research article interaction between chromosomal protein...

Research ArticleInteraction between Chromosomal Protein HMGB1 andDNA Studied by DNA-Melting Analysis

Elena V Chikhirzhina1 Starkova J Tatiana12 and Alexander M Polyanichko12

1 Institute of Cytology of the Russian Academy of Sciences Saint Petersburg 194064 Russia2Department of Molecular Biophysics Faculty of Physics Saint Petersburg State University 1 Ulyanovskaya StreetStary Petergoff Saint Petersburg 198504 Russia

Correspondence should be addressed to Alexander M Polyanichko apolyanichkospburu

Received 27 October 2014 Accepted 15 December 2014 Published 28 December 2014

Academic Editor Khalique Ahmed

Copyright copy 2014 Elena V Chikhirzhina et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Interaction ofHMGB1 nonhistone chromosomal protein withDNAwas studied using circular dichroism spectroscopy and thermaldenaturation of DNA Melting DNA in the complex was shown to be a biphasic processThe characteristic melting temperatures ofunboundDNAand theDNAbound toHMGB1 in 025mMEDTA solutionswere found to be119879I

119898= 440plusmn05

∘C and119879II119898= 620plusmn1

∘Crespectively It was shown that the binding of the HMGB1 molecule affects the melting of the DNA region approximately 30 bplong

1 Introduction

HMG-box proteins (HMGB) are the most abundant nonhis-tone chromosomal proteins They belong to the superfamilyof HMGproteins (highmobility group) [1ndash3] One of the bestknownmembers of theHMGB family is HMGB1 proteinTheamino acid sequence of the protein consists of three regionsforming twoDNA-binding domains (HMGB-domains A andB) and an unordered regulatory C-terminal domain [2 4 5]HMGB-domains have a conservative L-shaped structure [4]in whichDNA-binding activity is regulated by the C-terminaldomain represented by 29AspGlu amino acid residues oftenreferred to as the ldquoacidic tailrdquo [5ndash8] Although the individualHMGB-domains consist predominantly of 120572-helical regions[4 9 10] their structure in the whole protein depends on theinteractions with the acidic tail and may change considerably[6 11]

HMGB-domains bindDNA in theminor groove demon-strating specificity to the prebent DNA structures ratherthan to the particular sequences [2 3 5] Moreover HMGB-proteins themselves are able to induce bends of the doublehelix upon binding Originally structural organization ofDNA in the chromatin was thought to be the major functionof HMGB-proteins [12ndash15] However it was proved recently

that these proteins performnumerous regulatory functions incell nucleus [5 16ndash18] in cytoplasm [19 20] and even outsidethe cell [21ndash24]

In spite of extensive experimental data on the interactionof the HMGB-domains with short DNA fragments [5 25ndash27] the exact mechanisms of the interactions between thefull HMGB1 and DNA are not clear yet However it isthese mechanisms that determine the diversity of functionsperformed by HMGB1 It was demonstrated that the modeof HMGB1-DNA interaction depends on the protein to DNAratio [7 8 28ndash30] Large supramolecular complexes wereobserved at high HMGB1DNA ratios which appeared dueto increasing contribution of the protein-protein interactions[7 8 29 30] Earlier experimental data suggest that binding toDNAmay induce different structural changes of the HMGB1itself depending on the particular DNA species HMGB1interacts with [31 32] In the present study we investigatethe interaction between HMGB1 and DNA based on DNA-melting analysis

2 Experimental

21 DNA-Protein Complexes Nonhistone chromosomal pro-tein HMGB1 (MW 26500) was extracted from the nuclei of

Hindawi Publishing CorporationJournal of SpectroscopyVolume 2014 Article ID 387138 5 pageshttpdxdoiorg1011552014387138

2 Journal of Spectroscopy

calf thymus as described earlier [29 33]Thepurity of the pro-tein was tested with sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) The protein concentrationwas determined by UV absorbance using extinction coeffi-cients 120576

280= 21805Mminus1 cmminus1 [34] Calf thymus DNA (type

II) was purchased from Sigma-Aldrich and used withoutfurther purificationTheDNA concentrationwas determinedby measuring difference in absorbance at 290 and 270 nm(119860270

ndash119860290

) after hydrolysis in 6 HClO4[35] The quality

of the DNA was checked by measuring the increase ofits absorbance at 260 nm after denaturation (hyperchromiceffect)

