Báo cáo khoa học: ThermoFAD, a ThermofluorÒ-adapted flavin ad hoc detection system for protein folding and ligand binding pdf

Thermofluor determines the unfolding temperature of a protein through evaluation of the fluorescence of a solvatochromic dye such as 1-anilino-8-naphthalenesulfonate [5] or SYPRO Orange [6

ThermoFAD, a Thermofluor -adapted flavin ad hoc

detection system for protein folding and ligand binding Federico Forneris, Roberto Orru, Daniele Bonivento, Laurent R Chiarelli and Andrea Mattevi Department of Genetics and Microbiology, University of Pavia, Italy

Identification of optimal purification and storage

con-ditions is one the most critical investigations in the

biochemical analysis of a protein Challenging projects

such as characterisation of macromolecular complexes,

membrane proteins or large multidomain human

pro-teins often do not provide the large amounts of sample

required by protein biochemistry techniques, restricting

the investigation to a very limited, sometimes not

reproducible, set of information In this respect, the

Thermofluor technique [1] (Fig 1A) is an example of

how it is possible to minimise the amounts of protein

and time used for analysis of various parameters such

as ligand stabilisation, pH effects, and storage

condi-tions [2–4] Thermofluor determines the unfolding

temperature of a protein through evaluation of the

fluorescence of a solvatochromic dye such as 1-anilino-8-naphthalenesulfonate [5] or SYPRO Orange [6], which have a low fluorescence quantum yield in water and a high quantum yield when bound to the hydro-phobic surface of denatured proteins (Fig 1A) Over recent years, several reports have described successful use of the Thermofluor technique for identification of the stabilising conditions of biochemically uncharacter-ised proteins [5–8], library screening of potential ligands for selected drug targets [9–11], or simple investigations of the behaviour of proteins under vari-ous conditions [12,13] Although dedicated instruments are commercially available for Thermofluor analysis [1], the experiment can be performed without any tech-nical adaptation, using even the cheapest available

Keywords

flavin; fluorescence screening; ligand

screening; protein stability; Thermofluor 

Correspondence

F Forneris and A Mattevi, Dipartimento di

Genetica e Microbiologia, Universita` di

Pavia, Via Ferrata 1, 27100 Pavia, Italy

Fax: +39 0382 528496

Tel: +39 0382 985534

E-mail: forneris@ipvgen.unipv.it;

mattevi@ipvgen.unipv.it

Website: http://www.unipv.it/biocry/

(Received 14 January 2009, revised 3 March

2009, accepted 16 March 2009)

doi:10.1111/j.1742-4658.2009.07006.x

In living organisms, genes encoding proteins that contain flavins as a pros-thetic group constitute approximately 2–3% of the total The fluorescence

of flavin cofactors in these proteins is a property that is widely employed for biochemical characterisation Here, we present a modified Thermofluor approach called ThermoFAD (Thermofluor-adapted flavin ad hoc detec-tion system), which simplifies identificadetec-tion of optimal purificadetec-tion and storage conditions as well as high-affinity ligands In this technique, the fla-vin cofactor is used as an intrinsic probe to monitor protein folding and stability, taking advantage of the different fluorescent properties of flavin-containing proteins between the folded and denatured state The main advantage of the method is that it allows a large amount of biochemical data to be obtained using very small amounts of protein sample and stan-dard laboratory equipment We have explored several cases that demon-strate the reliability and versatility of this technique when applied to globular flavoenzymes, membrane-anchored flavoproteins, and macro-molecular complexes The information gathered from ThermoFAD analysis can be very valuable for any biochemical and biophysical analysis, includ-ing crystallisation The method is likely to be applicable to other classes of proteins that possess endogenous fluorescent cofactors and prosthetic groups

Abbreviations

LSD1, lysine-specific histone demethylase 1; MAO, monoamine oxidase; ThermoFAD, Thermofluor-adapted flavin ad hoc detection system; FMO, flavin-dependent monooxygenase.

