Báo cáo Y học: Contribution of Lys276 to the conformational flexibility of the active site of glutamate decarboxylase from Escherichia coli pptx

The observed spectral changes suggest that the cofactor is present at the active site in its hydrated form.. The pH- and guanidine-dependent absorbance changes in the GadB-K276A mutant w

Contribution of Lys276 to the conformational flexibility of the active

Angela Tramonti1, Robert A John2, Francesco Bossa1and Daniela De Biase1

1

Dipartimento di Scienze Biochimiche ‘A Rossi Fanelli’ and Centro di Studio sulla Biologia Molecolare del CNR, Rome, Italy;

2

Cardiff School of Biosciences, Cardiff, UK

Glutamate decarboxylase is a pyridoxal

5¢-phosphate-dependent enzyme responsible for the irreversible

a-decar-boxylation of glutamate to yield 4-aminobutyrate In

Escherichia coli, as well as in other pathogenic and

non-pathogenic enteric bacteria, this enzyme is a structural

component of the glutamate-based acid resistance system

responsible for cell survival in extremely acidic conditions

(pH < 2.5) The contribution of the active-site lysine

residue (Lys276) to the catalytic mechanism of E coli

glutamate decarboxylase has been determined Mutation of

Lys276 into alanine or histidine causes alterations in the

conformational properties of the protein, which becomes less

flexible and more stable The purified mutants contain very

little (K276A) or no (K276H) cofactor at all However,

apoenzyme preparations can be reconstituted with a full

complement of coenzyme, which binds tightly but slowly

The observed spectral changes suggest that the cofactor is present at the active site in its hydrated form Binding of glutamate, as detected by external aldimine formation, occurs at a very slowrate, 400-fold less than that of the reaction between glutamate and pyridoxal 5¢-phosphate in solution Both Lys276 mutants are unable to decarboxylate the substrate, thus preventing detailed investigation of the role of this residue on the catalytic mechanism Several lines

of evidence showthat mutation of Lys276 makes the protein less flexible and its active site less accessible to substrate and cofactor

Keywords: glutamate decarboxylase; pyridoxal 5¢-phos-phate; active-site lysine; site-directed mutagenesis; Escherichia coli

In all pyridoxal 5¢ phosphate (PLP)-dependent enzymes

studied so far, the e-amino group of a conserved lysine

residue at the active site [1] binds the cofactor as a

Schiff’s base It has been suggested that the formation of

an internal aldimine between the coenzyme and a

primordial apoenzyme occurred early in the evolution

of PLP-enzymes because, before becoming catalytically

advantageous, it prevented rapid loss of PLP which was

precious because of its ability to catalyse a number of

reactions on a wide variety of biosubstrates by itself [2]

Site-directed mutagenesis supports the proposal that

participation of this lysine residue in an internal aldimine

with the cofactor also accelerates formation of the

external aldimine between the PLP 4¢-aldehyde and

substrate amino groups because transaldimination is

more rapid than de novo Schiff’s base formation [3–6]

It also facilitates the proton transfers essential to many

B6-dependent reactions [3–10] In the amino acid decar-boxylases so far investigated, the corresponding lysine appears not to be involved in reprotonation after decarboxylation, but mainly to play a role in the initial transaldimination, in proper positioning of the a-carb-oxylate for decarboxylation and in product release [11,12]

Bacterial glutamate decarboxylase (Gad, E.C 4.1.1.15) is one of the structural components of the glutamate-based acid resistance system, responsible for acid survival of enteric pathogens, such as Escherichia coli, Shigella flexneri and Listeria monocytogenes [13–15], and of other nonpatho-genic bacteria [16,17] E coli synthesizes two Gad isoforms, GadA and GadB, 98% identical in amino acid sequence and biochemically indistinguishable [18,19] Gad catalyses the irreversible a-decarboxylation ofL-glutamate to yield 4-aminobutyrate and CO2 It has been suggested that in this enzyme the active-site lysine is involved in the protonation

of the quinonoid intermediate at C-4¢ during the abortive decarboxylation–transamination reaction, while a histidine has been proposed as the residue responsible for the protonation at C-a which occurs during the physiological decarboxylation reaction [20] Site-directed mutagenesis established that His167 and His275, likely candidates as proton donors, are not responsible for the reprotonation after CO2elimination [21]

The present work has been undertaken with the aim of determining the contribution of the active-site lysine residue (Lys276) to coenzyme binding and to stages in the reaction catalysed by E coli Gad

Correspondence to D De Biase, Dipartimento di Scienze Biochimiche

A Rossi Fanelli, Piazzale Aldo Moro, 5-00185 Roma, Italy.