Protein-DNA complexes were prepared by direct mixingof equal volumes ofDNAand protein solutions of appropriateconcentrations to obtain desired input protein to DNA ratio119903 (ww) in the complex The HMGB1DNA ratio 119903 variedfrom 0 to 05 The final concentration of DNA in solutionswas 30 120583gmL All solutions contained 025mM EDTA and02mM NaCl

22 Spectroscopy Absorbance spectra were recorded withSpecord M 40 spectrophotometer (Karl Zeiss Germany) intemperature controlled quartz cells with 1 cm optical pathlength

CD spectra were recorded with Mark V dichrograph(Jobin-Yvon France) in a temperature controlled quartz cellwith 1 cmoptical path lengthThe spectra of each samplewererecorded in the region between 200 and 320 nm in a 1 nmstep mode The signal was averaged for 1000 readings with1msec intervals at each wavelength The presented spectraare the average of three sequential runs Circular dichroismis represented by Δ119860 = 119860

119871minus 119860119877values the difference

between absorbance of left and right circularly polarized lightby a sample The CD instrument was calibrated using D-10-camphorsulfonic acid

23 DNA-Melting Analysis Melting of DNA and proteinswas achieved by gradually heating a sample from 20 to95∘C DNA-melting curves were obtained by measuringoptical density at 260 nm 119863

260(119879) or circular dichroism at

275 nm Δ119860275(119879) The melting curves of the protein samples

were obtained by measuring circular dichroism at 222 nmΔ119860222(119879) Normalizedmelting curves119891(119879) for DNA samples

were obtained using the equation below

119891 (119879) =119863260(119879) minus 119863

min260

119863max260minus 119863

min260

(1)

where119863min260

and119863max260

are the minimal andmaximal values ofthe optical density at 260 nm corresponding to the native andmelted states of the DNA in the samples The first derivativeof the melting curves 119889119891(119879)119889119879 was deconvoluted usingGaussian profiles

The melting of DNA-protein complexes was character-ized using melting temperature 119879

119898 which was determined

as the position of the maximum of the first derivative of

25 30 35 40 45 50 55 6000

02

04

06

08

10

000

f

002

004

006

008

010

Temperature (∘C)

dfdT

Figure 1 The normalized melting curve 119891(119879) (bold line) and thecorresponding first derivative (119889119891119889119879) of pure DNA in 025mMEDTA02mM NaCl solution

the melting curve The hyperchromic effect 119866 was quantifiedaccording to the equation

119866 =119863

max260minus 119863

min260

119863min260

times 100 (2)

3 Results and Discussion

The DNA molecule is a polyanion at physiological condi-tions Decreasing ionic strength of the solution leads to thedestabilization of the double helix resulting in lowering themelting temperature of DNA To reduce the effect of ionicstrength and to achieve better separation of the peaks onthe derivative melting curve we studied the DNA melting inthe complexes with HMGB1 in solutions containing 025mMEDTA and 02mM NaCl [36] The concentration of DNA inall samples was 30 120583gmL The melted DNA in all complexesdemonstrated the same level of hyperchromicity at 260 nm asthe pure DNA (Figure 1)

The first derivative of the pure DNA-melting curvein unbound state has a characteristic single-peak profile(Figure 1) This peak is attributed to the melting of DNAand the position of peakrsquos maximum provides the value ofDNA-melting temperature 119879

119898 For the DNA in solutions

of 025mM EDTA02mM NaCl the melting temperaturewas found to be 440 plusmn 05∘C (Figure 1) When the proteinis added to the system the first derivative of the meltingcurve demonstrates a different profile having two separatepeaks (Figure 2) revealing a biphasic process One of thesepeaks still has the maximum at approximately 44∘C (119879I

119898)

and thus can be attributed to the melting of unbound DNAThe other one is shifted towards the higher temperatures andindicates the presence of some fraction of DNA with meltingtemperature of sim62∘C (119879II

119898) As it was shown earlier [36ndash40]

the second peak can be attributed to the melting of DNAregions bound to the protein Thus interaction of DNA withHMGB1 results in stabilization of the double helix in thevicinity of HMGB1 binding site

Journal of Spectroscopy 3

25 30 35 40 45 50 55 60 65

TIm

TIIm

dfdT

Temperature (∘C)

