The mixture

was dialyzed overnight against buffer B (50 mM imida-

Table 1

Strains and plasmids

Relevant characteristics Reference or source

Strains

B178 W3110 galE sup+ our collection [14]

BL20 B178 htrA:miniTn10 TetR our collection [14]

K38 (pGP1-2) HfrC(k), T7 polymerase, KanR [18,19]

Plasmids

High copy number

pT7-5 T7 expression vector [19]

pQE60 T4 expression vector, C-terminal His6 tag Qiagen, Germany

pJS13 pT7-5 carrying the wild-type htrA gene [16]

pJS14 same as above but with Ser210!Ala substitution [16]

pTA3 pQE60 carrying htrA gene with substitutions

Cys57! Ser, Cys69! Ser

M. Ehrmann

pJS18 pQE60 carrying the wild-type htrA gene this work

pJS20 same as pTA3 but with Ser210!Ala substitution this work

Low copy number

pGB2 general cloning vector [20]

pJS7 pGB2 carrying the wild-type htrA this work

pJS21 pGB2 carrying htrA gene with substitutions

Cys57! Ser, Cys69! Ser

this work

J. Skorko-Glonek et al. / Biochimica et Biophysica Acta 1649 (2003) 171–182 173

zole, pH = 6.8, 10% glycerol, 10 mM h-mercaptoethanol,

0.1 mM EDTA) and then applied to a Bio-Rex 70 (Bio-

Rad) column (20 ml of resin). The column was washed

with 5 volumes of buffer B and then S-HtrA was eluted

with a linear gradient of 0–1 M KCl in buffer B (400 ml).

Fractions containing truncated HtrA were concentrated

using Centricon 30 microconcentrators (Amicon) and fro-

zen in liquid nitrogen.

2.4. Electrophoresis of proteins and Western blotting

Proteins were analyzed by sodium dodecyl sulfate-poly-

acrylamide gel electrophoresis (SDS-PAGE) as described by

Laemmli [22]. Gels containing 10%, 12.5% or 15% (w/v)

acrylamide were used. In some cases (as indicated in the

text), nonreducing conditions were applied and h-mercap-

toethanol was omitted from the system. Native electropho-

resis was performed in 10% polyacrylamide gel, pH = 4.0 as

described by Goldenberg [23]. Western blotting was per-

formed as described in Ref. [24].

2.5. Protein assay

Protein concentration was estimated by staining with

Amido Black and spectrophotometric measurement as

described before [21].

2.6. HtrA autocleavage in vitro

Reaction mixtures (250 Al) containing 10 Ag of HtrA and

100 Ag of lysozyme, h-casein or BSA in 25 mM HEPES

(pH= 8.0) buffer with or without 1.5 mM DTT were incu-

bated at 37 jC. In some cases, DTT (as stated in the text) was

substituted with h-mercaptoethanol at concentration 50 or

100 mM, or by 30 mM glutathione (reduced form).

At indicated times, 25 Al samples were taken and placed

in 2� concentrated SDS-PAGE lysis buffer. The samples

were then resolved by 15% SDS-PAGE and the gels were

stained with Coomassie Brilliant Blue.

2.7. Autocleavage of HtrA in vivo

E. coli B178 cells and E. coli BL20 htrA� transformed

with high or low copy number plasmids carrying mutant

htrA C57S C69S (D-Cys htrA) or wild type htrA (wt) genes

were grown at 37 jC in Luria Bertani (LB) medium [25] to

an OD595 of 0.2–0.3 and were submitted to a heat shock (45

jC) or treated with oxidizing or reducing agents. Plasmid

pJS20 carrying htrA S210A C57S C69S was used as a

negative control of proteolysis (it lacks active site serine

210). To the cultures containing pQE60 derivatives, 1 mM

IPTG was added to induce HtrA synthesis. DTT was added

to a final concentration 10 mM, ferrous sulfate 200 AM, h-mercaptoethanol 50 mM. At the indicated times, samples

containing aliquots of cells were taken, centrifuged and cells

were resuspended in SDS-PAGE lysis buffer. Following

SDS-PAGE, the presence of full-length and truncated HtrA

was detected using Western analysis.

2.8. Redox properties of HtrA

The in vivo redox state of HtrA was assayed by

trapping the free thiols by iodoacetamide (IAA) essentially

as described by Jakob et al. [26].

The in vitro redox state of purified HtrA was determined

according to the method described by Hermanson [27]: 2

Fig. 1. Induction and autocleavage of HtrA protease in vivo. E. coli B178

was grown in LB medium at 30 jC to an OD595 of 0.2–0.3 and then

submitted to a heat shock or treated with various reducing or oxidizing

agents. Extracts from equal numbers of cells were resolved by SDS-PAGE

(10% gel) and subjected to Western analysis using anti-HtrA serum. The

lanes show HtrA protein levels of control cells grown at 30 jC (lanes 1 and

3), cells heat-shocked for 45 min at 45 jC (lane 2) and cells treated for 30

min with 50 mM h-mercaptoethanol (lane 4), 0.3 mM ferrous sulfate (lane

5) and 10 mM dithiothreitol (DTT) (lane 6). The arrows indicate positions

of the full-length mature HtrA protein (FL-HtrA) and the truncated HtrA (S-

HtrA).


mg of purified HtrA was incubated in a reducing buffer (25

mM HEPES, pH = 8.0, 5 mM DTT) for 60 min at 37 jC.The reduced HtrA was washed from the excess of DTT and

concentrated on Centricon 100 microconcentrator (Milli-

pore) to concentration of approximately 10 mg/ml. The

nonreduced HtrA was concentrated in the same way. The

free sulfhydryl groups were trapped by 5-iodoacetoamido-

fluorescein (IAF). The reduced and nonreduced HtrA was

incubated in 25 mM HEPES, pH= 8.0, in the presence of 10

mM IAF for 1 h at room temperature. Samples were

subsequently electrophoresed in nonreducing conditions,

stained with Coomassie B, and electrophoretic mobilities

of the reduced and nonreduced HtrA were compared.