Fax: + 39 06 49917566, Tel.: + 39 06 49917692,

E-mail: debiase@caspur.it

Abbreviations: Gad, glutamate decarboxylase; PLP, pyridoxal

5¢-phosphate.

Enzymes: glutamate decarboxylase (P28302) (E.C.4.1.1.15).

Note: a web site is available at http://w3.uniroma1.it/bio_chem/

homein.html

(Received 21 May 2002, revised 24 July 2002, accepted 26 July 2002)

Site-directed mutagenesis

Site-directed mutagenesis was performed by overlap

exten-sion polymerase chain reactions [22], following the

proce-dure described in Tramonti et al [21] Mutagenic primers

were 5¢-GGCCATGCATTCGGTCTG-3¢, for the

GadB-K276A mutant, 5¢-GGCCATCACTTCGGTCTG-3¢, for

the GadB-K276H mutant, and their complementary

sequences Fragments EcoRV/HindIII, generated by

digest-ing the amplicons from the second polymerase chain

reaction, were subcloned into pQgadB [19] The newly

inserted fragments of plasmid pQgadBK276A and

pQgadBK276H were sequenced on both strands

Purification of mutant forms of Gad

Expression and purification of mutant enzymes were as

described for wild-type enzyme [19] The E coli strain

JM109(pREP4), known to produce low levels of

endo-genous GadA/B was used as host [19] Preparations of the

mutant enzymes were treated with NaBH3CN to inactivate

any wild-type enzyme present

Calorimetric and spectroscopic analyses

Thermal unfolding of GadB-K276A and GadB-K276H

(1.5–2.0 mgÆmL)1) was analyzed under nitrogen pressure

on a MicroCal MC-2D differential scanning calorimeter

(MicroCal, Inc., Northampton, MA, USA) Results were

corrected for instrumental baseline and normalized for

protein concentration

Absorption spectra were measured on a Hewlett-Packard

model 8452 diode-array spectrophotometer CD spectra

were recorded as the average of three scans on a Jasco 710

spectropolarimeter equipped with a DP520 processor at

25C Fluorescence spectroscopy was performed with a

LS50B fluorimeter (Perkin Elmer) at the excitation

wave-length of 295 nm Curve fitting and statistical analysis were

carried out using the data manipulation softwareSCIENTIST

(Micromath, Salt Lake City, UT) The PLP content of all

the enzyme preparations was determined by treating the

protein with 0.1MNaOH and measuring absorbance at

388 nm (e388¼ 6550 LÆmol)1Æcm)1[23]) The

pH-depend-ent absorbance variation of wild-type and mutant enzymes

was analyzed using the following equation:

AbsHnE AbsE

Abs Abs  1 ¼

10npK

Gad activity assay Enzyme activity was assayed by quantitating the reaction product, 4-aminobutyrate, by HPLC [24] or using Gabase,

a commercial preparation containing 4-aminobutyrate aminotransferase and succinic semialdehyde dehydrogen-ase, as previously described [19] On some occasions, enzyme activity was assayed using the pH indicator bromocresol green (0.02% w/v) as described by De Biase

et al [25]

R E S U L T S

Physical properties of mutant enzymes Yields of the mutant enzymes, K276A and GadB-K276H, after the standard purification, i.e in absence of added PLP, were 50 mgÆL)1 of bacterial culture, as for wild-type GadB [19] The mutant forms were stable for several months at 4C As judged by CD spectroscopy in the far-UV region, mutations did not affect the overall protein conformation The transition temperature of reconstituted GadB-K276A was 62.3C, suggesting that this mutant enzyme adopts a significantly more stable conformation than wild-type GadB (51C) The transi-tion temperature of GadB-K276H (55.3C) was only slightly higher than that of the wild-type enzyme (Fig 1)

In support of the above observation, limited proteolysis

by trypsin showed that GadB-K276A is more resistant to proteolytic degradation than the wild-type enzyme (data not shown)