Figure 2 The first derivative of the melting curves 119889119891119889119879 of DNAin the complexes with HMGB1 at different protein to DNA ratios 119903(ww) ◼ 119903 = 0 (DNA) 998779 119903 = 015 X 119903 = 025 998795 119903 = 04 e119903 = 05 All curves were obtained in 025mM EDTA02mM NaClsolution

90∘C

90∘C

50∘C

50∘C

25∘C

25∘C2

1

0

minus1

minus2

minus3

minus4

Wavelength (nm)200 220 240 260 280 300

ΔAtimes10

4

Figure 3 Circular dichroism spectra of the HMGB1DNA complexobtained at different temperatures in the range of 20ndash90∘CThe con-centration of DNA in sample was 30120583gmL and the concentrationof HMGB1 was 15120583gmLThe complexes were prepared in 025mMEDTA02mM NaCl solution The optical path length was 1 cm

Thermal stability of the complexes was also studied usingcircular dichroism spectroscopy (Figure 3) CD spectroscopyis suitable for monitoring conformational changes both inDNA and in the protein during the helix-to-coil transitionAnalysis of the changes in DNA CD during the meltinggives the melting temperature equal to 119879I

119898 which is in

agreement with absorption spectroscopy data The meltingcurve of the pure HMGB1 Δ119860

222(119879) (Figure 4) gives the

protein melting temperature of sim42∘C Similar dependenceΔ119860222(119879) for the DNA-protein complex at 119903 = 05 gives

slightly higher melting temperatures at approximately 47∘Cwhich might reflect increasing melting temperature of theprotein in the complex orand superposition with the DNA-melting transition (Figure 4) It is interesting to note thatthe conformational changes in the protein are essentiallycompleted by 60∘C The first derivative of the melting curvesof the complexes (Figure 2) shows that the second meltingpeaks rise at the temperatures higher than 60∘C Since thesesecond peaks correspond to themelting of DNAbound to theprotein wemay conclude that themelting of the boundDNA

30 40 50 60 70 80minus25

minus20

minus15

minus10

minus05

00

Temperature (∘C)

ΔA

222times10

4

Figure 4Themelting curves of pureHMGB1 (squares) andHMGB1in the complex with DNA at 119903 = 05 (circles) obtained bymeasuring circular dichroism at 222 nmThe concentration of DNAin the sample was 30 120583gmL and the concentration of HMGB1 was15 120583gmL The complex was prepared in 025mM EDTA02mMNaCl solution Optical path length was 1 cm

00 01 02 03 04 05

Fb

r (WW)

040

035

030

025

020

015

010

005

000

Figure 5 The fraction of DNA bound to the nonhistone proteinHMGB1 119865

119887as a function of protein to DNA ratio 119903 (ww)

follows the conformational transition in the protein whichin turn most likely induces changes in the DNA-bindingproperties of the HMGB1

Assuming that the area under the graph of the first deriva-tive of the melting curve is proportional to the concentrationof the DNA in the sample [36ndash40] we may conclude thatthe area under each of the two peaks is proportional to theamount of unbound and bound DNA in the system respec-tively The areas under the individual peaks change upon theprotein binding revealing that increasing the protein to DNAratio in the complex results in decreasing amount of unboundDNA and increasing the amount of DNA regions bound tothe protein (Figure 2) Based on the comparison of the areasunder these two peaks we have estimated the fraction of DNAbase pairs participating in binding to HMGB1 depending onthe input protein to DNA ratio 119903 (Figure 5) The fraction ofDNA base pairs with higher melting temperature 119879II

119898as a

function of input protein toDNA ratio 119903 shows linear increasewith increasing 119903 Such a linear dependence indicates that


in the whole range of the studied protein to DNA ratios thebinding of a singleHMGB1molecule stabilizes approximatelythe samenumber of theDNAbase pairs (bp) To estimate thesize of this region we can estimate the ratio of the number ofthe bp bound to the protein ]DNAbound to the number of theprotein molecules in the system ]HMGB1

119873 =]DNAbound]HMGB1

=]DNA sdot 119891]HMGB1

=119872HMGB1119872DNA

sdot119891

119903

=119872HMGB1119872DNA

sdot tan120572(3)

where 119891 is the fraction of the bound DNA ]DNA is the totalnumber of DNA bp in the sample119872 is the molar mass ofDNA bp (660Da) or HMGB1 (265 kDa) respectively 119903 isthe protein to DNA ratio (ww) and tan120572 is the slope of the119891(119903) graph equal to sim08 (Figure 5)