2.9. Size exclusion chromatography

The reacting components (100 Al) were incubated at 37

jC for 30 or 60 min in a buffer containing 25 mM HEPES

(pH = 8.0), 50 mM NaCl and 1.5 mM DTT before loading

onto a Superdex 200 HR10/30 sizing column (Pharmacia)

equilibrated with the same buffer. The chromatography was

carried out at a flow rate of 0.3 ml/min (room temperature)

using Gold HPLC system (Beckman) equipped with a diode

array detector. Elution of the proteins was monitored using

absorption at 280 nm. Fractions were collected and proteins

were visualized following SDS-PAGE and Coomassie Blue

staining. The Superdex column was calibrated with the

following BioRad molecular weight standards: bovine thy-

roglobulin (670 kDa), bovine g-globulin (158 kDa), chicken

ovalbumin (44 kDa), equine myoglobulin (17.5 kDa); and

Sigma molecular weight standards: bovine thyroglobulin

(670 kDa), apoferritin from horse spleen (443 kDa), h-amylase from sweet potato (200 kDa), yeast alcohol dehy-

drogenase (150 kDa) and BSA (66 kDa).

2.10. Binding studies with the resonant mirror biosensor

Studies on interaction of FL-HtrA or S-HtrA with

phospholipids (PLs) were carried out by using the resonant

mirror biosensor IAsys plus (Affinity Sensors). The cuv-

ettes used were of the CMD type with two wells. One well

was used to immobilize FL-HtrA or S-HtrA, while the

other well was used as a control. The immobilization

procedure was essentially that described by Ref. [28].

The carboxyl groups of CMD were activated with EDC

and NHS for 7 min. Then, the proper amount of desired

protein, dissolved in 10 mM acetate pH 5.0, was incubated

with the activated CMD for 7 min. After blocking the

activated unreacted carboxyl groups with 1 M ethanol-

amine pH 8.5, and after several washings with 25 mM

HEPES buffer, the amount of FL-HtrA covalently linked to

the CMD matrix was 6 ng/mm2, whereas the amount of S-

HtrA immobilized was 8.5 ng/mm2. All experiments were

carried out at 25 jC; 25 mM HEPES, 125 mM NaCl, pH

7.4 and 40% ethanol were used as running and regener-

ation buffer, respectively. Binding studies were carried out

by adding into the cuvette 0.5–100 AM phosphatidylgly-

cerol (PG) or cardiolipin (CL) in form of small unilamellar

vesicles (SUV), made as described in Ref. [29]. Kinetics of

the liposome–protein interaction at the surface of the

optical biosensors was analyzed using FASTfit software

supplied with the Iasys-plus instrument according to

Edwards et al. [30,31] and as described in Ref. [32].

3. Results

3.1. The autocleavage of HtrA occurs in vivo under

reducing stress conditions

The most substantial cleavage of the wild-type HtrA

with formation of the 43-kDa truncated polypeptide(s) (S-

HtrA) was observed at the early steps of purification

procedure of the protein [16]. To exclude the possibility

that the autocleavage of HtrA was only a nonphysiological

process occurring in vitro, we monitored formation of the

truncated HtrA in bacterial cells submitted to various stress

conditions (heat shock, oxidative shock induced by ferrous

ions, presence of the reducing agents). The results pre-

sented in Fig. 1 clearly showed that treatment of a culture

with the reducing agents (DTT or h-mercaptoethanol)

induced the synthesis of HtrA protein and promoted the

autocleavage process. Both the induction and cleavage

were more efficient in the presence of a stronger reducer

DTT than h-mercaptoethanol. Heat shock and oxidative

stress did cause an increase in the HtrA level but not the

autocleavage (Fig. 1, lanes 2 and 5). A trace amount of

HtrA degradation product appeared in the case of oxidative

stress, but molecular weight of this product was lower than

that observed routinely under reducing conditions or dur-

ing purification. We conclude that the autodegradation of

HtrA is a natural process occurring when a living bacterial

cell is exposed to reducing stress conditions.


3.2. The autocleavage of HtrA occurs in vitro only under

certain conditions, including reducing stress

To test the conditions of the autocleavage of the purified

HtrA protein, we incubated it at 37 jC in the presence of

various reducing agents (DTT, h-mercaptoethanol and

glutathione, reduced form) with or without a substrate.

We observed a significant level of autocleavage only during

the proteolysis of a substrate (lysozyme, h-casein) under

reducing conditions (Fig. 2A–C). It is worth noticing that

though h-casein, contrary to lysozyme, could be digested

without reducing agents, the process of proteolysis was not

accompanied by the HtrA autocleavage (Fig. 2C, lane 2).