Spectral properties of GadB-K276A and -K276H mutants

Absorption spectra of purified GadB-K276A showed maxima at 280 and 328 nm (Fig 2A) However, the specific absorbance at 328 nm due to the cofactor was significantly lower than that of the wild-type enzyme and, correspond-ingly, the amount of PLP released by NaOH treatment was only 10% of that expected for a fully saturated holoenzyme Nevertheless, the small amount of cofactor present was not displaced by either gel filtration in 0.5MKH2PO4, or incubation with cysteine or dialysis in 2MguanidineÆHCl Complete removal of the cofactor was achieved by overnight dialysis against 1MKH2PO4, pH 4.2, w ith the resulting apoenzyme having the spectrum shown in Fig 2A, inset Purification of GadB-K276H following the standard

protocol yielded 100% apoenzyme Unlike the wild-type

apoenzyme, which precipitates instantaneously upon

coen-zyme removal, both GadB-K276A and GadB-K276H

apoenzymes remained stable for many weeks in 0.1M

sodium acetate, pH 4.6 For both mutant forms, the

holoenzyme was regenerated by treating the apoenzyme

(150 lM) overnight with a fivefold molar excess of PLP

(750 lM) Unbound cofactor was removed by extensive

dialysis against 0.1Msodium acetate, pH 4.6 The

recon-stituted mutant enzyme (Fig 2A) contained one molecule

of PLP per monomer, as judged by NaOH treatment The

absorption spectrum above 320 nm fitted well to the sum of

two log normal curves having kmaxvalues of 330 nm and

388 nm (Fig 2B) The great majority of the coenzyme was

present as 330 nm-absorbing chromophore, the

corres-ponding peak being broader, but less intense, in the

GadB-K276H mutant enzyme (Fig 2B) Continuous monitoring

of the absorbance changes associated with reconstitution of

GadB-K276A indicated that the absorbance decrease at

388 nm (free PLP) and the increase at 330 nm were biphasic

(data not shown) and the curve fitted well to the sum of

two exponentials (k1¼ 0.58 ± 0.001 min)1; k2¼ 0.059 ±

0.002 min)1) with the more rapid phase accounting for 70%

of the reaction

Treatment of both reconstituted mutants with

NaCNBH3 did not affect the spectra, demonstrating that

PLP is bound as the free aldehyde

Figure 2(C) shows CD spectra of wild-type and GadB

mutants in the 300–500 nm range, where the chromophore

of all enzymic forms absorbs maximally Notably, the

GadB-K276A mutant produced a much smaller CD signal

than the GadB-K276H mutant, despite the lower

absorb-ance of the latter (Fig 2B)

E coliGad undergoes well-established changes in activity

and in the absorption spectrum of the cofactor depending

on pH (Fig 3A) [26] At pH values higher than 5.3, the

enzyme absorbs maximally at 340 nm and is inactive,

whereas at lower pH values, the enzyme absorbs maximally

at 420 nm The change in activity parallels the absorbance

Fig 2 Absorption and CD spectra of GadB Lys276 mutants (A) Absorption spectra of GadB-K276A mutant The absorption spectra

of GadB-K276A (20 l M ) as it is purified under standard conditions (dotted line), in the apoenzymatic form (solid line) and after its reconstitution with PLP (dashed line) were determined in 0.1 M sodium acetate, pH 4.6, containing 0.1 m M dithiothreitol (B) Analysis of absorption spectra of GadB-K276A and GadB-K276H mutants The solid lines are those of best fit to the sum of two log normal curves [32] having k max values of 330 nm and 388 nm Only one in three of the data points collect for the GadB-K276A (d) and GadB-K276H (j) absorption spectra is shown (C) CD spectra of wild-type and mutant enzymes The CD spectra of wild-type GadB (solid line), and of GadB-K276A (dotted line) and GadB-K276H (dashed line) mutants, each at

a concentration of 184 l M , were determined in 50 m M sodium acetate,

pH 4.6, containing 0.1 m M dithiothreitol.