real-time PCR apparatus [13,14] The fluorescence

signal is increased when the dye partitions into the

hydrophobic patches of proteins that become

solvent-exposed during the denaturation process The presence

of compounds interacting with the protein molecules

at various levels, from solvation to covalent binding,

alters the unfolding behaviour of the protein under

analysis, and a shift in the unfolding temperature can

be directly associated with a stabilisation or a destabil-isation effect [4,11] However, use of dyes that bind hydrophobic surfaces, such as SYPRO Orange, suffers from the limitation that the detergents used to solubi-lize membrane proteins interfere with the analysis, cre-ating a hydrophobic environment due to micelle formation This dye-detergent interaction does not allow correct measurement of the unfolding tempera-ture of the sample, limiting the analysis to water-solu-ble proteins For the same reason, Thermofluor cannot be applied successfully to many proteins that expose hydrophobic patches to the solvent (e.g pro-teins that interact in macromolecular complexes), because the dyes produce a fluorescence signal due to binding to these regions, masking the signal associated with protein unfolding

In both prokaryotic and eukaryotic organisms, genes encoding proteins that contain flavins as prosthetic group are estimated to constitute approximately 2–3%

of the total Enzymes that employ flavins for catalysis are involved in a multitude of processes, from drug metabolism to gene regulation [15] Because of their spectroscopic features, flavoproteins form one of the most studied protein classes In particular, the fluores-cence of the flavin is an intrinsic property that is widely used for biochemical characterisation of flavo-proteins By comparing the emission and excitation ranges of the dyes typically used in Thermofluor experiments, we noticed that flavins have fluorescence properties that fall in the same wavelength range The conventional excitation wavelength used in RT-PCR instruments is 450–530 nm, whereas flavins show fluo-rescence excitation maxima at 373–375 and 445–

450 nm (Fig 1B,C) [16] This broad shape of the flavin excitation spectrum makes the RT-PCR excitation wavelengths suitable for generating sufficient cence intensity for detection With regard to fluores-cence emission, the highest intensity for flavins is at

535 nm [16] Depending on the instrumental setup, RT-PCR instruments have various optical ranges for fluorescence detection, from fixed intervals to a com-pletely customizable detection range [17] However, we found that most RT-PCR systems, even the cheapest ones available on the market, can generally be used to excite flavins and measure their fluorescence signal without any specific adaptation As the fluorescence of flavin cofactors in flavoproteins is usually quenched by the protein environment when the protein is properly folded [16], we realised that is possible to measure the unfolding temperature of a flavoprotein using Thermo-fluor by monitoring the increase in cofactor fluores-cence (Fig 1B) This approach allows fast and reliable

A

B

C

Fig 1 (A) Schematic representation of the Thermofluor  binding

assay A solvatochromic dye (i.e SYPRO Orange) is used as an

indicator of protein unfolding Binding of the dye to the unfolded

protein results in a significant increase in its intrinsic fluorescence.

(B) Schematic representation of ThermoFAD In this case, the

increase in fluorescence is generated by exposure of the flavin

cofactor to the solvent upon protein unfolding (C) Overview of

fluo-rescence properties of flavins and comparison with RT-PCR

instru-mental parameters Dashed line, flavin excitation spectrum;

continuous line, flavin emission spectrum; red, wavelength range

for RT-PCR fluorescence excitation; green, SYBR Green detection

range; orange, SYPRO Orange detection range Flavin fluorescence

emission can be measured using the SYBR Green fluorescence

filter on the RT-PCR instrument without any adaptation.

evaluation of many protein parameters using extremely

low amounts of sample Moreover, it is more versatile

than conventional Thermofluor because, by using

intrinsic fluorescence instead of that of an external

dye, it is not influenced by the noise generated by

hydrophobic compounds present in solution or

hydro-phobic patches that may interact with the dyes used in

Thermofluor We named this modified Thermofluor

approach ‘ThermoFAD’ (Thermofluor-adapted flavin

ad hocdetection system)