Hence the simple calculations give the estimate of the sizeof the stabilized DNA region equal to approximately 30 bpwhich is about twice bigger than the HMGB1 binding sitedetermined earlier [7 28ndash30 41 42]

Thus we may conclude that despite the considerabledeformation of the double helix HMGB1 stabilizes the basepairing in DNA in the vicinity of the binding site which ismanifested by 20∘C increase in melting temperature Bindingof theHMGB1 stabilizes approximately 30 bp which is abouttwice bigger than the binding site of a single HMGB-domain

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to the Russian Foundation for BasicResearches for a financial support (Grant 12-08-01134) andthe Government of Saint Petersburg

References

[1] S S Ner ldquoHMGs everywhererdquo Current Biology vol 2 no 4 pp208ndash210 1992

[2] M Bustin ldquoRegulation of DNA-dependent activities by thefunctional motifs of the high-mobility-group chromosomalproteinsrdquoMolecular andCellular Biology vol 19 no 8 pp 5237ndash5246 1999

[3] E Chikhirzhina G Chikhirzhina and A Polyanichko ldquoChro-matin structure the role of lsquolinkerrsquo proteinsrdquo Biomedical Spec-troscopy and Imaging vol 3 no 4 pp 345ndash358 2014

[4] CM Read PD Cary C Crane-Robinson P CDriscoll andDGNorman ldquoSolution structure of aDNA-binding domain fromHMG1rdquo Nucleic Acids Research vol 21 no 15 pp 3427ndash34361993

[5] M Stros ldquoHMGB proteins interactions with DNA and chro-matinrdquo Biochimica et Biophysica ActamdashGene Regulatory Mech-anisms vol 1799 no 1-2 pp 101ndash113 2010

[6] K Stott M Watson F S Howe J G Grossmann and J OThomas ldquoTail-mediated collapse of HMGB1 is dynamic and

occurs via differential binding of the acidic tail to the A and Bdomainsrdquo Journal of Molecular Biology vol 403 no 5 pp 706ndash722 2010

[7] A M Polyanichko Z V Leonenko D Cramb H Wieser V IVorobrsquoev and E V Chikhirzhina ldquoVisualization of DNA com-plexes with HMGB1 and its C-truncated form HMGB1(A+B)rdquoBiophysics vol 53 no 3 pp 202ndash206 2008

[8] E Chikhirzhina A Polyanichko Z Leonenko H Wieser andV VorobrsquoEv ldquoC-terminal domain of nonhistone proteinHMGB1 as a modulator of HMGB1-DNA structural interac-tionsrdquo Spectroscopy vol 24 no 3-4 pp 361ndash366 2010

[9] HMWeir P J Kraulis C SHill A R C Raine E D Laue andJ OThomas ldquoStructure of theHMGboxmotif in the B-domainof HMG1rdquo EMBO Journal vol 12 no 4 pp 1311ndash1319 1993

[10] Y Xu W Yang J Wu and Y Shi ldquoSolution structure of the firstHMG box domain in human upstream binding factorrdquo Bio-chemistry vol 41 no 17 pp 5415ndash5420 2002

[11] P L Privalov I Jelesarov C M Read A I Dragan and CCrane-Robinson ldquoThe energetics of HMG box interactionswith DNA thermodynamics of the DNA binding of the HMGbox from mouse Sox-5rdquo Journal of Molecular Biology vol 294no 4 pp 997ndash1013 1999

[12] M Bustin and R Reeves ldquoHigh-mobility-group chromosomalproteins architectural components that facilitate chromatinfunctionrdquo Progress in Nucleic Acid Research and MolecularBiology vol 54 pp 35ndash100 1996

[13] R Grosschedl K Giese and J Pagel ldquoHMG domain proteinsarchitectural elements in the assembly of nucleoprotein struc-turesrdquo Trends in Genetics vol 10 no 3 pp 94ndash100 1994

[14] J J Love X Li D A Case K Giese R Grosschedl and P EWright ldquoStructural basis for DNA bending by the architecturaltranscription factor LEF-1rdquo Nature vol 376 no 6543 pp 791ndash795 1995

[15] J O Thomas and A A Travers ldquoHMG1 and 2 and relatedldquoarchitecturalrdquo DNA-binding proteinsrdquo Trends in BiochemicalSciences vol 26 pp 167ndash174 2001