The cleavage, to occur efficiently, needed both the proteo-

lytic action of HtrA and reducing conditions (Fig. 2C, lane

Fig. 2. Autocleavage of HtrA during proteolysis under reducing conditions in vitro

presence or absence of DTT (1.5 mM), h-mercaptoethanol (50 or 100 mM) and glu

substrate were included (C, lanes 4 and 5). Samples were withdrawn at times ind

resolved by SDS-PAGE (15% gel) and stained with Coomassie Brilliant Blue. The

the truncated HtrA (S-HtrA) and h-casein or lysozyme. In panel B, the bottom

autocleavage of HtrA at elevated temperature. HtrA was incubated at 45 jC for 90

the figure. Reaction mixtures (250 Al) contained 10 Ag of HtrA (lanes 1, 2 and 5

(pH= 8.0). Control samples ( = samples withdrawn at time 0) and samples after 90 m

with Coomassie Brilliant Blue. The arrows indicate positions of BSA, full-length

3). After 30 min at 37 jC under such conditions, approx-

imately 25% HtrA was cleaved (Fig. 2A and not shown

densitometrical results). Incubation of HtrA alone at 37 jC,even in the presence of 1.5 mM DTT for 60 min (Fig. 2C,

lane 5), or incubation of HtrA with lysozyme or h-caseinwithout a reducing agent did not result in autocleavage

(Fig. 2A, lane 2 and C, lane 2).

We observed that the speed of autocleavage was strictly

correlated with the rate of degradation of a substrate.

Increase in the rate of proteolysis (by increasing the temper-

ature) or slowing down the process (by partial aggregation

of a substrate or by lowering the pH of the reaction mixture)

resulted in proportional changes of the yield of truncated

HtrA: the faster the proteolysis, the more efficient the

autocleavage (data not shown).

. HtrA was incubated with lysozyme (A and B) or with h-casein (C) in the

tathione (reduced form, 30 mM) (GSH) at 37 jC. Control reactions withouticated in the figure (A) and after 90 min (B) or 60 min (C), proteins were

arrows indicate positions of the full-length mature HtrA protein (FL-HtrA),

fragment of the gel, containing lysozyme, was deleted. Panel D shows

min in the presence or absence of DTT (1.5 mM) and BSA, as indicated in

, 6) or HtrA and 100 Ag of BSA (lanes 3, 4 and 7, 8) in 25 mM HEPES

in incubation at 45 jC were resolved by SDS-PAGE (10 % gel) and stained

HtrA (FL-HtrA) and the truncated HtrA (S-HtrA).

Fig. 3. In vivo and in vitro redox state of HtrA. (A) In vivo: E. coli bacteria

BL20(pJS7), expressing wt HtrA and BL20(pJS21), expressing D-Cys

HtrA were grown in LB medium at 37 jC to OD595 = 0.4. One culture of

BL20(pJS7) was treated with 10 mM DTT for 15 min. Portions of 1.4 ml of

each culture were withdrawn and mixed with 0.4 ml of 0.45 M IAA in 100

mM Tris, 10 mM EDTA, pH= 9.5. The samples were incubated at 37 jCfor 2 min and the reaction was stopped with TCA (10% final

concentration). The cells were centrifuged, washed twice with ethanol

and resuspended in a nonreducing Laemmli lysis buffer. The samples were

lyzed, resolved by 10% SDS-PAGE and analyzed by Western blotting using

anti-HtrA polyclonal antibodies. (B) In vitro: Purified wt HtrA protein (200

Al, 10 mg/ml) was reduced (treated for 1 h with 5 mM DTT) and then

subjected to IAF treatment (as described in Materials and methods), along

with a nonreduced protein sample. The resulting samples were resolved by

nonreducing 10% SDS-PAGE. HtrA protein not treated with DTT/IAF was

used as a control (flanking lanes).


We noticed HtrA autodegradation when it was incubated

in the absence of any other protein at 45 jC for 90 min, with

or without DTT (Fig. 2D, lanes 5 and 6). However, when

bovine serum albumin (BSA) was present in the mixture

without the reducing agent, the autodegradation was

inhibited (Fig. 2D, lane 7). Our interpretation is that, most

probably, HtrA in a diluted solution at higher temperature is

not stable, undergoes denaturation and becomes a substrate

for itself. BSA, which without DTT is not a substrate for

HtrA (Fig. 2D, lane 7), may exert a stabilizing effect on HtrA

and prevent its denaturation and self-cleavage. Under reduc-

ing conditions at 45 jC, BSA became a substrate for HtrA,

and HtrA autocleavage occurred. This is visible in Fig. 2D,

lane 8, as a decrease in the amount of the full-length HtrA.

The formation of the truncated S-HtrA in this case was

confirmed by Western blotting (results not shown).

It may be interesting to point out that the rates of the

HtrA autocleavage in the in vivo and in vitro experiments

were comparable (see Fig. 1, lane 6 and Fig. 2A, lane 5).

3.3. In vivo and in vitro redox state of HtrA

As shown above, HtrA undergoes autocleavage only in

the presence of reducing agents. This fact raises the

possibility that the redox active amino acids might be

implicated in this process. Mature HtrA contains only two

cysteines, located in the N-terminal region, at the positions

57 and 69 of the protein, which presumably could form a

disulfide bridge. To test this possibility, we examined the

redox status of HtrA in vivo and in vitro. We incubated

cells or purified protein in the presence or absence of DTT

and then blocked free sulfhydryl groups to prevent their

uncontrolled reoxidation, using IAA in vivo (IAA can

penetrate cell membranes) and 5-iodoacetamidofluorescein

(IAF) in vitro. As shown in Fig. 3A, the wild-type HtrA,

expressed in cells and then reduced, had a decreased

electrophoretic mobility when compared to nonreduced

HtrA, similar to the mobility of the mutant, D-Cys HtrA.

