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荧光能量共转移
荧光能量共转移

M OLECULAR AND C ELLULAR B IOLOGY,Feb.2003,p.1025–1033Vol.23,No.3 0270-7306/03/$08.00?0DOI:10.1128/MCB.23.3.1025–1033.2003

Interaction of Histone Acetylases and Deacetylases In Vivo Satoshi Yamagoe,1?Tomohiko Kanno,1Yuka Kanno,1Shigakazu Sasaki,1?

Richard M.Siegel,2Michael J.Lenardo,2Glen Humphrey,1

Yonghong Wang,1Yoshihiro Nakatani,3Bruce H.Howard,1

and Keiko Ozato1*

Laboratory of Molecular Growth Regulation,National Institute of Child Health and Human Development,1

and Laboratory of Immunology,National Institute of Allergy and Infectious Diseases,2National Institutes

of Health,Bethesda,Maryland20892,and Dana Farber Cancer Institute and Harvard

Medical School,Boston,Massachusetts021153

Received12June2002/Returned for modi?cation1August2002/Accepted29October2002

Having opposing enzymatic activities,histone acetylases(HATs)and deacetylases affect chromatin and

regulate transcription.The activities of the two enzymes are thought to be balanced in the cell by an unknown

mechanism that may involve their direct https://www.docsj.com/doc/1f10654937.html,ing?uorescence resonance energy transfer analysis,we

demonstrated that the acetylase PCAF and histone deacetylase1(HDAC1)are in close spatial proximity in

living cells,compatible with their physical interaction.In agreement,coimmunoprecipitation assays demon-

strated that endogenous HDACs are associated with PCAF and another acetylase,GCN5,in HeLa cells.We

found by glycerol gradient sedimentation analysis that HATs are integrated into a large multiprotein HDAC

complex that is distinct from the previously described HDAC complexes containing mSin3A,Mi-2/NRD,or

CoREST.This HDAC-HAT association is partly accounted for by a direct protein-protein interaction observed

in vitro.The HDAC-HAT complex may play a role in establishing a dynamic equilibrium of the two enzymes

in vivo.

Speci?c lysines on the core histones are acetylated by a series of histone acetylases(HATs).The status of acetylation constitutes one basis for the histone code,an important basis of chromatin-mediated regulatory processes,including tran-scription,replication,and chromosome dynamics(12,23,29). Acetylation of histone tails can be reversed by a diverse series of histone deacetylases(5,15).

Both HATs and histone deacetylases are classi?ed into sev-eral distinct groups.The GNAT family of HATs,one of the best-studied families,is conserved throughout eukaryotes(23, 28).In mammalian species there are two GNAT members, GCN5and PCAF.They are structurally similar to each other and are expressed in overlapping sets of cells and tissues.They predominantly regulate acetylation of histone H3and are gen-erally involved in transcriptional activation(12,25,39).Both GCN5and PCAF form large multiprotein complexes whose compositions are also conserved(9,21,23,28).Among histone deacetylases,class I histone deacetylases(HDACs)were the ?rst to be identi?ed and are conserved from yeasts such as Saccharomyces cerevisiae to humans.Four HDACs are cur-rently known in humans:HDAC1,HDAC2,HDAC3,and HDAC8(15).These histone deacetylases are generally associ-ated with transcriptional repression mediated by various DNA binding transcription factors(5,15,20,24).HDAC1and HDAC2form at least three distinct complexes that contain representative factors,mSin3A,Mi-2/NRD,and CoREST/ kiaa0071(13,32,40,44,45,46).HDAC3also forms a complex that contains N-CoR and SMRT,among other components (10,16,36).

Although rapid progress has been made on understanding the structure and function of individual HATs and HDACs,a remaining question is how the activities of these two enzymes, which exert opposite functions,are mutually balanced in the cell(23).A series of genetic studies and promoter analyses suggest that the two enzymes may not act independently and that their activities in some cases may be linked to one another (22,34,37).Other lines of evidence indicate that some HATs and HDACs occupy the common space in the nucleus and coordinately regulate the same set of target genes.For exam-ple,transcriptional activation mediated by nuclear receptors such as retinoic acid receptor and retinoid X receptor involves ligand-dependent association and dissociation of HAT and HDAC,respectively,on a given promoter(2,38).Coordinated activity of the two enzymes may also be inferred for cell growth-regulated genes controlled by E2F,whose promoter activity is repressed by the HDAC-associated retinoblastoma protein but is activated by subsequent HAT recruitment(5). Furthermore,YY1and Sp1interact with both HATs and HDACs,thereby acquiring an activator or repressor function depending on promoter context and other factors(5).A close interrelationship between the two enzymes may also be pre-sumed,based on the earlier observation that histones are rap-idly acetylated and deacetylated with a half-life of less than10 min in some regions of a nucleus while in other regions histone acetylation is turned over more slowly(4,6).More-recent

*Corresponding author.Mailing address:Laboratory of Molecular Growth Regulation,National Institute of Child Health and Human Development,Bldg.6,Rm.2A01,National Institutes of Health,Be-thesda,MD20892-2753.Phone:(301)496-9184.Fax:(301)480-9354.

E-mail:ozatok@https://www.docsj.com/doc/1f10654937.html,.

?Present address:Department of Bioactive Molecules,National Institute of Infectious Diseases,Tokyo,Japan.

?Present address:Second Division of Internal Medicine, Hamamatsu University School of Medicine,Hamamatsu,Japan.

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studies demonstrate that HATs and HDACs are engaged in a rapid cycle of global histone acetylation and deacetylation that affects the whole yeast genome(1,14,34).The global,untar-geted alteration of histone acetylation is likely to be critical for rapid reversal of targeted chromatin modi?cation in a speci?c promoter associated with transcriptional activation and/or re-pression(14,34).

In this work we wished to address the mechanisms that may balance the activities of the two enzymes.We surmised that among the mechanisms that help coordinate their activities, one might involve a physical interaction between the two en-zymes.To search for evidence indicating the presence of HDAC-HAT interaction in vivo,we?rst employed a novel ?ow cytometry technique based on?uorescence resonance en-ergy transfer(FRET)(26,30).This technique allows an as-sessment of molecular interactions between two proteins in the living cell.Although not heretofore applied extensively to the analysis of nuclear events,this approach provides a powerful new tool to investigate the molecular behavior of transcription factors and chromatin modi?ers in the nucleus.By introducing PCAF and HDAC1labeled with distinct?uorochromes into HeLa cells,we observed clear FRET signals ascribable to their physical https://www.docsj.com/doc/1f10654937.html,ing coimmunoprecipitation assays,it was shown that HDAC1,HDAC2,and HDAC3are all associated with GCN5and PCAF in HeLa cells.Glycerol gradient sedi-mentation analysis of HDAC1complexes revealed that GCN5 is contained in a large multiprotein complex(es)distinct from the three HDAC complexes reported before.In vitro binding analysis indicated that the HATs are incorporated into the HDAC complex(es)at least in part by directly binding to HDACs.Finally,we present evidence suggesting that HDAC-HAT interactions occur in a dynamic fashion depending on the physiological state of cells.Taken together,these results point to a mechanism that internally maintains HDAC-HAT equi-librium in the cell.

MATERIALS AND METHODS

FRET analysis.Full-length hPCAF(42),hHDAC1(31),and ICSBP(17)were cloned into cyan?uorescent protein(CFP)and yellow?uorescent protein(YFP) expression vectors from Clontech,creating ECFP-C1-PCAF,EYFP-N1-HDAC1,and https://www.docsj.com/doc/1f10654937.html,ing Polyfect(Qiagen),HeLa cells(8?105) were transfected with a total of3?g of plasmid DNA encoding ECFP and YFP fusion proteins.At24h following transfection,cells were harvested in phos-phate-buffered saline and FRET analysis was performed on a FACS Vantage cytometer(Becton Dickinson)as previously described(26).EYFP was excited with an air-cooled argon laser tuned to514nm,and signals were detected with a546-to-10-nm bandpass?lter.ECFP was separately excited with a krypton laser tuned to413nm,and signals derived from ECFP and EYFP due to FRET were simultaneously detected with470-to-20-nm and546-to-10-nm bandpass?lters, respectively.Data were analyzed by FlowJo software.Microscopic images of live cells were obtained with a Leica TCS SP2confocal microscope. Immunoprecipitation and sedimentation analysis of HDAC complexes.HeLa cells were infected with a retrovirus vector expressing Flag-tagged HDAC1, HDAC2,or HDAC3as previously described(13).Nuclear extracts were pre-pared from?107to109cells as previously described(7)and were dialyzed against a buffer containing20mM Tris-HCl(pH7.5),100mM NaCl,0.2mM EDTA,10%glycerol,1mM phenylmethylsulfonyl?uoride,and10mM?-mer-captoethanol and readjusted to300mM NaCl supplemented with0.1%Tween 20before use.Extracts(10to30mg)were absorbed to anti-Flag M2af?nity gel (Sigma),washed,and eluted with Flag peptides(200?g/ml).For immunopre-cipitation of endogenous HDAC1,extracts from uninfected HeLa cells prepared in the same buffer as described above and containing150mM NaCl were precipitated with anti-HDAC1antibody conjugated to agarose beads.Bound proteins were eluted with100mM glycine(pH2.5)–0.1%Tween20(13).Eluted samples were resolved by sodium dodecyl sulfate–4to20%polyacrylamide gel electrophoresis(SDS–4to20%PAGE)and immunoblotted with rabbit antibod-ies to GCN5and PCAF(21),HDAC1,Mta-L1,MBP2,Kiaa0601and Kiaa0071 (13),Mi-2(Upstate Biotechnology),RbAp48(GeneTex),mSin3A(Santa Cruz), or Flag peptide(Sigma).The same procedure was used for analysis of U937cell extracts.Sedimentation analysis of Flag-HDAC1complexes was performed with 200?l of anti-Flag antibody eluates obtained from HeLa cells expressing Flag-tagged HDAC1in a10to35%glycerol gradient in20mM Tris-HCl(pH 8.0)–100mM NaCl as previously described(13).A total of26fractions,contain-ing170?l each,were collected and analyzed by silver staining and immunoblot-ting.Chromatographic fractionation of Flag-HDAC1immunoprecipitates was performed as follows.Nuclear extracts from HeLa cells(3?109to6?109cells) were af?nity puri?ed on anti-Flag M2agarose.Bound materials were eluted in buffer A(20mM Tris-HCl[pH8.0],100mM NaCl,5mM MgCl2,1mM dithiothreitol,10%glycerol)containing Flag peptides(200?g/ml).They were diluted in20mM NaCl and fractionated on a DEAE-Sepharose column(bed volume,100?l)(Amersham Pharmacia Biotech)with a stepwise elution in buffer A(containing100mM,300mM,500mM,and1M NaCl).Each fraction was dialyzed against20mM Tris-HCl(pH8.0),concentrated with a SpeedVac Con-centrator(Sovant),and analyzed by Western blotting.