[16] J M Song S Hwang W Wong et al ldquoThe DNA architecturalprotein HMGB1 facilitates RTA-mediated viral gene expressionin gamma-2 herpesvirusesrdquo Journal of Virology vol 78 no 23pp 12940ndash12950 2004

[17] Y Liu R Prasad and S H Wilson ldquoHMGB1 roles in baseexcision repair and related functionrdquo Biochimica et BiophysicaActamdashGene Regulatory Mechanisms vol 1799 no 1-2 pp 119ndash130 2010

[18] T Ueda and M Yoshida ldquoHMGB proteins and transcriptionalregulationrdquo Biochimica et Biophysica ActamdashGene RegulatoryMechanisms vol 1799 no 1-2 pp 114ndash118 2010

[19] H Rauvala and A Rouhiainen ldquoPhysiological and pathophys-iological outcomes of the interactions of HMGB1 with cellsurface receptorsrdquo Biochimica et Biophysica ActamdashGene Regu-latory Mechanisms vol 1799 no 1-2 pp 164ndash170 2010

[20] D Tang R Kang H J Zeh III andM T Lotze ldquoHigh-mobilitygroup box 1 and cancerrdquo Biochimica et Biophysica Acta GeneRegulatory Mechanisms vol 1799 no 1-2 pp 131ndash140 2010

[21] S Guazzi A Strangio A T Franzi andM E Bianchi ldquoHMGB1an architectural chromatin protein and extracellular signallingfactor has a spatially and temporally restricted expressionpattern in mouse brainrdquo Gene Expression Patterns vol 3 no1 pp 29ndash33 2003

[22] D Yang P Tewary G de la Rosa F Wei and J J Oppen-heim ldquoThe alarmin functions of high-mobility group proteinsrdquo


Biochimica et Biophysica ActamdashGene Regulatory Mechanismsvol 1799 no 1-2 pp 157ndash163 2010

[23] H Yang and K J Tracey ldquoTargeting HMGB1 in inflammationrdquoBiochimica et Biophysica Acta vol 1799 no 1-2 pp 149ndash1562010

[24] R Palumbo M Sampaolesi F De Marchis et al ldquoExtracellularHMGB1 a signal of tissue damage induces mesoangioblastmigration and proliferationrdquo The Journal of Cell Biology vol164 no 3 pp 441ndash449 2004

[25] A Gelasco and S J Lippard ldquoNMR solution structure of a DNAdodecamer duplex containing a cis- diammineplatinum(II)d(GpG) intrastrand cross-link the major adduct of the anti-cancer drug cisplatinrdquo Biochemistry vol 37 no 26 pp 9230ndash9239 1998

[26] M van Beest D Dooijes M van deWetering et al ldquoSequence-specific high mobility group box factors recognize 10ndash12-basepair minor groove motifsrdquoThe Journal of Biological Chemistryvol 275 no 35 pp 27266ndash27273 2000

[27] K Stott G S F TangK-B Lee and JOThomas ldquoStructure of acomplex of tandemHMGboxes andDNArdquo Journal ofMolecularBiology vol 360 no 1 pp 90ndash104 2006

[28] A M Polianichko S G Davydenko E V Chikhirzhina andV I Vorobrsquoev ldquoInteraction of superhelical DNA with thenonhistone protein HMG1rdquo Tsitologiia vol 42 no 8 pp 787ndash793 2000

[29] E V Chikhirzhina A M Polyanichko A N Skvortsov E IKostyleva C Houssier and V I Vorobrsquoev ldquoHMG1 domains thevictims of the circumstancesrdquo Molecular Biology vol 36 no 3pp 412ndash418 2002

[30] A M Polyanichko E V Chikhirzhina A N Skvortsov et alldquoThe HMG1 ta(i)lerdquo Journal of Biomolecular Structure andDynamics vol 19 pp 1053ndash1062 2002

[31] T Y Rodionova E V Chikhirzhina V I Vorobrsquoyov and A MPolyanichko ldquoChanges in the secondary structure of HMGB1protein bonded to DNArdquo Journal of Structural Chemistry vol50 no 5 pp 976ndash981 2010

[32] A M Polyanichko T J Rodionova V I Vorobrsquoev and E VChikhirzhina ldquoConformational properties of nuclear proteinHMGB1 and specificity of its interaction with DNArdquo Cell andTissue Biology vol 5 no 2 pp 114ndash119 2011