The difference was small but significant and reproducible.

We did not expect a dramatic change in mobility, since the

disruption of the putative disulfide bridge binding two Cys

residues spaced by only 11 amino acids should not

introduce a big change in the polypeptide conformation.

Reduction of the purified HtrA resulted in a more pro-

nounced mobility shift, when compared to the nonreduced

protein (Fig. 3B), which is understandable, since binding

of two IAF molecules should increase molecular weight by

approximately 1 kDa (Mw of IAF = 515 Da). Additionally,

an in vitro test with the Ellman reagent [33] gave a

negative result confirming the lack of accessible SH

groups in HtrA molecule (data not shown). These results

indicate that the Cys57 and Cys69 form a disulfide bridge

both in vivo and in vitro. Disruption of the S–S bond

could be a reason why HtrA autocleavage occurs under

reducing conditions and vice versa. The presence of the

disulfide bridge could stabilize HtrA polypeptide.

3.4. HtrA lacking Cys residues in the N-terminal region is

less stable in vivo and in vitro

To test the possibility that the presence of the disulfide

bridge Cys57–Cys69 is important for the stability of HtrA

molecule, we monitored appearance of the truncated S-

HtrA form in strains expressing the mutated HtrA C57S

C69S, lacking both cysteines (D-Cys HtrA). We found that

in cells expressing D-Cys HtrA, the truncated S-HtrA was

produced in all tested conditions, including physiological,

whereas in strains expressing wild type HtrA, S-HtrA

appeared in bacteria grown in reducing conditions only

(Fig. 4A–C). The instability of D-Cys HtrA was observed

both in the case of a physiological level of HtrA (HtrA

expressed from a low-copy number plasmid) and an

increased level of HtrA (HtrA expressed from a high-copy

number plasmid), and was due to autocleavage, since the

D-Cys HtrA lacking catalytic Ser210 was not cleaved (Fig.

4B). This result indicates that the lack of the S–S bridge

stimulates autodegradation of HtrA molecule. This con-

clusion was further supported by an in vitro degradation

assay, in which we used h-casein as a substrate for the

wild-type and D-Cys HtrA, in the presence or absence of

DTT. We found that h-casein was cleaved by both HtrA

forms at a similar rate, but there were significant differ-

ences in the autocleavage behaviour. The wt HtrA autode-

graded in the presence of DTT only, whereas D-Cys HtrA

autodegraded also in the absence of DTT (Fig. 4D). It is

also worth mentioning that all strains grew well in the

presence of 10 mM DTT at 37 jC showing only approx-

imately 20% decrease in growth rate as compared to the

nontreated cultures (data not shown).

Fig. 4. The autocleavage of D-Cys HtrA. The E. coli BL20 cells transformed with low copy number plasmids pJS21 htrA C57S C69S (D-Cys) and pJS7 htrA

(wt) (A) or with high copy number plasmids pTA3 htrA C57S C69S (D-Cys), pJS20 htrA S210 AC57S C69S (S210A D-Cys) and pJS18 htrA (wt) (B) were

grown at 37 jC in LB medium. Extracts from equal numbers of cells were resolved by SDS-PAGE (10% gel) and subjected to Western analysis using anti-HtrA

serum. (C) The E. coli BL20 cells transformed with low copy number plasmids pJS7 (wt HtrA) and pJS21 (D-Cys HtrA) were grown at 30 jC and then

submitted to various stress conditions, as described in Materials and methods. The aliquots of cultures were withdrawn and treated as above. The lanes show

HtrA protein levels of control cells grown at 30 jC (lanes 1 and 6), cells treated for 60 min with 0.2 mM ferrous sulfate (lanes 2 and 7), 50 mM h-mercaptoethanol (lanes 3 and 8), 10 mM DTT (lanes 4 and 9) and cells shocked for 45 min at 45 jC (lanes 5 and 10). (D) Purified D-Cys HtrA and wt HtrA

were incubated with h-casein in the presence or absence of DTT (1.5 mM). At the indicated times, samples were withdrawn and proteins were resolved by

SDS-PAGE (10%).


3.5. During autocleavage, HtrA changes its oligomerization

status

Our finding that during proteolytic action under reduc-

ing conditions, the N-terminal fragment of HtrA is

cleaved, prompted us to investigate whether the N-termi-

nal region is involved in maintaining HtrA in oligomeric

form. We monitored the state of HtrA oligomerization

before and after proteolysis of a substrate under reducing

conditions, using size exclusion chromatography. The

reaction mixture containing FL-HtrA, substrate (lysozyme)

and DTT was incubated for various periods of time and

then applied onto a filtration column. The control FL-

HtrA was eluted at the position corresponding to molec-

ular mass larger than 200 kDa, but smaller than 450 kDa

(Fig. 5 and data not shown), confirming previously

published data [4,38] showing that HtrA exists mainly

in a hexameric form. Analysis of the eluted fractions

showed that HtrA, initially present only in a hexameric

form, disassembled partially during reaction as indicated

by the second elution peak at a position of approximately

44 kDa, corresponding to HtrA monomer (Fig. 5). After

30 min of reaction, approximately half of the HtrA

molecules were in a monomeric state (Fig. 5B) and after

60 min, about 60% of HtrA was monomeric (not shown).