Fig 1 Differentialscanning calorimetry of wild-type GadB and

active-site lysine mutants Thermal denaturation profiles of GadB wild-type

(solid line), of GadB-K276A mutant (dashed line) and GadB-K276H

mutant (dotted line) Protein samples (1.5–2.0 mgÆmL)1) w ere in 0.1 M

sodium acetate, pH 3.6, containing 0.1 m M dithiothreitol.

the mutant enzymes In both mutants, guanidineÆHCl in the range 0–2Minduced an absorbance change characterized

by an increase at 388 nm and a decrease at 328 nm (Fig 3C) The same behavior was also observed when sodium chloride was added (data not shown) In the same concentration range of guanidineÆHCl and sodium chloride, the absorbance spectrum of the wild-type enzyme remained unaffected

When excited at 295 nm in the absence of guanidineÆHCl, the reconstituted mutant enzymes exhibited two fluores-cence emission maxima (Fig 4A) The first, at 332 nm, also present in the wild-type (Fig 4B), is due to the intrinsic fluorescence of the protein It is likely that the second, at

380 nm, is due to energy transfer to the 330-nm absorbing form of PLP at the active site At 2MguanidineÆHCl the emission spectrum of both mutants showed exclusively a peak at 332 nm, because the PLP in the active site has been converted into a form mainly absorbing at 388 nm In the range 2–5M guanidineÆHCl the change in fluorescence emission spectra indicated that the unfolding profiles of wild-type and mutant enzymes are superimposable, with the transition point (50% unfolding) centered at 3.4M guani-dineÆHCl Upon unfolding, a blue-shifted emission maxi-mum at 360 nm in both wild-type and mutant enzymes was observed (Fig 4)

Reaction with glutamate Addition of 20 mMglutamate to GadB-K276A produced

an increase in absorbance at 412 nm and a decrease at

328 nm each with the same half-time of approximately 2 h (Fig 5A) The change was characterized by an isosbestic point at 342 nm The 412 nm contribution was completely abolished by adding NaCNBH3, a reagent known to reduce exclusively the protonated Schiff bases After 7 h, an additional slowspectral change occurred which was com-plete within 30 h This spectral change is characterized by a decrease at 412 nm and an increase at 340 nm, with an isosbestic point at 375 nm (Fig 5B) The change observed

at 412 nm conformed to an equation describing two consecutive irreversible reactions (see Materials and meth-ods, Eqn 2) Increasing the glutamate concentration produced a linear increase in the value observed for k1 whereas k2did not change significantly (0.07 ± 0.01 h)1)

No 4-aminobutyrate was detected at the end of the reaction although the method used was sensitive enough to detect this compound at 10% of the enzyme concentration

Fig 3 Effect of pH and guanidineÆHClon the absorbance spectra (A)

Absorption spectra of wild-type GadB (11.4 l M ) were determined in

0.1 M sodium acetate in the pH range 3.5–6.2 Only relevant spectra are

shown In the inset, the pH variation at 420 nm is represented The

solid line is that of best fit to Eqn (1) (Materials and methods), with

pK ¼ 5.292 ± 0.007, Abs E ¼ 0.0069 ± 0.0008, Abs HnE ¼ 0.1058 ±

0.0007 and n ¼ 5.1 ± 0.4 (B) Absorption spectra of GadB-K276H

(10.3 l M ) were determined as above In the inset, the pH variation at

388 nm is represented The solid line is that of best fit to Eqn (1)

(Materials and methods), with pK ¼ 5.60 ± 0.01, Abs E ¼ 0.032 ±

0.001, Abs HnE ¼ 0.0060 ± 0.0006 and n ¼ 8.4 ± 4.6 (C) Absorption

spectra of GadB-K276H mutant (19 l M ) measured in the presence of

0, 0.1, 0.2, 0.4, 0.6, 1 and 2 M guanidineÆHCl in 0.1 M sodium acetate,

pH 4.6 The pH- and guanidine-dependent absorbance changes in the

GadB-K276A mutant were identical with those in GadB-K276H

mutant, and therefore the data are omitted.

Moreover, no pH increase was detected during the reaction

with glutamate when using the pH indicator bromocresol

green in an unbuffered solution, thus indicating that there

was no consumption of protons Treatment of the reaction

mixture with 0.2M NaOH at the times 0, 5 and 20 h

released the full complement of cofactor as PLP (detected

and measured by its 388 nm absorbance)