Results

The ThermoFAD technique

A ThermoFAD analysis requires only 20 lL of protein

sample, in a concentration range from 0.3 to

4.0 mgÆmL)1, and an RT-PCR instrument The whole

experiment takes < 2 h and allows evaluation of

1–384 samples at the same time (depending on the

set-up of the PCR instrument) In a typical

experi-ment, 1–2 lL of a concentrated sample are added

together with the buffers and ligands for analysis

directly into the wells of the RT-PCR instrument

Next, a temperature gradient is applied, starting from 15–20 C and increasing to 90 C, measuring the fluorescence signal every 0.5 min As in a standard RT-PCR Thermofluor experiment, a sigmoidal curve (thermogram) is obtained by plotting the fluorescence intensity against the temperature The unfolding tem-perature is then determined as the maximum of the derivative of this sigmoidal curve (Figs 2 and 3) [1] By comparing various thermograms for the same protein under various conditions, it is possible to evaluate which compounds stabilise (or destabilise) the sample under analysis and to screen many conditions with a minimum consumption of protein [5]

In order to validate our ThermoFAD technique, we have chosen a set of flavoproteins with various features

in terms of biological activity, size, and type of interac-tion (covalent and non-covalent) with the flavin cofac-tor (Table 1) As an indication of the efficiency and sensitivity of our approach, we compared the results obtained with ThermoFAD with conventional Thermo-fluormeasurements obtained using SYPRO Orange as the fluorescent probe for denaturation (Fig 2) The results are in perfect agreement for the whole set of flavoproteins under analysis (Table 1) The sensitivity

Fig 2 Comparison between Thermofluorand ThermoFAD for various flavoproteins The selected flavoproteins differ with respect to the type of flavin cofactor, flavin linkage to the protein, and source organism of the protein (for details see Table 1) Thermal stability curves are plotted against normalised fluorescence signal Green lines, Thermofluor  experiments using SYPRO Orange as fluorescent dye; red lines, ThermoFAD experiments measured without addition of any dye The detector filter of the RT-PCR instrument for ThermoFAD is the one that

is commonly used for SYBR Green dye (fluorescence emission of 523–543 nm; see Fig 1C).

and specificity of ThermoFAD make the technique

extremely versatile, in that it allows evaluation of the

stability of a flavoprotein even in partially purified

sam-ples (data not shown), which is impossible to detect

using dyes that bind nonspecifically to all the

hydro-phobic patches present in solution Here, we report on

the application of the modified Thermofluorapproach

to a few of our investigated flavoproteins (Table 1 and

Fig 2) with the intention of demonstrating the

advan-tages offered by the ThermoFAD technique

Comparison of the stability of a soluble and

globular flavoenzyme (FMO) in the presence of

various ligands

We tested a number of conditions for optimal

stabili-sation of a flavin-containing monooxygenase (FMO)

from Methilophaga sp strain SK1 FMOs are

involved in the metabolism of several drugs,

cataly-sing the oxygenation of many nitrogen-, sulphur-,

phosphorus- and selenium-containing nucleophilic

compounds using molecular oxygen and NADPH as

substrates [18] Using ThermoFAD, we compared the

stability of FMO in various buffers and evaluated the

effect of addition of NADP(H) analogues on protein

stability Our buffer screening led to identification of

optimal stabilisation conditions for FMO that

corre-spond to the buffer that was successfully used for

crystallisation of this flavoprotein [18] (Fig 3A)

Moreover, the ThermoFAD analysis allowed us to

identify NADP analogues with higher affinity to

FMO compared to NADP:3-acetylpiridine ADP,

thio-NADP and nicotinic acid ADP These compounds

were then tested as FMO crystallisation additives,

leading to high-quality crystals, with a significant

increase in the diffraction quality and resolution of

the data (F Forneris and A Mattevi, unpublished results)