[33] AM Polyanichko E V Chikhirzhina V V Andrushchenko VI Vorobrsquoev and H Wieser ldquoThe effect of manganese(II) on thestructure of DNAHMGB1H1 complexes electronic and vibra-tional circular dichroism studiesrdquoBiopolymers vol 83 no 2 pp182ndash192 2006

[34] C N Pace F Vajdos L Fee G Grimsley and T Gray ldquoHow tomeasure and predict the molar absorption coefficient of aproteinrdquo Protein Science vol 4 no 11 pp 2411ndash2423 1995

[35] A S Spirin ldquoSpectrophotometric determination of total nucleicacidsrdquo Biokhimiia vol 23 no 5 pp 656ndash662 1958

[36] E I Ramm G S Ivanov V I Vorobrsquoev S N Kadura and SN Khrapunov ldquoStructure of illexine I2 and its complexes withDNArdquo Molekulyarnaya Biologiya vol 21 no 6 pp 1590ndash15991987

[37] R M Santella and H Jei Li ldquoInteraction between poly(L-Lysine48 L-Histidine52) and DNArdquo Biopolymers vol 16 no 9pp 1879ndash1894 1977

[38] D E Olins ldquoInteraction of lysine-rich histones and DNArdquoJournal of Molecular Biology vol 43 no 3 pp 439ndash460 1969

[39] E V Chikhirzhina T Y Starkova E I Kostyleva G IChikhirzhina V I Vorobiev and A M Polyanichko ldquoInterac-tion of DNAwith sperm-specific histones of the H1 familyrdquo Celland Tissue Biology vol 5 no 6 pp 536ndash542 2011

[40] E Chikhirzhina T Starkova E Kostyleva and A PolyanichkoldquoSpectroscopic study of the interaction of DNA with the linkerhistone H1 from starfish sperm reveals mechanisms of theformation of supercondensed sperm chromatinrdquo Spectroscopyvol 27 no 5-6 pp 433ndash440 2012

[41] E C Murphy V B Zhurkin J M Louis G Cornilescu andG M Clore ldquoStructural basis for SRY-dependent 46-XY sexreversal modulation of DNA bending by a naturally occurringpoint mutationrdquo Journal of Molecular Biology vol 312 no 3 pp481ndash499 2001

[42] M E A Churchill A Changela L K Dow and A J KriegldquoInteractions of high mobility group box proteins with DNAand chromatinrdquo Methods in Enzymology vol 304 pp 99ndash1331999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


calf thymus as described earlier [29 33]Thepurity of the pro-tein was tested with sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) The protein concentrationwas determined by UV absorbance using extinction coeffi-cients 120576

280= 21805Mminus1 cmminus1 [34] Calf thymus DNA (type

II) was purchased from Sigma-Aldrich and used withoutfurther purificationTheDNA concentrationwas determinedby measuring difference in absorbance at 290 and 270 nm(119860270

ndash119860290

) after hydrolysis in 6 HClO4[35] The quality

of the DNA was checked by measuring the increase ofits absorbance at 260 nm after denaturation (hyperchromiceffect)

Protein-DNA complexes were prepared by direct mixingof equal volumes ofDNAand protein solutions of appropriateconcentrations to obtain desired input protein to DNA ratio119903 (ww) in the complex The HMGB1DNA ratio 119903 variedfrom 0 to 05 The final concentration of DNA in solutionswas 30 120583gmL All solutions contained 025mM EDTA and02mM NaCl

22 Spectroscopy Absorbance spectra were recorded withSpecord M 40 spectrophotometer (Karl Zeiss Germany) intemperature controlled quartz cells with 1 cm optical pathlength

CD spectra were recorded with Mark V dichrograph(Jobin-Yvon France) in a temperature controlled quartz cellwith 1 cmoptical path lengthThe spectra of each samplewererecorded in the region between 200 and 320 nm in a 1 nmstep mode The signal was averaged for 1000 readings with1msec intervals at each wavelength The presented spectraare the average of three sequential runs Circular dichroismis represented by Δ119860 = 119860

119871minus 119860119877values the difference

between absorbance of left and right circularly polarized lightby a sample The CD instrument was calibrated using D-10-camphorsulfonic acid

23 DNA-Melting Analysis Melting of DNA and proteinswas achieved by gradually heating a sample from 20 to95∘C DNA-melting curves were obtained by measuringoptical density at 260 nm 119863