Recombinant HDACs and PCAF deletions.Full-length HDAC1tagged with the Flag epitope at the C terminus and PCAF tagged with the Flag epitope at the N terminus and their deletions were cloned by a BaculoGold(Pharmingen)or a Bac-to-Bac(GIBCO-BRL)system into baculovirus vectors.Recombinant pro-teins were af?nity puri?ed from extracts prepared from infected Sf9cells(mul-tiplicity of infection?10).Brie?y,cells suspended in a buffer containing50mM Tris-HCl(pH8.0),20%glycerol,0.2mM EDTA,600mM NaCl,and0.1% Triton X-100were subjected to freezing and thawing.Supernatants were applied on M2anti-Flag antibody beads and eluted with buffer supplemented with100 mM NaCl containing100?g of Flag peptide/ml.Puri?cation of recombinant histidine-tagged HDAC2was as previously described(24).

In vitro binding assays.Sf9extracts containing recombinant PCAF(100ng) were incubated with in vitro transcribed and translated35S-labeled HDAC1at 4°C for1h and precipitated with anti-PCAF antibody conjugated to protein G Sepharose beads(2).Bound materials were resolved on SDS–10%PAGE and visualized by?uorography.To test binding of full-length and truncated PCAF, recombinant HDAC2(24)was conjugated to Ni2?-nitrilotriacetic acid(NTA) agarose beads(Qiagen)in NS-1-10buffer(20mM Tris-HCl[pH8.0],10% glycerol,1mM EDTA,0.01%Triton X-100,10mM imidazole,0.5mM phenyl-methylsulfonyl?uoride,10mM?-mercaptoethanol)containing2%bovine se-rum albumin and incubated with50ng or500ng of full-length or truncated recombinant PCAF at4°C for1h.Proteins were eluted with NS-1-10buffer with 200mM imidazole and were evaluated by immunoblot analysis using anti-PCAF antibody.

HAT and deacetylase enzymatic activities.HAT assays were performed as previously described(2,25).[3H]acetyl coenzyme A(Amersham)(5nmol)was incubated with2?g of core histones(Sigma)in30?l of each reaction mixture at30°C for10to30min,and the reaction mixtures were separated on SDS–4to 20%PAGE.Deacetylase assays were performed as previously described(13,24).

RESULTS

FRET analysis reveals spatial proximity of PCAF and HDAC1in living cells.The previous observations that HATs and HDACs generate a dynamic equilibrium of chromatin acetylation across the genome(14,34)suggest the possibility

that HATs and HDACs are closely associated with each other in at least some regions of the nucleus.To investigate the molecular proximity of HAT and HDAC in real time in living cells,we have employed a novel,?ow cytometry-based FRET technique(19,30).This type of FRET analysis utilizes two proteins fused to green?uorescent protein variants CFP and YFP that are transfected into appropriate cells.When a CFP protein and a YFP are in proximity with each other,excitation of the CFP triggers a?uorescent emission of the YFP.CFP and YFP interact with maximal energy transfer at approxi-mately50to60A?(26)(for reference,the diameter of a nu-cleosome is110A?).Given this fact and the assumption that a globular form of HDAC1and PCAF would have a diameter of

1026YAMAGOE ET AL.M OL.C ELL.B IOL.

?50to 60A

?,the presence of FRET would indicate a direct interaction of the two proteins.We have used the ?ow cytom-etry-based FRET procedure for this analysis,rather than a single-cell-based microscopic procedure,since it enables the detection of FRET in a large,heterogeneous population of cells (26).

HeLa cells were cotransfected with CFP-PCAF along with YFP-HDAC1.As a control,CFP-PCAF was also transfected along with YFP-ICSBP.The latter protein is a nuclear tran-scription factor that forms a multiprotein complex but does not interact with PCAF (18).Fluorescence microscopy analysis of transfected cells (Fig.1A)showed that CFP-CAF and YFP-HDAC1localized to the nucleus as ?ne speckles,with the CFP and YFP signals apparently colocalizing in most areas of the nucleus except for those of the nucleoli.Figure 1D to H show the results of ?ow cytometry analysis of CFP and YFP signals detected by the emission spectrum characteristic of each ?uo-rochrome.YFP-HDAC1and YFP-ICSBP were detected in

?40to 50%of transfected cells,irrespective of whether cells were cotransfected with CFP-PCAF or transfected alone.Fig-ure 1C and E show that CFP-PCAF and YFP-HDAC1(or YFP-ICSBP)were coexpressed in most of the transfected cells (the transfection ef ?ciency of CFP-PCAF was also ?40to 45%).FRET signals were monitored,as shown in the lower middle panels (I to M)of Fig.1.As expected,when CFP-PCAF or YFP-HDAC1was expressed alone,no FRET signals were detected (Fig.1I and J).In contrast,when CFP-PCAF was coexpressed with YFP-HDAC1,6.5%of YFP-positive cells exhibited FRET signals (panel K),indicating that PCAF and HDAC1were spatially close to each other in these cells.The relatively low percentage of cells that showed FRET sig-nals may be due to the fact that CFP-PCAF was expressed at a somewhat low level and/or to the possible interaction of CFP-PCAF with endogenous HDACs abundantly expressed in HeLa cells (see below).To ascertain whether the FRET signals detected by PCAF and HDAC1were attributable to

their

FIG.1.FRET detection of PCAF-HDAC1interaction in living cells.HeLa cells were transfected with CFP-PCAF and YFP-HDAC1or YFP-ICSBP (control).Microscopic images of live cells are shown in panels A to C.In panels D to H,relative ?uorescence levels of CFP and YFP in individual transfected cells are presented as contour plots re ?ecting the number of cells exhibiting either one or both ?uorochrome signals.YFP-positive populations (shown in rectangles within panels)were subjected to YFP-FRET plotting (I to M).Each number represents the percentage of cells in the compartment.The FRET signals detected are presented in panel K.Panels N and O show overlays of the FRET histograms obtained with HDAC1-or ICSBP-positive cells.

V OL .23,2003THE HAT-HDAC COMPLEX IN VIVO 1027

physical proximity rather than mere coexpression in the nu-cleus,a similar analysis was performed with CFP-PCAF and YFP-ICSBP(Fig.1L and M).No FRET signals were observed with this combination.The absence of FRET signals from ICSBP was not due to the amounts of protein expressed,since YFP-ICSBP was expressed at levels higher than those of YFP-HDAC1.Likewise,YFP-ICSBP did not exhibit FRET signals with CFP-HDAC1(data not shown).An overlay of the corre-sponding histograms of the FRET signals elicited by PCAF and HDAC1is shown in panels N and O in Fig.1,in which a prominent FRET-positive population is evident in the cells coexpressing CFP-PCAF and YFP-HDAC1but not in the cells coexpressing CFP-PCAF and YFP-ICSBP.We also found FRET signals by analysis of PCAF and HDAC1labeled with the alternate?uorochrome(i.e.,YFP-PCAF and CFP-HDAC1)(data not shown).These results led us to conclude that a signi?cant fraction of transfected PCAF and HDAC1 are in close proximity to each other in living cells. Endogenous HDAC1forms a complex with GCN5and PCAF in HeLa cells.Because the FRET results relied on ectopically expressed HAT and HDAC,it was of importance to assess whether endogenous HDACs and HATs interact with each other.To investigate biochemical evidence for HDAC-HAT interaction,coimmunoprecipitation analysis was performed with antibody speci?c for HDAC1.For this purpose,HeLa cell nuclear extracts adjusted in a buffer with150mM to300mM NaCl were tested.To preserve complexes of weak associations, extracts were not processed by a urea-based chromatography method previously employed for puri?cation of stable HDAC complexes(13).Precipitated materials were immunoblotted with antibody for the endogenous GCN5.As shown in Fig.2A, HDAC1antibody coprecipitated GCN5along with HDAC1, RbAp48,Sin3A,and other components known to be contained in the stable HDAC1complexes(13,32,44–46).Preimmune sera did not precipitate HDAC1or GCN5,indicating that GCN5was speci?cally associated with HDAC1.We assessed whether the association of HDAC1with GCN5described above is mediated by DNA by including ethidium bromide in immunoprecipitation experiments(43).A comparable level of GCN5was coprecipitated in the presence of1.2?g of ethidium bromide/ml(data not shown).In addition,immunoblot analy-sis of precipitated materials did not reveal an appreciable amount of histone H3or H4,indicating that this association is not dependent on the presence of chromatin.