Incubation of HtrA with DTT alone (without a substrate)

did not result in any change in oligomerization status (not

shown). To check if the formed monomers and remaining

hexamers were stable, we collected fractions containing

separated monomers and hexamers, concentrated them

and applied on the same filtration column. We found that

the monomers were eluted in the position of monomers

and the hexamers were eluted in a peak corresponding to

hexameric form of HtrA. No additional peaks were

detected (not shown). Our results indicate that during

proteolysis of a substrate under reducing conditions, HtrA

hexamers undergo irreversible dissociation to a mono-

meric form. The hexameric fraction contained FL-HtrA

and S-HtrA almost in equal amounts, while the mono-

meric fraction consisted mainly of the truncated S-HtrA

(Fig. 5C). Dissociation of HtrA oligomers during proteol-

ysis was confirmed by polyacrylamide gel electrophoresis

under nondenaturing conditions. As shown in Fig. 5D,

dissociation of HtrA occurred during proteolysis of lyso-

Fig. 5. Autocleavage causes dissociation of HtrA oligomers. The full-length HtrA protein (100 Ag) was incubated with lysozyme (500 Ag), in the presence of

1.5 mM DTT at 37 jC for 30 min and then size exclusion chromatography was performed (B). Panel A shows elution profile of full-length HtrA. The hexamer

and monomer fractions indicated in B were analyzed by SDS-PAGE (15% gel) and stained with Coomassie Brilliant Blue (C). The arrows indicate positions of

full-length HtrA (FL-HtrA) and the truncated HtrA (S-HtrA). (D) HtrA wild type and HtrAS210A mutant proteins were incubated with lysozyme at 37 jC for

60 min, with or without 1.5 mM DTT, as described in Materials and methods. Control reactions without HtrA (lane 1) or without lysozyme (lanes 2 and 3) were

included. Reaction mixtures were analyzed by native gel electrophoresis. The arrow indicates position of dissociated HtrA (probably monomers).


zyme under reducing conditions. HtrAS210A, proteolyti-

cally inactive mutant protein, did not dissociate in the

presence of lysozyme and DTT (Fig. 5D, lane 5). This

confirms that the dissociation process requires proteolysis

and not only formation of a complex of HtrA with a

substrate (such complexes are seen as the high molecular

weight bands in Fig. 5D, lane 5).

To test if proteolysis of a substrate without HtrA auto-

cleavage may lead to dissociation of oligomers, we have

analyzed HtrA after proteolysis of h-casein with and with-

out DTT, using native polyacrylamide gel electrophoresis.

HtrA dissociation was observed only in the presence of

DTT, when the autocleavage occurred, but not without DTT,

when the proteolysis but not autocleavage occurred (not

shown). The coincidence of the cleavage of the N-terminus

and dissociation of HtrA hexamers indicates that the N-

terminal part of protein participates in formation of

oligomers.

3.6. S-HtrA possesses a lower affinity for CL and PG than

FL-HtrA

It has been shown previously that HtrA is a peripheral

protein of the inner membrane and that it interacts with

phosphatidylglycerol (PG) and cardiolipin (CL) in vitro

[34]. Having found that proteolytial activity of HtrA under

reducing conditions leads to autocleavage followed by

dissociation of hexamers and formation of monomeric S-

HtrA, we set out to check if this process would change the

interaction of HtrA with phospholipids.

The interaction of PG or CL liposomes with FL-HtrA

and S-HtrA was followed in real time by the IAsys

resonant mirror biosensor. We obtained a series of asso-

ciation curves (Fig. 6) and the association profiles were

used to calculate the association (Kass) constants, the

dissociation constants (Kdiss) and KD (KD =Kdiss/Kass).

The results of calculations are summarized in Table 2.

Fig. 6. Association curves of PG or CL liposomes binding to immobilized FL-HtrA or S-HtrA. The figures reported beside each curve represent the micromolar

concentration of phospholipid added.


In the case of FL-HtrA, small differences were found in

the values of KD, Kass and Kdiss related to the interaction

of the protein with PG or CL. KD resulted slightly higher

in the presence of PG, while both Kass and Kdiss resulted

slightly lower in the presence of PG than in the presence

of CL. These results indicate that FL-HtrA possesses a

similar affinity for CL and PG. In the case of S-HtrA, the

KD value obtained in the presence of PG was 6.5 times

higher that that obtained in the presence of CL, indicating

that S-HtrA has a lower affinity for PG than for CL.

Table 2

Kinetic and equilibrium parameters for the interaction between immobilized

FL-HtrA or S-HtrA and liposomes made of PG or CL

PG CL

FL-HtrA

KD (AM) 3.37F 0.40 2.64F 0.41

Kass (M� 1 s� 1) 785.8F 53.0 1193.2F 74.9

Kdiss (s� 1)� 104 26.4 31.5

S-HtrA

KD (AM) 36.5F 10.7 5.60F 1.55

Kass (M� 1 s� 1) 10.61F 3.7 316.0F 61.8

Kdiss (s� 1)� 104 3.87 17.7

The association (Kass), dissociation (Kdiss) rate constants and the

dissociation equilibrium constants (KD) were calculated from the curves

reported in Fig. 6 as described [30–32].

Moreover, when comparing the data with those related to

FL-HtrA, it appears that S-HtrA shows a significantly

lower affinity for both phospholipids than FL-HtrA. Our

findings suggest that the autocleavage at the N-terminal

region of HtrA may lead to a weaker interaction of the

truncated form of HtrA with the inner membrane. Indeed,

we found a significant amount of S-HtrA in the fraction

of soluble cellular proteins, whereas FL-HtrA copurified

almost exclusively with the membrane fraction (data not

shown).