Reaction of GadB-K276H with 20 mMglutamate resulted

in a spectral change similar to that occurring in the

GadB-K276A mutant (data not shown) The only difference

observed between the two mutants was the amplitude of

the change in absorbance at 412 nm, which at 20 lMprotein

was much smaller in GadB-K276H (total absorbance change

of 0.03) than in GadB-K276A (total absorbance change of

0.12; Fig 5A) As observed for the alanine mutant, the

histidine mutant did not produce 4-aminobutyrate

In the active-site lysine mutants of aspartate

aminotrans-ferase [7], tryptophan synthase [4],D-amino acid

transam-inase [5] and alanine racemase [27] it was observed that

exogenous amines can partially or totally substitute for

the catalytic role of the active-site lysine In order to

study the effect of exogenous amines on GadB-K276A, 2,2,

2-trifluoroethylamine (pKa¼ 5.7) [7] and aminoacetonitrile

bisulfate (pKa¼ 5.3) [7] were added to the enzyme in

presence of sodium glutamate

When 1M 2,2,2-trifluoroethylamine or 0.2M amino-acetonitrile bisulfate were included in the reaction mixture containing 20 lM GadB-K276A and 20 mM sodium glutamate, the enzyme underwent spectral changes identical

to those already described, but the increase in absorbance at

412 nm occurred approximately six times faster The devel-opment of turbidity however, prevented analysis of the later phases of the reaction Even in the presence of exogenous amines 4-aminobutyrate production was undetectable (data not shown)

When both mutant enzymes were incubated with glutamate in the presence of a lowconcentration of guanidineÆHCl (0.4M), spectral changes identical to those previously described occurred, even though the initial spectrum was different, and at the end of reaction a species absorbing at 340 nm could be detected (data not shown)

D I S C U S S I O N

Many of the alterations produced by mutating the active-site Lys276 of GadB can be attributed to changes in the conformational properties of the protein The findings that the mutation to alanine increases the unfolding temperature

Fig 5 Reaction of GadB-K276A with glutamate The absorbance spectra of GadB-K276A mutant (60 l M ) were recorded with 20 m M

sodium glutamate in 0.1 M sodium acetate, pH 4.6, from 0 to 6 h (A) and from 6 to 30 h (B).

Fig 4 Effect of guanidine on fluorescence emission spectra (A)

Emis-sion spectra (k exc ¼ 295 nm) of GadB-K276H mutant (0.82 l M ) in

0.1 M sodium acetate, pH 4.6, containing 0, 1, 2, 3 and 5 M

guani-dineÆHCl (B) Emission spectra of wild-type GadB (0.77 l M ) in 0.1 M

sodium acetate, pH 4.6, containing 0, 2, 4 and 5 M guanidineÆHCl.

[11,12], non–covalent interactions between protein and

cofactor are sufficient to ensure tight binding This is also

in line with the finding that His275 contributes to cofactor

binding via an ionic interaction with the phosphate group of

PLP [21]

Because the mutant forms of GadB cannot form an

internal aldimine, it is not surprising that the 420 nm

chromophore, characteristic of the wild-type enzyme at

pH 4.6, is absent However, the spectrum of PLP bound to

the Lys276 mutants is quite different from the spectrum of

the same compound when it is free in solution Absorption

bands at 388 nm and 330 nm are present in the spectra of

both free PLP and PLP bound to the mutant enzymes, but in

PLP free in solution the 388 nm chromophore is the most

abundant species, whereas it is only a minor component of

the spectrum of the mutant enzymes In free PLP, the

388 nm and 330 nm chromophores are attributed to the

unsubstituted and hydrated aldehydes, respectively [28] It

seems likely that the absorbance changes observed are due to

an increase in the proportion of the PLP hydrate when the

cofactor binds A similar structure has also been suggested to

be formed in the active-site lysine mutant of aromatic

L-amino acid decarboxylase [11] The biphasic nature of

the changes in spectrum occurring upon PLP binding,

reported also with the wild-type enzyme [29], suggests that

initial cofactor binding is followed by a slower

confor-mational adjustment The CD spectra (Fig 2C) show

that GadB-K276H retains much more asymmetry than

GadB-K276A probably because movement of the

cofac-tor within the active site is more restricted by the histidine

side-chain

Both wild-type GadB and its active-site lysine mutants

showabrupt pH-dependent spectral changes involving

simultaneous transfer of multiple protons Other

PLP-dependent enzymes undergo similar pH-PLP-dependent changes

which are related to activity and are attributed to

protona-tion of the internal aldimine formed with lysine and the

cofactor aldehyde For example, aspartate aminotransferase

is converted from an inactive 430 nm-absorbing protonated

internal aldimine to an active unprotonated 362

nm-absorbing form with a pK of 6.2 in a process that fits well

to the ionization of a single proton [30] It has been

suggested that in wild-type Gad, the ionization responsible

for the absorbance change does not take place on the

internal aldimine and much evidence indicates that the

change in spectrum of the wild-type enzyme is due to a

conformational transition in the protein induced by shifting

conformation favors the unsubstituted internal aldimine [31] However, because in the mutant enzymes the high