Another GNAT member,PCAF,was not detected in the HDAC1immunoprecipitates at an appreciable level,most likely because PCAF is expressed at a very low level in HeLa cells(27,39).Because PCAF and GCN5are75%identical to each other in their amino acid sequences(39),it was of interest to test whether ectopically expressed PCAF associates with the HDAC1complexes.As shown in Fig.2B,extracts from HeLa cells transduced with a retrovirus vector with Flag-tagged PCAF(21)were immunoprecipitated with antibody for HDAC1as described above and precipitated materials were tested for Flag-PCAF.Flag-PCAF was clearly coprecipitated

along with HDAC1,as detected by both anti-PCAF antibody and anti-Flag antibody.As expected,Flag-PCAF was not pre-cipitated when tested with preimmune sera.

To further establish the association of GCN5with HDAC1, coimmunoprecipitation analysis was performed with HeLa cells expressing Flag-tagged HDAC1(13).In Fig.2C,extracts were immunoprecipitated with M2anti-Flag antibody and blotted with antibody for GCN5.GCN5was coprecipitated by anti-Flag antibody,along with Flag-HDAC1and its

compo-FIG.2.Endogenous HDAC1and Flag-tagged HDAC1associate with GCN5and PCAF in HeLa cells.(A)Nuclear extracts were pre-cipitated with preimmune sera or anti-HDAC1antibody conjugated to agarose beads.Eluted materials were immunoblotted with antibody for GCN5(top panel)or the indicated proteins(bottom panels).The input represents immunoblot analysis of5%of total extracts.(B)Ex-tracts from HeLa cells expressing Flag-tagged PCAF were immuno-precipitated with anti-HDAC1antibody and subjected to blotting for PCAF(top)or other proteins as described for panel A.(C)Extracts from HeLa cells infected with Flag-tagged HDAC1(Flag-HDAC1)or control virus(Control)were immunoprecipitated with M2anti-Flag antibody,eluted with Flag peptide,and analyzed by immunoblotting as described for panel A.(D)Enzymatic activity of HDAC1immunopre-cipitates.HAT(top panel)and HDAC(bottom panel)activities were measured with30?l and100?l,respectively,of eluates obtained from the experiments described for panel C.Increasing the amounts of control eluates revealed neither HAT nor HDAC activity.

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nents RbAp48,Sin3A,and Mta-L1.However,cells transduced with control vector did not precipitate HDAC1or GCN5. Coomassie blue staining of the precipitated materials indicated that HDAC1was in large excess over GCN5in the precipi-tates.In addition,a relatively small proportion(?5%)of total GCN5in the cells associated with HDAC1complex(data not shown).

Given that HATs are associated with HDAC1,it was of interest to examine whether HAT activity is associated with the HDAC1immunoprecipitates.As shown in Fig.2D,extracts from cells expressing Flag-HDAC1were precipitated with an-ti-Flag antibody and the precipitated materials were tested for HAT and deacetylase enzymatic activities.Interestingly,a sig-ni?cant HAT activity was detected with precipitates from cells expressing Flag-HDAC1;the levels of activity detected in30?l of eluates in Fig.2D corresponded to?3ng of recombinant PCAF.In contrast,no acetylase activities were detected in eluates from control cells(Fig.2D,top panel).As expected, deacetylase activity was also detected in the same Flag-HDAC1samples but not in control samples(Fig.2D,lower panel).These results indicate that HDAC1complexes possess both HAT and deacetylase activities,further supporting the association of HDAC1and HAT.

PCAF and GCN5interact with members of another acety-lase family,p300and CBP(28,41).Since p300and CBP display a slight structural similarity to GNAT family members (23),we examined whether these proteins are also coprecipi-tated with HDAC1.Tests with various available antibodies against p300and CBP did not reveal either protein in the HDAC1immunoprecipitates(data not shown).PCAF forms a large multiprotein complex that contains a set of conserved proteins,such as ADA2and ADA3,and GCN5forms a similar complex(9,21,33).To assess whether these GCN5-and PCAF-containing complexes associate with HDAC1,we searched for ADA2and ADA3,as well as for PAF65?and

hSPT3,components of the HAT complex(33),in the HDAC1 and Flag-HDAC1immunoprecipitates.These proteins were not detected in either precipitate(data not shown),indicating that GCN5and PCAF incorporated into the HDAC1complex are independent of the previously described stable HAT com-plexes.It should also be noted that reciprocal immunoprecipi-tation in which Flag-PCAF was immunoprecipitated?rst,fol-lowed by immunoblotting for HDAC1,did not reveal a detectable level of HDAC1in the eluates(data not shown). Although the basis for the absence of HDAC1in Flag-PCAF immunoprecipitates is not entirely clear,it is possible that PCAF incorporated into HDAC1complexes is sequestered and not readily accessible to the antibody.

Other class I histone deacetylases also form a complex with GCN5.Class I deacetylases share a common structural fold(5, 15).HDAC1and HDAC2form stable multiprotein complexes that are similar to each other;in addition,HDAC1and HDAC2complexes can form a dimer(13).HDAC3,although it also forms stable complexes,appears to associate with unique components SMRT and N-CoR(16).To determine whether association with HATs is a general property among class I deacetylases,coimmunoprecipitation analysis was per-formed with Flag-tagged HDAC2and HDAC3expressed in HeLa cells.As shown in Fig.3,GCN5was coprecipitated along with Flag-HDAC2and Flag-HDAC3.These data show that all class I deacetylases can associate with GCN5. Sedimentation analysis of the HDAC1-GCN5complex. HDAC1and HDAC2form at least three distinct complexes containing,respectively,(i)mSin3A(11,20,45),(ii)Mi-2/ NRD(32,35,40,46),and(iii)CoREST/Kiaa0071(13,44). These complexes are each at minimum200to400kDa and are thought to assume distinct functions,e.g.,transcriptional re-pression by the mSin3complex and chromatin remodeling by the NRD complex.To assess whether HDAC-associated GCN5is contained in any of the above complexes or whether it exists in a separate complex,immunoprecipitated Flag-HDAC1complexes were subjected to velocity sedimentation analysis in glycerol gradient(13).Sediments were separated into26fractions corresponding to sedimentation coef?cients of 4to28S.Figure4illustrates the results of silver staining(Fig. 4A,top two panels)and immunoblot analysis(4B and remain-ing panels of4A)of each of the fractions.The majority of HDAC1was found in fractions8through12,containing RbAp48,mSin3A,Mta-L1and Mi-2,and CoREST/kiaa0071. Importantly,HDAC1was also found in the fractions exhibiting more-rapid sedimentation(factions13through17),with sed-imentation coef?cients ranging from15to26S.The bulk of the GCN5detected was found in this most rapidly

sedimenting FIG.3.Flag-tagged HDAC2and HDAC3associate with GCN5in HeLa cells.Extracts from HeLa cells infected with control virus or with recombinant virus with Flag-tagged HDAC1,HDAC2,or HDAC3 were immunoprecipitated with M2anti-Flag antibody as described for Fig.2and subjected to blotting analysis for GCN5and other indicated proteins.

V OL.23,2003THE HAT-HDAC COMPLEX IN VIVO1029

fraction(s),peaking roughly at 21S.The GCN5-containing fractions may represent a previously unidenti ?ed large HDAC1complex,because they have little RbAp48,mSin3A,MtaL1,and Kiaa0071and because silver staining reveals sev-eral protein bands not present in fractions below 13S.The predicted average molecular mass of the GCN5-containing fractions is ?1MDa.Considering the broad spread over mul-tiple fractions,the GCN5-containing fractions may represent several heterogeneous complexes.These results indicate that HDAC1can form a large complex(es)distinct from the previ-ously described complexes that contain GCN5.Although re-producibly detected in glycerol gradient analyses,the GCN5-containing complex(es)appears to represent a relatively small component of the total HDAC1(?5%).We also noted that a certain amount of HDAC1was detected in the fractions rep-resenting a smaller molecular mass corresponding to ?5S (Fig.4).Some of these components likely represent the HDAC core recruitment complex described by Zhang et al.(46).