4. Discussion

The achievement of the proper conformation by a

protein is crucial for its activity and may have a great

impact on the molecule stability. Misfolded proteins are

usually very unstable and are rapidly degraded. The

correct folding of many exocytoplasmic proteins often

requires the formation of disulfide bridges between two

cysteine residues. In Gram-negative bacteria, disulfide

bond formation normally occurs following export into

periplasmic space, where environment is more oxidizing

than in cytoplasm [35]. The crosslink introduced by

formation of the S–S bond provides for increased protein

stability [36].


HtrA protease plays an important role in the removal of

misfolded proteins formed in bacterial envelope (composed

of the inner and outer membranes and periplasm) as the

effect of various stress conditions, including reducing

environment [4]. It has been shown that HtrA is responsible

for degradation of alkaline phosphatase lacking proper S–S

bridges in vivo [9] and its expression is strongly stimulated

by mutations disturbing native S–S bonds formation

[9,37].

The results presented in this paper confirm that HtrA

is able to cleave reduced substrates, like lysozyme and

BSA, and show that reducing environment induces its

level in the cell. Previously, increase of the HtrA level

has been demonstrated during heat shock and oxidative

stress [8].

It has been shown previously that the mature, 48-kDa

HtrA protein undergoes partial degradation by autocata-

lytic cleavage occurring after Cys69 or after Gln82, result-

ing in the truncated, approximately 43-kDa proteins (S-

HtrA) [16]. Here we found that this autodegradation took

place in cells exposed to reducing environment (DTT or

h-mercaptoethanol) but was not observed in physiological

conditions or during other stresses (heat shock, oxidative

stress). This led to conclusion that the autocleavage of

HtrA is a natural process occurring when a living bacterial

cell is exposed to reducing stress conditions. Experiments

performed with purified HtrA protein showed a significant

level of autocleavage only during proteolysis of a sub-

strate (lysozyme, h-casein) under reducing conditions, but

not during proteolysis without reducing agents or in the

presence of such agents without simultaneous proteolytic

process. The in vivo and in vitro results taken together

indicate that in a cell exposed to reducing conditions,

HtrA degrades misfolded proteins (most probably those

with reduced disulfide bonds) and in this process cleaves

itself.

The fact that autocleavage of HtrA occurred in the

presence of reducing agents only suggested the involve-

ment of the redox active amino acids. Mature HtrA

contains only two cysteines, located in the N-terminal

region, at the positions 57 and 69 of the protein, close to

the cleavage sites. The redox state of these residues has

not been estimated previously; furthermore, any structural

data describing the region of the two cysteines is absent

[17]. We found that addition of DTT caused reduction of

both cellular and purified HtrA, as judged by the differ-

ences in the electrophoretic mobility of the protein.

Furthermore, there were no accessible SH groups in

purified HtrA protein when tested with Ellman’s reagent.

These results indicate that the Cys57 and Cys69 form a

disulfide bridge both in vivo and in vitro. Since the

differences in the electrophoretic mobility of the reduced

and oxidized HtrA were not big, the cleavage of the S–S

bond probably does not cause significant changes in the

tertiary structure of protein. The reduction of the disulfide

bridge in HtrA molecule did not require high concentra-

tions of reducing agents or treatment with strong denatur-

ants, therefore we assume that the S–S bridge is well

exposed to the solvent. Disruption of this S–S bond could

be a reason why HtrA autocleavage occurs under reducing

conditions and vice versa, presence of the disulfide bridge

could stabilize HtrA polypeptide. To test this hypothesis,

we checked stability of the mutant HtrA, lacking both

cysteines (D-Cys HtrA), and showed that the autocleavage

occurred in cells under all tested conditions (not only in

reducing environment), independently of the level of D-

Cys HtrA. Analogically—in the in vitro assays—S-HtrA

was produced at the same rate in the presence or absence

of DTT. These results further confirm stabilizing role of

the Cys57 and Cys69 residues. Our finding is in agreement

with the hypothesis of Clausen et al. [4] that proteolytic

activation of HtrA requires conformational changes, in

particular disruption of the structure formed by three

loops, the LA* loop (aa 44–79) and the L1–L2 loops

participating in formation of the active site (the LA* loop

originates from a different subunit than the L1–L2 loops).

Theoretically, reduction of the disulfide bond in the LA

loop might cause such activating conformational change,

resulting in autocleavage.

Very intriguing was the observation that the autocleavage

process led to destabilization of HtrA hexamer, resulting in

formation of protein monomers. The mechanism of this

phenomenon could be explained on the basis of the recently

published crystal structure of HtrA molecule [4,17]. The

cleavage sites (after Cys69 and Gln82) are located at the C-

terminal end of a linker loop (LA loop) connecting h-strand1 (of the N-terminal region) and h-strand 2 (of proteolytic

domain). This loop, together with the PDZ domains of C-

terminus, plays an important role in connecting the trimeric

rings within the hexameric molecule of HtrA (see Introduc-

tion). The cleavage disconnecting the N-terminal part of

HtrA from the rest of the polypeptide should deprive the

hexamer of one of its binding pillars. Our results are in

agreement with the crystallography data, further confirming

the involvement of the N-terminal region in maintaining the

oligomeric structure.