pH favors the 388 nm-absorbing unsubstituted aldehyde, formation of a covalent bond between PLP and a cysteine side-chain can be excluded as the basis of the pH-dependent absorbance changes observed with the Lys276 mutant enzymes An explanation that unites observations from both wild-type and mutant enzymes is that the pH-dependent conformational change induces an alteration in the polarity of the active site In this hypothesis, the environment of the cofactor is more hydrated in the

low-pH conformation Thus, the increased polarity favors the

420 nm-ketoenamine tautomer in the wild-type enzyme and the 330 nm-absorbing hydrated form of PLP in the mutant enzymes Conversely, the less hydrated environ-ment of the high-pH conformation favors the 340 nm-absorbing enolimine tautomer of the wild-type cofactor and the 388 nm-absorbing unhydrated aldehyde of PLP in the mutant enzymes (Fig 6)

Fig 6 Chemicalstructures of the chromophore proposed to be present

at the active site of GadB wild-type and GadB-K276 mutants at low and high pH, respectively.

In the presence of lowconcentrations of guanidineÆHCl

or of sodium chloride, there is a spectral change in the

mutant enzymes similar to that which occurs upon changing

the pH We suggest that lowconcentrations of solutes, by

subtracting water molecules, cause a change in polarity of

the active site and favor the 388 nm-absorbing unhydrated

aldehyde of PLP

The failure of all the methods used to detect enzymatic

activity in GadB-K276A and GadB-K276H mutants

shows that GadB mutated at the active-site lysine loses

reactivity towards the substrate, though the mutants are

still capable of slowly bindingL-glutamate and forming an

external aldimine The increase in absorbance at 412 nm

observed when GadB K276A was mixed with glutamate,

together with the observation that this chromophore

converted to one absorbing at 340 nm when NaCNBH3

was added provides strong evidence that an external

aldimine is formed between the cofactor and the amino

acid The linear dependence on glutamate concentration of

the observed rate constant governing this phase shows

that there is no detectable saturation of the mutant

enzyme with substrate, even at high concentrations The

second order constant calculated from this experiment

(3.6· 10)3± 0.3 · 10)3M )1Æs)1) is much lower than

that calculated for the reaction between free PLP and

glutamate in 0.1M sodium acetate, pH 4.6 (1.47 ±

0.13M )1Æs)1) The 400-fold reduction in reaction rate

contrasts markedly with the observation that PLP bound

to the corresponding mutant of aspartate

aminotransfer-ase forms an external aldimine with glutamate or aspartate

at least three orders of magnitude more rapidly than does

free PLP [3] It seems likely that at least part of this major

reduction in reactivity of the enzyme-bound PLP is due to

the extensive hydration we propose to be responsible for

the predominant 330 nm chromophore, as well as to the

reduced flexibility discussed earlier

The cause of the subsequent and even slower change in

spectrum from 412 nm to 340 nm is unknown, although

the failure to detect 4-aminobutyrate shows that it is not

due to decarboxylation of the substrate A possible

explanation could be that Lys276 is, either directly or

indirectly, involved in correctly positioning the Ca-COO–

bond orthogonal to the plane of the delocalized cofactor

imine system Moreover, the release of cofactor as PLP

when the enzyme was treated with NaOH after

comple-tion of this reaccomple-tion shows that it is not due to

transamination to pyridoxamine phosphate A similar

but much more rapid spectral change occurs in the

reaction catalyzed by the wild-type enzyme, where it has

been attributed to a reversible conformational change in

the enzyme leading to a form that does not undergo

further reaction [19,21]

A C K N O W L E D G M E N T S

This work was partially supported by grants from the Italian Ministero

dell’Istruzione, dell’Universita` e della Ricerca and from the Istituto

Pasteur-Fondazione Cenci Bolognetti (to DDB) The Centro di

Eccellenza di Biologia e Medicina Molecolare (BEMM), Universita`

di Roma La Sapienza, is also acknowledged.

We thank Professor A Giartosio for DSC measurements and

Professor D Barra for critical reading of the manuscript.

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