To obtain additional evidence of an HDAC1complex(es)containing GCN5,we have fractionated the Flag-HDAC1im-munoprecipitates by DEAE-Sepharose chromatography.Fig-ure 4B shows the results of immunoblot analysis of materials eluted stepwise with increasing concentrations of NaCl.HDAC1was mostly found in two fractions,namely,the ?ow-through and the 300mM NaCl eluates,but not in the 100mM,500mM,or 1M NaCl eluates.The HDAC-associated compo-nents,Sin3A,Mi-2,Mta-L1,and Kiaa0071,were predomi-nantly eluted with 300mM NaCl,indicating that this fraction corresponds to the previously described stable HDAC com-plexes (13,32,40,44–46).In contrast,GCN5was found almost totally in the ?ow-through fraction with a trace amount in the 300mM NaCl eluates.A signi ?cant amount of RbAp48ap-peared to be present in the ?ow through,as well as in 300mM NaCl eluates.These results support the presence of a GCN5-

containing HDAC1complex that is distinct from the previously described HDAC complexes.

HDACs interact with PCAF in vitro.It was of importance to determine whether the association of HAT with HDAC is attributable to a direct protein-protein interaction or to an indirect association involving a third protein.As seen in the results shown in Fig.5A,we tested whether HDAC1binds to PCAF.35S-labeled,in vitro-translated HDAC1was incubated with recombinant PCAF and immunoprecipitated with anti-PCAF antibody.Radiolabeled HDAC1,but not radiolabeled luciferase,run as a control was coimmunoprecipitated with PCAF.Anti-PCAF antibody did not precipitate HDAC1when incubated with an unrelated recombinant protein,ICSBP,ver-ifying speci ?c immunoprecipitation.A direct interaction be-tween PCAF and HDAC2was also observed (Fig.5B).Histi-dine-tagged,recombinant HDAC2conjugated to Ni 2?agarose beads (24)was incubated with increasing amounts of recom-binant PCAF in these experiments,and bound PCAF was detected in immunoblot analysis.PCAF bound to HDAC2in a dose-dependent manner but not to control beads conjugated to control protein,ICSBP.

Using deletion analysis,we assessed domains of PCAF in-volved in the interaction with HDAC2.As shown in Fig.5C,recombinant PCAF deletion mutants were incubated with full-length HDAC2conjugated to Ni 2?beads.While constructs ?444-512and ?511-656bound to HDAC2,constructs 1-529and ?65-464did not,indicating that an N-terminal region encompassing amino acids 65to 464and the C-terminal region encompassing amino acids 656to 832are required for inter-action with HDAC2.These results indicate that HDACs and PCAF can interact with each other through protein-protein interaction.

Alteration of the HDAC1-HAT complex during U937cell differentiation.In an effort to understand the potential role of the HDAC-HAT complex(es),we sought to examine whether the formation of the HDAC-GCN5complex changes during differentiation.For this purpose,a macrophage differentiation model using U937cells was tested in which the

myelomono-

FIG.4.Sedimentation analysis of HDAC1-GCN5complex(es).(A)Using M2anti-Flag antibody agarose beads,Flag-HDAC1com-plexes in HeLa cells were isolated and subjected to 10to 35%glycerol gradient centrifugation.Each fraction was analyzed on SDS –10%PAGE.Upper two large panels:silver-staining patterns.Markers used were aldolase (7.3S),catalase (11.3S),thyroglobulin (19S),and 28S rRNA,as indicated at the bottom of the two large panels.Lower panels:immunoblot detection of indicated proteins.(B)Flag-HDAC1complexes were fractionated on a DEAE-Sepharose column and eluted stepwise with buffer containing the indicated NaCl concentra-tions,and proteins were identi ?ed by immunoblotting analysis.

1030YAMAGOE ET AL.M OL .C ELL .B IOL .

cytic cells undergo differentiation in response to phorbol ester tetradecanoyl phorbol acetate (TPA)(3,17).TPA treatment induced differentiation within 3to 4days,coinciding with in-creased adherence of cells to the petri dish and growth arrest.As shown in Fig.6,nuclear extracts were prepared from cells treated with TPA for 4days and tested for expression of HDAC1and GCN5.As seen in the input lane,expression levels of HDAC1and GCN5were markedly reduced follow-ing TPA treatment in U937cells,as measured on a protein content basis.Additionally,other proteins,including RbAp48,mSin3A,Mta-L1,and Kiaa0601,were also proportionally re-duced in their levels,indicating that relative levels of these components were unchanged after TPA treatment.As shown in Fig.6A,extracts were immunoprecipitated with antibody for the endogenous HDAC1and tested for GCN5and other pro-teins by immunoblotting.Endogenous GCN5was coprecipi-tated along with HDAC1as well as other HDAC1components from untreated U937cells,establishing that the association of

HAT with HDAC1is not restricted to HeLa cells.Surprisingly,HDAC1precipitates from TPA-treated cells contained little GCN5,with levels almost undetectable by immunoblot assays (see quanti ?cation in Fig.6A).The paucity of GCN5was striking,since other HDAC1components such as mSin3A and Mta-L1as well as Kiaa0601were coprecipitated from TPA-treated cells at levels comparable to or exceeding that in un-treated cells.Quanti ?cation of the coprecipitated proteins (shown as the ratios in Fig.6A)veri ?ed a selective reduction of GCN5,suggesting that the association of GCN5with HDAC1is modulated upon differentiation in U937cells.PCAF ap-peared to be expressed at low levels in U937cells and was not coprecipitated with HDAC1at a detectable level before or after TPA treatment,suggesting that the reduction of GCN5in the HDAC1complex(es)was not due to the replacement of GCN5by PCAF.These results were reproducibly observed with samples treated with TPA for 3and 5days.To assess whether the reduction in the HDAC1-GCN5complex is a result of general growth arrest rather than differentiation,qui-escent and exponentially growing WI38?broblasts were tested by a similar coimmunoprecipitation analysis.No discernible difference was detected for the relative amounts of GCN5in the HDAC1precipitates in these materials (data not shown),indicating that the selective reduction of GCN5-HDAC1asso-ciation is not a mere result of growth inhibition.These results suggest that interaction of HDAC1with GCN5is dynamic and is in ?uenced by physiological processes such as differentiation.

DISCUSSION

The aim of this study was to explore how HATs and deacety-lases might mutually balance their activities in vivo.Our study began with the use of the novel ?ow cytometry-based FRET technique to search for an interaction of ?uorescent protein-tagged PCAF and HDAC1in living cells.Results

demon-

FIG.5.HDAC-PCAF interactions in vitro.(A)35S-labeled HDAC1or 35S-labeled luciferase was incubated with Flag-tagged re-combinant PCAF or a control protein (His-ICSBP)and coimmuno-precipitated with anti-PCAF antibody.Precipitated materials were de-tected by ?uorography.The input represented 10%of the total reaction mixture.(B)Sf9extracts containing recombinant His-HDAC2(?)or the control (His-ICSBP)(?)were conjugated to Ni 2?-NTA agarose beads and incubated with 50ng or 500ng of recombinant PCAF.Bound materials were analyzed by immunoblotting with anti-PCAF antibody.(C)PCAF domain analysis.HDAC2(?)or control protein (ICSBP)(?)immobilized to Ni 2?-NTA agarose was incubated with Flag-tagged PCAF deletions.Bound materials were detected by immunoblotting analysis with anti-FCAF

antibody.

FIG.6.Modulation of HDAC1-GCN5interaction in differentiat-ing U937cells.(A)U937cells were treated with TPA (20ng/ml)for 4days.Nuclear extracts were immunoprecipitated with anti-HDAC1antibody and evaluated by immunoblotting analysis for the indicated proteins as described for Fig.2A.The numbers indicate the ratios of precipitated proteins in untreated cells (assigned a value of 1)versus TPA-treated cells.(B)Model of an HDAC-HAT complex(es).A majority of HDACs and HATs form their own multiprotein complexes (top).A fraction of the two enzymes interact with each other and form an independent complex(es)(bottom),which may have a distinct func-tion.

V OL .23,2003THE HAT-HDAC COMPLEX IN VIVO 1031

strated that in some cells,the acetylase and deacetylase reside within50to60A?of each other,a distance close enough to form a direct protein-protein interaction.The physical prox-imity of tagged PCAF and HDAC1under these conditions is unambiguous,since another nuclear protein ICSBP displayed no FRET signals with either PCAF or HDAC1.To our knowl-edge this is the?rst demonstration of an in situ nuclear pro-tein-protein interaction detected by FRET on a cell population basis.These observations provided a strong impetus to further study the interaction of the endogenous enzymes and its bio-chemical characteristics.

Coimmunoprecipitation experiments(Fig.2and3)fur-nished a biochemical con?rmation that endogenous HDAC1is associated with endogenous GCN5.Furthermore,GCN5was coprecipitated with Flag-tagged HDAC1,as well as HDAC2 and HDAC3,the latter having been shown to associate uniquely with N-CoR and SMRT(16).In addition,PCAF, another GNAT family member,was coprecipitated with HDAC1when ectopically expressed in HeLa cells,indicating that the HDAC-HAT association is a general feature of the two enzymes.Supporting the presence of HAT in the com-plexes,Flag-tagged HDAC1immunoprecipitates exhibited both HAT and deacetylase enzymatic activities.