ThermoFAD on a membrane-anchored flavoenzyme in the presence of detergents When working with membrane proteins, it is necessary

to use detergents after membrane extraction through-out the purification and characterisation process The choice of detergent is the most critical parameter in obtaining a stable and active protein suitable for biochemical and structural characterisation For this reason, effective detergent screening methods are required (see [19] for a recent development in this area) Thermofluor is an excellent candidate for this type of analysis, but suffers from the limitation that the fluo-rescent dyes used to determine the protein unfolding temperature interact with the detergent lipophilic moiety This limitation makes the analysis difficult, if not impossible [20] However, ThermoFAD allowed unfolding temperature analysis of a membrane-anchored flavoprotein to be performed in the presence

of detergents, because the flavin cofactor fluorescence is not influenced by these amphipathic molecules As a test case, we used human monoamine oxidase B, a membrane-bound flavoenzyme that catalyses the oxida-tion of arylalkylamine neurotransmitters and bears a FAD cofactor covalently attached to a cysteine residue [21] We performed both Thermofluor (using SYPRO Orange as a dye) and ThermoFAD experiments on the same sample in order to compare the two techniques The Thermofluor experiment did not produce a sigmoidal curve, most likely because of interaction of SYPRO Orange with the detergent and⁄ or the hydro-phobic membrane-binding region of the enzyme On the other hand, ThermoFAD produced a clear result

Table 1 Comparison of unfolding temperature using Thermofluorand ThermoFAD for various flavoproteins ND, not determined.

Protein concentration (mgÆmL)1)

T m (C)

Thermofluor  ThermoFAD Lysine-specific demethylase

+ CoREST complex

L -Galactono-c-lactone dehydrogenase Plant (Arabidopsis thaliana) Non-covalent FAD [26] 1.3 58.2 58.6 Flavin-dependent

monooxygenase

Bacterial (Methylophaga sp.) Non-covalent FAD [18] 2.0 43.0 43.3

Alditol oxidase Bacterial [Streptomyces

coelicolor A3(2)]

Vanillyl-alcohol oxidase Fungus; (Penicillium

simplicissimum)

with a nice sigmoidal curve indicating an unfolding temperature of 51.2C (Fig 2) The significance of this result was further verified by circular dichroism spec-tropolarimetry By means of this technique, we mea-sured a value for the unfolding temperature (57 C) that is slightly higher than that measured by Thermo-FAD, probably reflecting the inherent differences between the two methodologies ThermoFAD senses the exposure of flavin to water, which is likely to be an earlier event in the denaturation process than the loss

of secondary structures, as probed by circular dichro-ism Our study of human monoamine oxidase B shows that ThermoFAD can be efficiently used in the case of flavoproteins that require detergents for stabilisation or that contain hydrophobic patches on their surface

Evaluation of in vitro reconstitution of a protein complex using ThermoFAD

A more complicated case is an investigation conducted

on the human flavin-dependent histone demethylase LSD1 This flavoenzyme catalyses removal of a methyl group from a protein substrate (histone H3) with a highly specific substrate specificity (Lys4) LSD1 is a partially non-globular, multidomain protein that is known to interact with a co-repressor protein named corepressor of the neural receptor REST (CoREST) LSD1 and CoREST assemble to generate a hetero-dimeric sub-complex that is part of several nuclear multiprotein complexes [22] Using ThermoFAD, we were able to measure the stabilising effect induced by association of CoREST with purified LSD1 (Fig 3B) Binding of CoREST to LSD1 shifts the unfolding tem-perature by 4C, consistent with a tight association between the two proteins Thus, the experiment allowed

us to quickly establish using a very limited amount of protein that the complex could be reconstituted in vitro Moreover, we confirmed that various histone H3 pep-tides bind tightly to LSD1, in perfect agreement with biochemical enzymatic assays [23] Importantly, the increases in protein stability are proportional to the inhibitory power of the analysed peptides (Fig 3C) Especially interesting is the finding that the histone H3 peptide with the Lys4Met mutation has the highest stabilising effect This peptide is a tight nanomolar inhibitor, which was successfully used for crystal structure determination of the LSD1⁄ CoREST ⁄ histone peptide ternary complex [24]