Since the cleaved form of HtrA, S-HtrA, seems to

have a different quaternary structure, we wondered if this

fact might influence the localization of S-HtrA. In phys-

iological conditions, HtrA is attached to the inner mem-

brane on its periplasmic side [34]. However, we found

that following the DTT treatment, more HtrA was present

in the soluble protein fraction and approximately 50% of

S-HtrA did not copurify with the membranes (data not

shown). The biosensor affinity studies showed that S-

HtrA possessed significantly lower affinity towards the

acidic phospholipids, PG and CL, when compared to the

full-length HtrA (FL-HtrA), which led to a conclusion

that truncated HtrA may be more loosely bound to the

membranes.

To summarize the presented results, we would like to

propose a model of HtrA autocleavage. In physiological


conditions, HtrA contains a disulfide bridge in the N-

terminal region, which makes the molecule slightly more

compact. In the presence of reducing agents, the S–S bridge

is disrupted. Subsequently, degradation of a substrate leads

to autocatalytical cleavage after Cys69 and Gln82. Similar

effect is achieved during degradation of a substrate by the

D-Cys HtrA mutant lacking S–S bond. This cleavage

causes destabilization of the whole hexamer, possibly due

to disconnection of the LA loop from the remaining cata-

lytic domain. After prolonged autocleavage, truncated forms

of HtrA are released from hexamer and because they show a

lower affinity to the membrane, remain in the soluble

fraction of the periplasm.

The physiological meaning of the autocleavage still

remains unclear. One possibility is that in this way, the excess

of HtrA is removed from the cell. However, our studies on

HtrA stability following chloramphenicol treatment showed

that the levels of FL-HtrA and S-HtrA remained rather stable

at least for 30min (data not shown). There is also a possibility

that the autocleavage may influence the proteolytic activity of

HtrA. Our preliminary results showed that S-HtrA remained

fully active; moreover, partially cleaved HtrA was more

active towards h-casein than FL-HtrA. Our present work is

focused on understanding the physiological role of HtrA

autocleavage.

Acknowledgements

This work was supported by grants from the Polish

Committee for Scientific Research (KBN, No 1008/P04/

2000; from the Foundation for Polish Science (‘‘Immuno’’

103/99), from University of Gdansk (BW/1160-5/0142-0)

and by grants from Ancona University (F.T. and E.B). We

would like to thank Michael Ehrmann for generous gift of

plasmid pTA3.

References

[1] C. Georgopoulos, K. Liberek, M. Zylicz, D. Ang, Properties of the

heat shock proteins of Escherichia coli and the autoregulation of the

heat shock response, in: R.I. Moromoto, A. Tissieres, C. Georgopou-

los (Eds.), The Biology of Heat Shock Proteins and Molecular Chap-

erones, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

NY, 1994, pp. 209–249.

[2] S. Gottesman, Proteases and their targets in Escherichia coli, Annu.

Rev. Genet. 30 (1996) 465–506.

[3] M.J. Pallen, B.W. Wren, The HtrA family of serine proteases, Mol.

Microbiol. 26 (1997) 209–221.

[4] T. Clausen, C. Southan, M. Ehrmann, The HtrA family of proteases:

implications for protein composition and cell fate, Mol. Cell 10

(2002) 443–455.

[5] K.I. Kim, S. Park, S.H. Kang, G. Cheong, C.H. Chung, Selective

degradation of unfolded proteins by the self-compartmentalizing HtrA

protease, a periplasmic heat shock protein in Escherichia coli, J. Mol.

Biol. 294 (1999) 1363–1374.

[6] H. Kolmar, P.R.H. Waller, R.T. Sauer, The DegP and DegQ pe-

riplasmic endoproteases of Escherichia coli: specificity for cleav-

age sites and substrate conformation, J. Bacteriol. 178 (1996)

5925–5929.

[7] C. Spiess, A. Beil, M. Ehrmann, A temperature-dependent switch

from chaperone to protease in a widely conserved heat shock protein,

Cell 97 (1999) 339–347.

[8] J. Skorko-Glonek, D. Zurawa, E. Kuczwara, M. Wozniak, Z. Wypych,

B. Lipinska, Escherichia coli heat shock HtrA protease participates in

the defense against oxidative stress, Mol. Gen. Genet. 262 (1999)

342–350.

[9] M. Sone, S. Kishigami, T. Yoshihisa, K. Ito, Roles of disulfide

bonds in bacterial alkaline phosphatase, J. Biol. Chem. 272 (1997)

6174–6178.

[10] S.-I. Hu, M. Carozza, M. Klein, P. Nantermet, D. Luk, R.M. Crowl,

Human HtrA, an evolutionarily conserved serine protease identified

as a differentially expressed gene product in ostheoarthritic cartilage,

J. Biol. Chem. 273 (1998) 34406–34412.

[11] C.W. Gray, R.V. Ward, E. Karran, S. Turconi, A. Rowles, D.

Viglienghi, C. Southan, A. Barton, K.G. Fantom, A. West, J.

Savopoulos, N.J. Hassan, H. Clinkenbeard, C. Hanning, B. Ame-

gadzie, J.B. Davis, C. Dingwall, G.P. Livi, C.L. Creasy, Charac-

terization of human HtrA2, a novel serine protease involved in the

mammalian cellular stress response, Eur. J. Biochem. 267 (2000)

5699–5710.

[12] D.H. Ly, D.J. Lockhart, R.A. Lerner, P.G. Schultz, Mitotic misregu-

lation and human aging, Science 287 (2000) 2486–2492.