Glycerol gradient sedimentation analysis revealed that GCN5occurs in the fractions comprising a large protein com-plex(es)of an average size minimally estimated to be?1MDa. This complex(es)appeared distinct from the previously re-ported stable HDAC complexes of200to400kDa that contain representative factors,mSin3,Mi-2/NRD,and Co-REST/ Kiaa0071(13,32,44–46).The presence of a distinct GCN5-HDAC complex(es)was further supported by the cofraction-ation of GCN5and HDAC1on an ion-exchange column(Fig. 4B).The GCN5-containing complex(es)may be heteroge-neous in size and may contain a number of additional factors not included in the previously described HDAC complexes. GCN5and PCAF contained in the HDAC complex(es)were associated neither with P300/CBP nor with other components of the previously characterized,stable HAT complexes(21, 33).Thus,GCN5in the HDAC complex most likely represents an entity separate from the known HAT complexes and may have a distinct function(Fig.6B).The HDAC-HAT associa-tion is in part attributed to a direct interaction of the two proteins,since recombinant HDACs and PCAF bound to each other in vitro.However,considering that the two proteins are in a large complex(es)and are capable of complexing with various other proteins,interaction of the two enzymes is most likely stabilized by other proteins in vivo.

The existence of a HDAC-HAT complex(es)appears com-patible with the long-held notion that there is a mechanism for establishing and maintaining an equilibrium between HAT and deacetylase activities(23).It has been known that in vivo core histones are rapidly acetylated and deacetylated with a half-life of less than10min in?15%of chromatin(4).The chromatin regions that support rapid histone acetylation turnover are thought to coincide with the regions of high transcriptional activity where HATs and deacetylases can coexist(6).Thus, the HDAC-HAT complex(es)found in the present study may reside in this compartment of chromatin and participates in transcription.For example,the complex may play a role in ligand-dependent transcription by nuclear receptors,providing timely activation and repression of hormone-dependent gene expression.Consistent with this idea,HDACs and HATs both interact with a number of nuclear receptors(2,38).In a similar context,signal-induced transcription is often followed by a rapid reversal of transcription leading to the restoration of basal gene expression.In these situations the HDAC-HAT complexes may help?nely coordinate acetylation and deacety-lation of local promoters.In support of this idea,there are other transcription factors such as YY1and Sp1(5)which interact with both HDAC and HAT and which may play a part in coordinated activation and repression of speci?c promoters. On the other hand,the HDAC-HAT complexes may act in a more global manner,affecting a genome-wide state of his-tone acetylation(1,14,34).It has been shown that yeast core histones are rapidly and globally acetylated and deacetylated, creating a constant,dynamic?ux in histone acetylation status. This rapid cycle of histone acetylation and deacetylation is dependent on the activity of gcn5and rpd3,a yeast HDAC (34).It is possible that the HDAC-HAT complex(es)found in the present study contributes to this untargeted,globally acting enzymatic activity.This global acetylation and deacetylation is shown to confer a rapid return of transcription to a ground state,following activation or repression,occurring within a matter of minutes(14).Although this rapid,untargeted acet-ylation and deacetylation is likely to be important for transcrip-tional regulation,it may also be involved in a wider range of biological activities such as DNA replication and differentia-tion(1).

Lastly,we have shown that the GCN5-HDAC1association is markedly reduced in U937cells following TPA-induced differ-entiation.This result is interesting,since global alterations of chromatin structures and changes in transcriptional patterns are the main features of cellular differentiation(8).Our data indicate that assembly of HAT into the HDAC complex(es)is a dynamic process and is closely tied to the state of chromatin and cellular activities.

In conclusion,this study demonstrates that through a phys-ical interaction,a certain fraction of HAT is integrated into a large HDAC complex(es)distinct from those described previ-ously.The HDAC-HAT complex(es)may have a role in coor-dinating acetylation and deacetylation of chromatin affecting many cellular activities.

ACKNOWLEDGMENTS

We thank S.Schreiber and E.Seto for HDAC plasmids,T.Howard for viral transduction of HeLa cells,and R.Swofford and K.Holmes for?ow cytometry analysis.

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1荧光共振能量转移 原理 如果两个荧光团相距在1~10 nm之间,且一个

1.荧光共振能量转移 原理 如果两个荧光团相距在1~10 nm之间,且一个荧光团的发射光谱与另一个荧光团的吸收光谱有重叠,当供体被入射光激发时,可通过偶极-偶极耦合作用将其能量以非辐射方式传递给受体分子,供体分子衰变到基态而不发射荧光,受体分子由基态跃迁到激发态,再衰变到基态同时发射荧光。这一过程称为荧光共振能量转移(fluorescence resonance energy transfer,FRET)。 优点 1.适用于活细胞和固定细胞的各类分子, 2.灵敏度和分辨率高,并能清晰成像, 3.准确度高,操作简便 4.最直观地提供蛋白质相互作用的定位和定量信息, 缺点 首先,FRET对空间构想改变十分敏感,其测量范围在1~10 nm,但如果待测蛋白原本就相当接近, FRET信号已经达到最大值,此时一些刺激引起的微小的构想改变就可能无法引起FRET信号的很大改变; 其次,存在光漂白作用, FRET需要起始激发光激发D,这时就很难避免对A的间接激发,这样的交叉激发降低了分析的灵敏性; 第三,存在其他一些本底荧光的干扰; 另外,起始激发光可能会破坏一些光敏的组织和细胞,产生光毒性。这些缺点很大程度上限制了FRET的进一步发展。

2.蛋白质双杂交技术 原理 以与调控SUC2基因有关的两个蛋白质Snf1和Snf2为模型, 将前者与Gal4的DB结构域融合, 另外一个与Gal4的AD结构域的酸性区域融合。由DB和AD形成的融合蛋白现在一般分别称之为“诱饵”(bait)和“猎物”或靶蛋白(prey or target protein)。如果在Snf1和Snf2之间存在相互作用, 那么分别位于这两个融合蛋白上的DB和AD就能重新形成有活性的转录激活因子, 从而激活相应基因的转录与表达。这个被激活的、能显示“诱饵”和“猎物”相互作用的基因称之为报道基因(reporter gene)。通过对报道基因表达产物的检测, 反过来可判别作为“诱饵”和“猎物”的两个蛋白质之间是否存在相互作用。 酵母双杂交系统的优点及局限 双杂交系统的另一个重要的元件是报道株。报道株指经改造的、含报道基因(reporter gene)的重组质粒的宿主细胞。最常用的是酵母细胞,酵母细胞作为报道株的酵母双杂交系统具有许多优点: 〈1〉易于转化、便于回收扩增质粒。〈2〉具有可直接进行选择的标记基因和特征性报道基因。〈3〉酵母的内源性蛋白不易同来源于哺乳动物的蛋白结合。一般编码一个蛋白的基因融合到明确的转录调控因子的DNA-结合结构域(如GAL4-bd,LexA-bd);另一个基因融合到转录激活结构域(如GAL4-ad,VP16)。激活结构域融合基因转入表达结合结构域融合基因的酵母细胞系中,蛋白间的作用使得转录因子重建导致相邻的报道

荧光共振能量转移技术的基本原理和应用

荧光共振能量转移技术的基 本原理和应用 -标准化文件发布号:(9456-EUATWK-MWUB-WUNN-INNUL-DDQTY-KII

荧光共振能量转移技术的基本原理和应用荧光共振能量转移(fluorescence resonance energy transfer,FRET)作为一种高效的光学“分子尺”,在生物大分子相互作用、免疫分析、核酸检测等方面有广泛的应用。在分子生物学领域,该技术可用于研究活细胞生理条件下研究蛋白质-蛋白质间相互作用。蛋白质-蛋白质间相互作用在整个细胞生命过程中占有重要地位,由于细胞内各种组分极其复杂,因此一些传统研究蛋白质-蛋白质间相互作用的方法如酵母双杂交、免疫沉淀等可能会丢失某些重要的信息,无法正确地反映在当时活细胞生理条件下蛋白质-蛋白质间相互作用的动态变化过程。FRET技术是近来发展的一项新技术,为在活细胞生理条件下对蛋白质-蛋白质间相互作用进行实时的动态研究提供了便利。 荧光共振能量转移是指两个荧光发色基团在足够靠近时,当供体分子吸收一定频率的光子后被激发到更高的电子能态,在该电子回到基态前,通过偶极子相互作用,实现了能量向邻近的受体分子转移(即发生能量共振转移)。FRET是一种非辐射能量跃迁,通过分子间的电偶极相互作用,将供体激发态能量转移到受体激发态的过程,使供体荧光强度降低,而受体可以发射更强于本身的特征荧光(敏化荧光),也可以不发荧光(荧光猝灭),同时也伴随着荧光寿命的相应缩短或延长。能量转移的效率和供体的发射光谱与受体的吸收光谱的重叠程度、供体与受体的跃迁偶极的相对取向、供体与受体之间的距离等因素有关。作为共振能量转移供、受体对,荧光物质必须满足以下条件: ①受、供体的激发光要足够分得开;②供体的发光光谱与受体的激发光谱要重叠。 人们已经利用生物体自身的荧光或者将有机荧光染料标记到所研究的对象上,成功地应用于核酸检测、蛋白质结构、功能分析、免疫分析及细胞器结构功能检测等诸多方面。(传统有机荧光染料吸收光谱窄,发射光谱常常伴有拖尾,这样会影响供体发射光谱与受体吸收光谱的重叠程度,而且供、受体发射光谱产生相互干扰。相对于传统有机荧光染料分子,量子点的发射光谱很窄而且不拖尾,减少了供体与受体发射光谱的重叠,避免了相互间的干扰;由于量