Discussion

Our method shows that it is possible to exploit the intrinsic fluorescence of flavin cofactor to determine the

A

B

C

Fig 3 (A) Evaluation of FMO stability using ThermoFAD against

var-ious buffers at varvar-ious pH values (B) ThermoFAD comparison of

LSD1 stability with (red) and without (green) addition of the protein

CoREST The T m shift corresponds to formation of a heterodimeric

complex between the two proteins The Thermofluor  profile of

iso-lated CoREST is shown in blue; in this case it is not possible to

calcu-late a T m value because of the many exposed hydrophobic patches

of CoREST that bind to the dye before complete unfolding of the

pro-tein (C) ThermoFAD profiling of LSD1 ⁄ CoREST stability towards

known inhibitor peptides All data are in good agreement with the

biochemical analysis [23] In particular, the Lys4Met (K4M) peptide

shows the highest stabilising effect, in agreement with the fact that

it is the peptide that allowed us to solve the crystal structure of the

LSD1 ⁄ CoREST complex with a bound peptide substrate analogue.

unfolding temperature of flavoproteins, instead of using

the fluorescent dyes commonly used in Thermofluor

experiments This approach simplifies the screening and

identification of optimal conditions for protein stability,

storage and ligand binding In addition, this technique

does not require any customised procedure or specific

chemical compound, and can also be used in the

pres-ence of compounds that are known to interfere

signifi-cantly with the dyes used in the conventional

Thermofluorapproach, such as detergents or

contami-nants We have provided some examples of the

versatil-ity of this technique, which can be used with proteins

with covalently and noncovalently bound flavin

cofac-tors to identify stabilising agents, high-affinity ligands,

protein complex formation, and other factors that can

affect protein stability This information is obviously

very valuable for any biochemical and biophysical

analysis, including crystallisation In all cases, the

exper-iments were performed in just a few hours using

standard laboratory equipment with minimal sample

consumption As flavoproteins are among the most

widely studied protein classes because of their

abun-dance, variety and biological importance, we believe

that this fast, cheap and reliable method will be of

great help for the many groups that study new and

uncharacterised flavoproteins Moreover, it is likely to

be applicable to other classes of proteins that possess

endogenous fluorescent cofactors and prosthetic groups

Experimental procedures

Protein samples

All flavoproteins used for our analysis were expressed and

purified as described in the original papers reporting their

biochemical and structural characterisation (Table 1) Their

purity was checked by SDS–PAGE analysis, and protein

concentration was evaluated by measuring the UV⁄ vis

absorbance of the bound flavin cofactor using published

extinction coefficients

ThermoFAD experimental setup

Experiments were performed using a MiniOpticon real-time

PCR detection system, using 48-well RT-PCR plates

(Bio-Rad Laboratories, Hercules, CA, USA) Measurements were

performed using an excitation wavelength range between 470

and 500 nm and a SYBR Green fluorescence emission filter

(523–543 nm), which falls within the same fluorescence range

as the isoalloxazine ring of FAD or flavin mononucleotide

(470–570 nm) (Fig 1C) The flavoprotein concentration

required for optimal signal-to-noise ratio was initially

evalu-ated using LSD1 as a benchmark Unfolding curves were

generated using a temperature gradient from 20 to 90C, performing a fluorescence measurement after every 0.5C increase after a 10-s delay for signal stabilisation All experi-ments were performed at least three times, and the reported