260(119879) or circular dichroism at

275 nm Δ119860275(119879) The melting curves of the protein samples

were obtained by measuring circular dichroism at 222 nmΔ119860222(119879) Normalizedmelting curves119891(119879) for DNA samples

were obtained using the equation below

119891 (119879) =119863260(119879) minus 119863

min260

119863max260minus 119863

min260

(1)

where119863min260

and119863max260

are the minimal andmaximal values ofthe optical density at 260 nm corresponding to the native andmelted states of the DNA in the samples The first derivativeof the melting curves 119889119891(119879)119889119879 was deconvoluted usingGaussian profiles

The melting of DNA-protein complexes was character-ized using melting temperature 119879

119898 which was determined

as the position of the maximum of the first derivative of

25 30 35 40 45 50 55 6000

02

04

06

08

10

000

f

002

004

006

008

010

Temperature (∘C)

dfdT

Figure 1 The normalized melting curve 119891(119879) (bold line) and thecorresponding first derivative (119889119891119889119879) of pure DNA in 025mMEDTA02mM NaCl solution

the melting curve The hyperchromic effect 119866 was quantifiedaccording to the equation

119866 =119863

max260minus 119863

min260

119863min260

times 100 (2)

3 Results and Discussion

The DNA molecule is a polyanion at physiological condi-tions Decreasing ionic strength of the solution leads to thedestabilization of the double helix resulting in lowering themelting temperature of DNA To reduce the effect of ionicstrength and to achieve better separation of the peaks onthe derivative melting curve we studied the DNA melting inthe complexes with HMGB1 in solutions containing 025mMEDTA and 02mM NaCl [36] The concentration of DNA inall samples was 30 120583gmL The melted DNA in all complexesdemonstrated the same level of hyperchromicity at 260 nm asthe pure DNA (Figure 1)

The first derivative of the pure DNA-melting curvein unbound state has a characteristic single-peak profile(Figure 1) This peak is attributed to the melting of DNAand the position of peakrsquos maximum provides the value ofDNA-melting temperature 119879

119898 For the DNA in solutions

of 025mM EDTA02mM NaCl the melting temperaturewas found to be 440 plusmn 05∘C (Figure 1) When the proteinis added to the system the first derivative of the meltingcurve demonstrates a different profile having two separatepeaks (Figure 2) revealing a biphasic process One of thesepeaks still has the maximum at approximately 44∘C (119879I

119898)

and thus can be attributed to the melting of unbound DNAThe other one is shifted towards the higher temperatures andindicates the presence of some fraction of DNA with meltingtemperature of sim62∘C (119879II

119898) As it was shown earlier [36ndash40]

the second peak can be attributed to the melting of DNAregions bound to the protein Thus interaction of DNA withHMGB1 results in stabilization of the double helix in thevicinity of HMGB1 binding site


25 30 35 40 45 50 55 60 65

TIm

TIIm

dfdT

Temperature (∘C)


90∘C

90∘C

50∘C

50∘C

25∘C

25∘C2

1

0

minus1

minus2

minus3

minus4

Wavelength (nm)200 220 240 260 280 300

ΔAtimes10

4



119898 which is in





30 40 50 60 70 80minus25

minus20

minus15

minus10

minus05

00

Temperature (∘C)

ΔA

222times10

4


00 01 02 03 04 05

Fb

r (WW)

040

035

030

025

020

015

010

005

000





119898as a






=119872HMGB1119872DNA

sdot119891

119903

=119872HMGB1119872DNA

sdot tan120572(3)






Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


25 30 35 40 45 50 55 60 65

TIm

TIIm

dfdT

Temperature (∘C)


90∘C

90∘C

50∘C

50∘C

25∘C

25∘C2

1

0

minus1

minus2

minus3

minus4

Wavelength (nm)200 220 240 260 280 300

ΔAtimes10

4



119898 which is in





30 40 50 60 70 80minus25

minus20

minus15

minus10

minus05

00

Temperature (∘C)

ΔA

222times10

4


00 01 02 03 04 05

Fb

r (WW)

040

035

030

025

020

015

010

005

000





119898as a






=119872HMGB1119872DNA

sdot119891

119903

=119872HMGB1119872DNA

sdot tan120572(3)






Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





=119872HMGB1119872DNA

sdot119891

119903

=119872HMGB1119872DNA

sdot tan120572(3)






Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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