荧光共振能量转移(FRET)技术在生物研究探究中的运用资料精

荧光共振能量转移(FRET)技术在生物研究中的应用 高裕锋分析化学B200425012 摘要:简要综述了荧光共振能量转移(FRET)技术在生物研究中的一些应用。核酸的结构、DNA测序、核酸杂交、蛋白质结构和蛋白质相互作用等的研究是生命科学研究重要组成部分,相关工作一直备受关注,而FRET技术被广泛应用于相关领域研究中,并取得了较突出的结果。 关键词:荧光共振能量转移(FRET),核酸结构,DNA测序,核酸杂交,蛋白质结构,蛋白质相互作用。 生命科学被誉为21世纪的科学,为了揭示生命的奥妙,人们投入了大量的工作。其中对于核酸和蛋白质的研究备受关注,大量的新技术与新方法被用于该领域的研究中。荧光共振能量转移技术是一项经典的荧光技术,但是随着荧光成像技术的发展,二者相互结合,成为了生命科学领域的一个重要研究手段[1,2]。本文简单介绍了基于FRET原理的新技术在生物研究中的一些应用。 一、FRET基本原理[3] FRET现象是Perrin在20世纪初首先发现的,1948年,Foster[4,5]创立了理论原理。FRET 指荧光能量给体与受体间通过偶极-偶极耦合作用以非辐射方式转移能量的过程,又称为长距离能量转移。产生FRET的条件(图1)主要有三个:(1)给体与受体间在合适的距离(1~10 nm);(2)给体的发射光谱与受体的吸收光谱有一定的重叠,这是能量匹配的条件;(3)给体与受体的偶极具一定的空间取向,这是偶极-偶极耦合作用的条件。 图1 产生FRET条件示意图

FRET 的效率用E 表示,E 用式(1)计算:其中R 0为Foster 距离,表示某一给定给体与受 60660R E R R =+ (1) 240D DA R const n J κφ?=???? (2) 体间能量转移效率为50%时的距离;R 为给体与受体的实际距离。R 0可由式(2)计算:其中κ2 是方向因素,n 是溶剂的反射系数,φD 是供体探针结合到蛋白的量子效率, J DA 是供体发射波长和受体吸收波长的交叠系数。 由式(2)可看出R 0对于给定的给-受体是一个特征值,因此,式(1)中E 值与 R 的关系紧密,这也成为了FRET 用于测定分子间或基团间距离的重要理论依据。 E 值可由以下几种方式测定:用荧光强度表征( E=1- I DA / I D ,I DA 表示A 存在时D 的荧光强度);用量子产率表征( E=1-φDA /φD );用荧光寿命表征(E=1-τDA /τD )。这表示研究FRET 可以通过不同的实验设备,既可以用普通光谱仪测定其荧光强度、量子产率,也可以用时间分辨仪测定其荧光寿命。 随着成像技术的发展,用显微成像的方法可更直观地观测FRET 地发生。 二、FRET 探针 FRET 需要两个探针,即荧光给体与荧光受体,要求是给体的发射光谱与受体的吸收光谱有一定交叠,而与受体的发射光谱尽量无交叠。 常用的探针主要有三种:荧光蛋白、传统有机染料和镧系染料。 荧光蛋白[6]是一类能发射荧光的 天然蛋白及其突变体,常见的有绿色荧光蛋白(GFP )、蓝色荧光蛋白(BFP )、 青色荧光蛋白(CFP )和黄色荧光蛋白(YFP )等。不同蛋白的吸收和发射波长不同,可 根据需要组成不同的探针对。荧光蛋白的突出优点是自身为生物分子,可有效地与目标分子融 合,更易于在生物环境中使用,且其种类多,可满足不同光谱需要。其缺陷是分子体积大, 空间分辨率较低,且可能与目标分子作用产生化学发光,需要较长地时间来确定荧光形式, 不利于动力学研究。为克服这些缺陷,常使用荧光蛋白与其他染料联用或用其他染料对来研究。 传统有机染料是指一些具有特征吸收和发射光谱地有机化合物组成地染料对。常见的有荧光素、罗丹明类化合物和 青色染料Cy3、Cy5等。该类染料分子体积较小,种类较多且大部分为商品化的分子探针染料,因此应用较为广泛。 镧系染料[7]一般与有机染料联用分 别作为FRET 的给-受体,其主要优点是:测量量可

荧光共振能量转移

FRET技术研究PEDF和目标蛋白 之间在小鼠神经元(神经胶质细胞)的 相互作用 一、FRET技术基本原理 荧光共振能量转移是指两个荧光发色基团在足够靠近时,当供体分子吸收一定频率的光子后被激发到更高的电子能态,在该电子回到基态前,通过偶极子相互作用,实现了能量向邻近的受体分子转移(即发生能量共振转移)。FRET是一种非辐射能量跃迁,通过分子间的电偶极相互作用,将供体激发态能量转移到受体激发态的过程,使供体荧光强度降低,而受体可以发射更强于本身的特征荧光(敏化荧光),也可以不发荧光(荧光猝灭),同时也伴随着荧光寿命的相应缩短或延长。能量转移的效率和供体的发射光谱与受体的吸收光谱的重叠程度、供体与受体的跃迁偶极的相对取向、供体与受体之间的距离等因素有关。作为共振能量转移供、受体对,荧光物质必须满足以下条件: ①受、供体的激发光要足够分得开; ②供体的发光光谱与受体的激发光谱要重叠。 人们已经利用生物体自身的荧光或者将有机荧光染料标记到所研究的对象上,成功地应用于核酸检测、蛋白质结构、功能分析、免疫分析及细胞器结构功能检测等诸多方面。(传统有机荧光染料吸收光谱窄,发射光谱常常伴有拖尾,这样会影响供体发射光谱与受体吸收光谱的重叠程度,而且供、受体发射光谱产生相互干扰。最新的一些报道将发光量子点用于共振能量转移研究,克服了有机荧光染料的不足之处。相对于传统有机荧光染料分子,量子点的发射光谱很窄而且不拖尾,减少了供体与受体发射光谱的重叠,避免了相互间的干扰;由于量子点具有较宽的光谱激发范围,当它作为能量供体时,可以更自由地选择激发波长,可以最大限度地避免对能量受体的直接激发;通过改变量子点的组成或尺寸,可以使其发射可见光区任一波长的光,也就是说它可以为吸收光谱在可见区的任一生色团作能量供体,并且保证了供体发射波长与受体吸收波长的良好重叠,增加了共振能量转移效率。) 以GFP的两个突变体CFP(cyan fluorescent protein)、YFP(yellow fluorescent protein)为例简要说明其原理:CFP的发射光谱与YFP的吸收光谱有相当的重叠,当它们足够接近时,用CFP的吸收波长激发,CFP的发色基团将会把能量高效率地共振转移至YFP的发色基团上,所以CFP的发射荧光将减弱或消失,主要发射将是YFP的荧光。两个发色基团之间的能量转换效率与它们之间的空间距离的6次方成反比,对空间位置的改变非常灵敏。例如要研究两种蛋白质a和b间的相互作用,可以根据FRET原理构 建融合蛋白,这种融合蛋白由三部分组成:CFP(cyan fluorescent protein)、蛋白 质b、YFP(yellow fluorescent protein)。用CFP吸收波长433nm作为激发波长,实验灵巧设计,使当蛋白质a与b没有发生相互作用时,CFP与YFP相距很远不能发生荧光共振能量转移,因而检测到的是CFP的发射波长为476nm的荧光;但当蛋白质a与b