Tmvalues are based on the mean values determined from the peaks of the derivatives of the experimental data In a typical experiment, 1–2 lL of a concentrated protein were mixed together with the ligands for analysis directly into the wells

of the RT-PCR instrument and diluted with reaction buffer (50 mm KPi, pH 7.5) to a final volume of 20 lL The best concentrations for ThermoFAD analysis were between 0.5 and 4 mgÆmL)1, and all subsequent experiments were carried out using protein concentrations in this range

Evaluation of the reliability of Thermo FAD versus Thermofluorfor various flavoproteins

To compare the results of the ThermoFAD analysis with conventional Thermofluor, we performed experiments in parallel with the same amounts of flavoproteins, with and without the addition of 3 lL of 5000· SYPRO Orange (Sigma-Aldrich, St Louis, MO, USA) The experimental setup, gradients and methods were identical in the Thermo-FAD and Thermofluoranalyses Detection was performed using the SYPRO Orange and SYBR Green fluorescence filters for both techniques to evaluate the interference possi-bly caused by superposition of the flavin fluorescence on that of the SYPRO Orange No interference was detected (data not shown)

Determination of stabilisation conditions for FMO FMO was concentrated using an Amicon concentrator (Millipore Corp., Billerica, MA, USA) with a 30 kDa cutoff to a final concentration of 20 mgÆmL)1 A set of 15 buffers at 50 mm concentration in the pH range 4.2–10.6 was prepared in RT-PCR plates, and 2 lL of flavoenzyme were added to each well (final protein concentration of 2.0 mgÆmL)1) Buffers that showed a significant stabilisa-tion effect are reported in Fig 3A

Determination of the unfolding temperature of human monoamine oxidase B

Human monoamine oxidase B, stored at 3 mgÆmL)1 in

50 mm KPi pH 7.0 supplemented with 0.8% w⁄ v octyl-glucoside, was diluted in the same buffer to a final concen-tration of 1 mgÆmL)1 and used for thermal unfolding assays The unfolding temperature was also measured by circular dichroism spectropolarimetry For this purpose, we used a Jasco J-710 spectropolarimeter (Jasco Europe, Cre-mella, Italy) equipped with a Neslab RT-11 programmable water bath (Thermo Fisher Scientific, Waltham, MA, USA) and a 1 mm path-length cuvette Thermal denaturation was

followed by continuous measurements of ellipticity at

222 nm in the temperature range 25–70C with a constant

heating rate of 1CÆmin)1

LSD1⁄ CoREST reconstitution and inhibition

assays

Human LSD1, 8 mgÆmL)1 in 50 mm KPi buffer

supple-mented with 5% v⁄ v glycerol pH 7.2, was diluted with the

same buffer to a final concentration of 1 mgÆmL)1

Experi-ments were performed using the LSD1 alone or supplied

with human CoREST in stoichometric amounts to

deter-mine the Tmincrease associated with formation of the

hete-rodimeric protein complex For inhibition assays, a

tandem-affinity purified LSD1⁄ CoREST complex [24] was

used instead of LSD1 alone for better comparison with

pre-viously published biochemical data [23] The complex was

used at a final concentration of 1 mgÆmL)1, and 3 lL of

2 mm histone peptide inhibitors were added to each well

Copyright notice

The Thermofluor assay was developed by

3-Dimen-sional Pharmaceuticals Inc., which is now part of

Johnson & Johnson Pharmaceutical Research &

Devel-opment (Raritan, NJ, USA) ‘Thermofluor’ is a

trade-mark registered in the USA and certain other

countries

Acknowledgements

Financial support from the Italian Ministry of Science

(PRIN06 and FIRB programmes), the Fondazione

Cariplo, the Italian Association for Cancer Research,

and the American Chemical Society Petroleum

Research Fund (46271-C4) is gratefully acknowledged

We thank Drs Dale E Edmondson (Emory University,

Atlanta, GA, USA), Claudia Binda (University of

Pavia, Italy), Willem J van Berkel (University of

Wageningen, the Netherlands) and Marco W Fraaije

(University of Groningen, the Netherlands) for

provid-ing us with protein material and helpful advice

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