[13] A. Baldi, A. De Luca, M. Morini, T. Battista, A. Felsani, F. Baldi,

C. Catrigal, A. Amaantea, D.M. Noonan, A. Albini , P.G. Natali,

D. Lombardi, M.G. Paggi, The HtrA1 serine protease is down-

regulated during human melanoma progression and represses

growth of metastatic melanoma cells, Oncogene 21 (2002)

6684–6688.

[14] B. Lipinska, S. Sharma, C. Georgopoulos, Sequence analysis and

transcriptional regulation of the HtrA gene of Escherichia coli, Nu-

cleic Acids Res. 16 (1988) 10053–10067.

[15] B. Lipinska, O. Fayet, L. Baird, C. Georgopoulos, Identification,

characterization and mapping of the Escherichia coli htrA gene,

whose product is essential for bacterial growth only at elevated tem-

peratures, J. Bacteriol. 171 (1989) 1574–1584.

[16] J. Skorko-Glonek, A. Wawrzynow, K. Krzewski, K. Kurpierz, B.

Lipinska, Site-directed mutagenesis of the HtrA (DegP) serine pro-

tease, whose proteolytic activity is indispensable for Escherichia coli

survival at elevated temperatures, Gene 163 (1995) 47–52.

[17] T. Krojer, M. Garrido-Franco, R. Huber, M. Ehrmann, T. Clausen,

Crystal structure of DegP (HtrA) reveals a new protease-chaperone

machine, Nature 416 (2002) 455–459.

[18] M. Russel, P. Model, Replacement of the fip gene of Escherichia coli

by an inactive gene cloned on a plasmid, J. Bacteriol. 159 (1984)

1034–1039.

[19] S. Tabor, C.C. Richardson, A bacteriophage T7 RNA polymerase/

promoter system for controlled exclusive expression of cloned genes,

Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 1074–1078.

[20] G. Churchward, D. Belin, Y. Nagamine, A pSC01-derived plasmid

which shows no sequence homology to other commonly used vectors,

Gene 31 (1984) 165–171.

[21] B. Lipinska, M. Zylicz, C. Georgopoulos, The HtrA (DegP) protein,

essential for Escherichia coli survival at elevated temperatures, is an

endopeptidase, J. Bacteriol. 172 (1990) 1791–1797.

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

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

[23] D.P. Goldenberg, in: T.E. Creighton (Ed.), Protein Structure: A Prac-

tical Approach, IRL Press, Oxford University Press, Oxford, 1989,

pp. 225–250.

[24] R. Oberfelder, Detection of proteins on filters by enzymatic methods,

in: G. Howard (Ed.), Methods in Nonradioactive Detection, C. Ap-

pleton & Lange, Norwalk, CT, 1993, pp. 83–85.

[25] J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY, 1972.


[26] U. Jakob, W. Muse, M. Eser, J.C.A. Bardwell, Chaperone activity

with a redox switch, Cell 96 (1999) 341–352.

[27] G.T. Hermanson, Bioconjugate Techniques, Academic Press, San

Diego, CA, 1996, pp. 307–309.

[28] R.J. Davies, P.R. Edwards, H.J. Watts, C.R. Lowe, P.E. Buckle,

D. Yeung, T.M. Kinning, D.V. Pollard-Knight, The resonant mir-

ror: a tool for the study of biomolecular interactions, Techniques

in Protein Chemistry vol. V, Academic Press, San Diego, 1994,

pp. 285–292.

[29] F. Szoka, D. Papahadjopoulos, Liposomes: preparation and character-

isation, in: C.G. Knight (Ed.), Liposomes, Elsevier/North-Holland

Biomedical Press, Amsterdam, 1981, pp. 51–82.

[30] P.R. Edwards, A. Gill, D.V. Pollard-Knight, M. Hoare, P.E. Buckle,

P.A. Lowe, R.J. Leatherbarrow, Kinetics of protein–protein interac-

tions at the surface of an optical biosensor, Anal. Biochem. 231

(1995) 210–217.

[31] P.R. Edwards, P.A. Lowe, R.J. Leatherbarrow, Ligand loading at

the surface of an optical biosensor and its effect upon the ki-

netics of protein–protein interactions, J. Mol. Recognit. 10 (1997)

128–134.

[32] A. Scire, F. Tanfani, F. Saccucci, E. Bertoli, G. Principato, Specific

interaction of cytosolic and mitochondrial glyoxalase II with acidic

phospholipids in form of liposomes results in the inhibition of the

cytosolic enzyme only, Proteins 41 (2000) 33–39.

[33] G.T. Hermanson, Bioconjugate Techniques, Academic Press, San

Diego, CA, 1996, pp. 132–133.

[34] J. Skorko-Glonek, B. Lipinska, K. Krzewski, G. Zolese, E. Bertoli, F.

Tanfani, HtrA heat-shock protease interacts with phospholipid mem-

branes and undergoes conformational changes, J. Biol. Chem. 272

(1997) 8974–8982.

[35] R.B. Freedman, The formation of protein disulfide bonds, Curr. Opin.

Struct. Biol. 5 (1995) 85–91.

[36] H.F. Gilbert, Thiol/Disulfide equilibria and disufide bond stability,

Methods Enzymol. 251 (1995) 8–28.

[37] S. Raina, D. Missiakas, C. Georgopoulos, The rpoE gene encoding

the jE (j24) heat shock sigma factor of Escherichia coli, EMBO J.

14 (1995) 1043–1055.

[38] N. Sassoon, J.-P. Arle, J.-M. Betton, PDZ domains determine the

native oligomeric structure of the DegP(HtrA) protease, Mol. Micro-

biol. 33 (1999) 583–589.

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