荧光共振能量转移技术的基本原理和应用

荧光共振能量转移技术的基本原理和应用荧光共振能量转移(fluorescence resonance energy transfer,FRET)作为一种高效的光学“分子尺”,在生物大分子相互作用、免疫分析、核酸检测等方面有广泛的应用。在分子生物学领域,该技术可用于研究活细胞生理条件下研究蛋白质-蛋白质间相互作用。蛋白质-蛋白质间相互作用在整个细胞生命过程中占有重要地位,由于细胞内各种组分极其复杂,因此一些传统研究蛋白质-蛋白质间相互作用的方法如酵母双杂交、免疫沉淀等可能会丢失某些重要的信息,无法正确地反映在当时活细胞生理条件下蛋白质-蛋白质间相互作用的动态变化过程。FRET技术是近来发展的一项新技术,为在活细胞生理条件下对蛋白质-蛋白质间相互作用进行实时的动态研究提供了便利。 荧光共振能量转移是指两个荧光发色基团在足够靠近时,当供体分子吸收一定频率的光子后被激发到更高的电子能态,在该电子回到基态前,通过偶极子相互作用,实现了能量向邻近的受体分子转移(即发生能量共振转移)。FRET是一种非辐射能量跃迁,通过分子间的电偶极相互作用,将供体激发态能量转移到受体激发态的过程,使供体荧光强度降低,而受体可以发射更强于本身的特征荧光(敏化荧光),也可以不发荧光(荧光猝灭),同时也伴随着荧光寿命的相应缩短或延长。能量转移的效率和供体的发射光谱与受体的吸收光谱的重叠程度、供体与受体的跃迁偶极的相对取向、供体与受体之间的距离等因素有关。作为共振能量转移供、受体对,荧光物质必须满足以下条件: ①受、供体的激发光要足够分得开;②供体的发光光谱与受体的激发光谱要重叠。 人们已经利用生物体自身的荧光或者将有机荧光染料标记到所研究的对象上,成功地应用于核酸检测、蛋白质结构、功能分析、免疫分析及细胞器结构功能检测等诸多方面。(传统有机荧光染料吸收光谱窄,发射光谱常常伴有拖尾,这样会影响供体发射光谱与受体吸收光谱的重叠程度,而且供、受体发射光谱产生相互干扰。相对于传统有机荧光染料分子,量子点的发射光谱很窄而且不拖尾,减少了供体与受体发射光谱的重叠,避免了相互间的干扰;由于量子点具有较宽的光谱激发范围,当它作为能量供体时,可以更自由地选择激发波长,可以最大限度地避免对能量受体的直接激发;通过改变量子点的组成或尺寸,可以使其发射可见光区任一波长的光,也就是说它可以为吸收光谱在可见区的任一生色团作能量供体,并且保证了供体发射波长与受体吸收波长的良好重叠,增加了共振能量转移效率。)

荧光漂白恢复_荧光共振能量转移和荧光相关光谱检测的技术特点

ZHONGGUO YIXUEZHUANGBEI 于 淼① 高 建① [文章编号] 1672-8270(2009)06-0008-02 [中图分类号] R 197 [文献标识码] B Characteristics of application and technology on FRAP , FRET and FCS/Yu Miao , Gao Jian//China Medical Equipment,2009,6(6):8-9. [Abstract] Fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are three experimental techniques based on the fluorescence analysis that are commonly used to study molecular interaction. In this article, we will discuss and compare the application and technical specifications for FRAP , FRET and FCS.[Key words] FRAP; FRET; FCS; Fluorescence Analysis [First-author's address] Laboratory Center, China Medical University, Shenyang 110001, China. 荧光漂白恢复、荧光共振能量转移和荧光相关光谱检测的技术特点 [摘要] 荧光漂白恢复(FRAP)、荧光共振能量转移(FRET)和荧光相关光谱(FCS)是三种以荧光为基础的检测技术,常用来研究分子间相互作用。对三种技术的特点做以比较和讨论。 [关键词] 荧光漂白恢复;荧光共振能量转移;荧光相关光谱;荧光检测 作者简介 于淼,女,(1980- ),硕士,助教。现就职于中国医科大学实验技术中心,主要从事激光扫描共聚焦显微镜工作。 FRAP:经荧光素标记的某一区域被光照射后,荧光物质的光化学结构被破坏,荧光强度下降,但随之此处荧光强度会逐渐恢复,荧光强度与恢复强弱及快慢代表周围分子扩散的速率或分子运动速度[1]。 FRET:受激态荧光素(供体)将其能量向另一个荧光素(受体)传递,使后者被激发,这一过程称荧光能量共振转移。测定FRET程度的参数,包括供体淬灭、受体发射、供体荧光寿命、供体荧光去极化等[2]。 FCS:是一种通过检测微区内(共焦体积)分子 的荧光信息(强度、波动、波长等)来分析样品特性的检测 技术,类似于传统的荧光分光光度计,主要用于液态样品的成份分析[3]。 以上三种技术的主要参数有: 扩散率:测量扩散的速率,通常表现在分子和分子络合物的扩散系数。 多组分扩散:用来检测和区别单个和多组分之间扩散的能力。 运动分量:检测能够自由扩散的组分。 ①中国医科大学实验技术中心 辽宁 沈阳 110001

荧光共振能量转移技术的基本原理和应用

荧光共振能量转移技术的基本原理和应用 荧光共振能量转移(fluorescence resonance energy transfer,FRET)作为一种高效的光学“分子尺”,在生物大分子相互作用、免疫分析、核酸检测等方面有广泛的应用。在分子生物学领域,该技术可用于研究活细胞生理条件下研究蛋白质-蛋白质间相互作用。蛋白质-蛋白质间相互作用在整个细胞生命过程中占有重要地位,由于细胞内各种组分极其复杂,因此一些传统研究蛋白质-蛋白质间相互作用的方法如酵母双杂交、免疫沉淀等可能会丢失某些重要的信息,无法正确地反映在当时活细胞生理条件下蛋白质-蛋白质间相互作用的动态变化过程。FRET技术是近来发展的一项新技术,为在活细胞生理条件下对蛋白质-蛋白质间相互作用进行实时的动态研究提供了便利。 一、FRET技术基本原理 荧光共振能量转移是指两个荧光发色基团在足够靠近时,当供体分子吸收一定频率的光子后被激发到更高的电子能态,在该电子回到基态前,通过偶极子相互作用,实现了能量向邻近的受体分子转移(即发生能量共振转移)。FRET是一种非辐射能量跃迁,通过分子间的电偶极相互作用,将供体激发态能量转移到受体激发态的过程,使供体荧光强度降低,而受体可以发射更强于本身的特征荧光(敏化荧光),也可以不发荧光(荧光猝灭),同时也伴随着荧光寿命的相应缩短或延长。能量转移的效率和供体的发射光谱与受体的吸收光谱的重叠程度、供体与受体的跃迁偶极的相对取向、供体与受体之间的距离等因素有关。作为共振能量转移供、受体对,荧光物质必须满足以下条件: ①受、供体的激发光要足够分得开; ②供体的发光光谱与受体的激发光谱要重叠。 人们已经利用生物体自身的荧光或者将有机荧光染料标记到所研究的对象上,成功地应用于核酸检测、蛋白质结构、功能分析、免疫分析及细胞器结构功能检测等诸多方面。(传统有机荧光染料吸收光谱窄,发射光谱常常伴有拖尾,这样会影响供体发射光谱与受体吸收光谱的重叠程度,而且供、受体发射光谱产生相互干扰。最新的一些报道将发光量子点用于共振能量转移研究,克服了有机荧光染料的不足之处。

单分子荧光共振能量转移技术

研究生光谱 技术与应用课程 作业 河南大学 单分子荧光共振能量转移技术 学生:郭爱宇 学号:104753120870 学院:物理与电子学院 年级专业:2012级光学工程 课程名称:光谱技术及应用

指导老师:郭立俊教授

单分子荧光共振能量转移技术 摘要:单分子荧光共振能量转移技术(single molecule fluorescence resonance energy transfer, smFRET) 通过检测单个分子内的荧光供体及受体间荧光能量转移的效率,来研究分子构象的变化。在单分子探测技术发展之前,大多数的分子实验是探测分子的综合平均效应(ensemble averages),这一平均效应掩盖了许多特殊的信息。单分子探测可以对体系中的单个分子进行研究,得到某一分子特性的分布状况,也可研究生物分子的动力学反应。介绍了近来单分子荧光共振能量转移技术的进展。 关键词:单分子;荧光共振能量转移;荧光基团 1 引言 光谱技术是研究生物分子最常用的方法之一。在单分子光谱(single molecule spectroscopy, SMS)探测技术发展以前,大多数的实验是探测分子的综合平均效应,得到的是由大量对象组成的一个整体所表现出的平均响应和平均值,这一平均效应掩盖了许多特殊的信息。而单分子探测可对体系中的单个分子进行研究,通过与时间相关过程的探测,能实时了解生物大分子构象变化的信息。2002年美国第46届生物物理年会表明单分子仍是生物物理学目前和今后重点发展的研究领域。主要的技术手段包括生物大分子荧光光谱,单分子荧光能量转移谱、与原子力显微镜结合进行单分子水平的分子间相互作用力的测量,以及可进行单分子操作的激光光钳,高时间分辨率的单分子轨迹追踪等[1]。由此可见,单分子荧光技术具有重要的地位。 标记在生物大分子上单个荧光基团的各种特性变化能够提供有关分子间相互作用、酶活性、反应动力学、构象动力学、分子运动自由度(molecular freedom of motion)及在化学和静电环境下活性改变的信息。近年,在动态结构生物学研究领域,用单分子荧光光谱技术研究生物分子构象变化的方法主要有两种:一是通过单分子荧光偏振的各向异性(single molecule fluorescence polarization anisotropy,smFPA)研究生物分子的构象动力学(conformational dynamics)和旋转运动(rotational motions)。另一个是单分子对荧光共振能量转移(single pair

荧光共振能量转移动态检测蛋白质相互作用的研究进展

*[基金项目]国家自然科学基金资助项目(30971081;30870932; 81070961);山东省自然科学基金资助项目(No. ZR2011CM027;ZR2009DZ004);山东省泰山学者专 项基金 △[通信作者]陈京,E-mail:jingchen@warwick.ac.ukJ Jining Med Univ,February 2012,Vol.35,No.1 doi:10.3969/j.issn.1000-9760.2012.01.020·综述·荧光共振能量转移动态检测蛋白质相互作用的研究进展 王 宏1 蔡 欣1 白 波2 陈 京2Δ (1泰山医学院基础医学院,山东泰安271016;2济宁医学院神经生物学研究所,山东济宁272067) 摘 要 荧光共振能量转移(FRET)技术是近10年来出现的一种新的检测蛋白质-蛋白质相互作用的技术。它的最大优势是能从“时间、空间、动态、连续”对活细胞中蛋白质之间的相互作用进行检测,该技术不但可以与其他技术结合来研究细胞膜上蛋白质之间的相互作用;而且还可用于分析细胞膜-细胞质-细胞核中所发生的信号转导途径。资料显示,FRET技术所发挥作用是最近出现的几种技术所无法企及的,这对于疾病发病机制的研究及新的药物靶点的发现具有深远意义。 关键词 荧光共振能量转移;蛋白质-蛋白质相互作用;信号转导 中图分类号:Q274 文献标识码:A 文章编号:1000-9760(2012)02-060-04 机体细胞中种类繁多的蛋白质各具不同的生理功能,从而使细胞对胞外信号做出不同的生物学反应。蛋白质的功能作用经常受不同条件下与不同配体间相互作用的调节。蛋白质之间的相互作用能够整合来自不同信号通路的信号并协调细胞内部的调节机制[1]。因此,研究蛋白质之间相互作用能够给这些研究提供新的见解。在过去的20年中,已经开发了很多用于探测蛋白质相互作用的技术和方法,这些技术一般分为两部分,1)体外方法,例如免疫共沉淀、far-western blot、GST融合蛋白沉降技术等;2)体内方法,例如酵母双杂交系统,但是这些方法都具有一定的局限性。一方面,基于机械的、离心力高的或去垢剂的细胞裂解,上述方法都可能会改变蛋白质-蛋白质相互作用的自然状态;另一方面,上述技术无法提供活细胞内的特定蛋白间相互作用的时空信息[2]。近年来研究开发的荧光共振能量转移(Fluorescence resonance en-ergy transfer,FRET)却能克服以上缺点,可以在活细胞中实时动态观测蛋白质之间的相互作用。更重要的是,FRET还可以与其他技术结合[3],如生物发光共振能量转移(BRET)、蛋白互补技术(PCAs)等,既能研究两个蛋白之间的相互作用,还能用来研究3个或更多蛋白之间的相互作用,甚至是对信号网络的研究。本文就FRET、FRET与其他一些技术的结合及其应用做一简要综述。1 荧光共振能量转移(FRET)技术的原理 FRET理论描述的是两个荧光团之间的能量转移(图1)。在能量传输过程中,其中的一个荧光团作为能量供体(D),另一荧光团作为能量受体(A)。以适当的激发光照射D,将会产生振荡偶极子,如果D的基态和A的第一激发态的振动能量差相当,或者D的发射光谱与A的吸收光谱能有效重叠时,当D与A靠近时,或者说D与A的偶极子之间的距离足够近时即能产生共振,通过偶极-偶极耦合作用将能量从D向A转移,即发生非放射能量共振转移;A接受能量从而发射出特异性荧光。在此共振能量转移过程当中,D特异性荧光的衰减或者A特异性荧光的增强即可被检测到,从而实现FRET检测[4]。 FRET中两个常用的荧光蛋白为青色荧光蛋白(cyan fluorescent protein,CFP)和黄色荧光蛋白(yellow fluorescent protein,YFP)。FRET具有广泛的应用前景,在蛋白质、DNA等复合物的研究方面发挥重要作用。Khan等[5]用FRET检测出溶血磷脂胆碱能够通过迅速磷酸化和内化来快速激活G2A,G2A的活化能促使Gαi-1和Gαq/11和Gβγ的释放,Gαi-1和Gαq/11会使胞质内的Ca2+浓度升高及G2A的激活促使GRK6和β-ar-restin的循环利用;而Gβγ能够与激活的Hck相互作用。Kazutoshi Yoshitake等[6]构建了一对融合的锌指结构蛋白,它们是一种具有N端二聚化序列和C端GFP突变体的荧光传感蛋白。他们分别在锌指结构的末端标记GFP的突变体CFP和YFP,将这一对锌指结构蛋白混合,并且加入特异 06

单分子对荧光共振能量转移(spFRET)

单分子对荧光共振能量转移(spFRET)
生物物理学系 郑晓惠 学号 10281034 一 引言 光谱技术是研究生物分子最常用的方法之一。在 单分子光谱(single molecule spectroscopy, SMS) 探测技术发展以前,大多数的实验是探测分子的 综合平均效应,得到的是由大量对象组成的一个 整体所表现出的平均响应和平均值,这一平均效 应掩盖了许多特殊的信息。而单分子探测可对体 系中的单个分子进行研究,通过与时间相关过程 的探测,能实时了解生物大分子构象变化的信息。 2002 年美国第 46 届生物物理年会表明单分子仍是 生物物理学目前和今后重点发展的研究领域。主 要的技术手段包括生物大分子荧光光谱,单分子 荧光能量转移谱、与原子力显微镜结合进行单分 子水平的分子间相互作用力的测量,以及可进行 单分子操作的激光光钳,高时间分辨率的单分子 轨迹追踪等[1]。由此可见,单分子荧光技术具有重 要的地位。 标记在生物大分子上单个荧光基团的各种特 性变化能够提供有关分子间相互作用、酶活性、 反应动力学、构象动力学、分子运动自由度 (molecular freedom of motion)及在化学和静电环境 下活性改变的信息。近年,在动态结构生物学研 究领域,用单分子荧光光谱技术研究生物分子构 象变化的方法主要有两种:一是通过单分子荧光 偏 振 的 各 向 异 性 ( single molecule fluorescence polarization anisotropy,smFPA)研究生物分子的 构象动力学(conformational dynamics)和旋转运动 (rotational motions)。 另一个是单分子对荧光共振能 量转移(single pair fluorescence resonance energy transfer, spFRET),在单分子水平测量一个分子内 或两个不同分子间的距离变化和相互作用。本文 主要就后者做简要介绍。 二 原理 荧光共振能量转移是指当两种不同的荧光生色团 离的较近,且其中一种生色团(供体, donor)的发 射谱与另一种生色团 (受体, acceptor) 的激发谱有 相当程度的重叠时,当供体被激发时,受体会因 供体激发能的转移而被激发。其直观表现就是供 体产生的荧光强度较其单独存在时要低的多,而 受体发射的荧光却大大增强,同时伴随它们荧光 寿命的相应缩短和延长。能量转移效率与两个生 色团激发谱和发射谱的重叠程度、供体与受体跃 迁偶极的相对取向、供受体间的距离有密切关系, 其经典公式为 E=R06/(R6+ R06), R0 为能量传递达到 50%的距离。选定供、受体对之后,可将 R0 看作 恒量。能量转移的发生将改变供、受体的去偏振 程度、荧光寿命、荧光强度等,测定这些参数值 就可得出 E。利用 R0 和实验测得的 E 就可测得供 受体间的距离 R。 用此方法测量生色团距离的方法 被称为光谱尺,可测量 1.0-10.0nm 之间的距离。 spFRET 是在一个生物大分子或两个相互作用的分 子上标记两个不同的荧光基团。同样,当供体的 发射光谱与受体的吸收光谱相重叠时,发生共振 能量转移。通过探测能量转移效率,就可确定两 点间的距离。由于相对取向和光谱重叠积分等不 确定因素,该方法不能准确确定绝对距离。但可 通过监测供体-受体的相对距离变化,结合其时间 变化推测生物大分子在生命活动中的构象变化。 该技术不仅有非常好的静态定位能力,也能提供 分子内或分子间两个荧光基团在距离和方向上的 动态变化。由于每次只观察一对供、受体系统, 因此能在毫秒时间尺度内观察这些变化,从而将 具有不同能量转移效率的亚群分开。将这种方法 扩展, 可用于研究生物多聚体的组成动力学、 DNA 限制性内切酶酶切反应,以及 DNA 发夹结构的去 折叠等。 三 方法 1 设备 单分子荧光探测必须满足两个基本要求,一是在 被照射的体积中只有一个分子与激光发生相互作 用;二是要确保单分子的信号大于背景的干扰信 号。背景信号来自于 Raman 散射、Rayleigh 散射、 溶剂中杂质、盖玻片产生的荧光和探测器的暗电 流。因此,进行单分子探测要求 (1) 激发容积要 小,因为背景的吸收与激发体积成正比,尽量减 小激发体积可降低背景干扰, (2)高效的收集光 学系统, (3)灵敏的探测器, (4)采用针孔装置, 或将溶剂中杂质预漂白以及用低荧光光学材料等 方法清除背景荧光。 常 用 的 装 置 是 近 场 光 学 扫 描 显 微 镜 (near-field scanning optical microscope NSOM),其最小激发 体积小于 10-2m3。该方法在单分子荧光的探测中 发挥了很重要的作用。可监测单个生物大分子的 构造变化,例如旋转和纳米水平上的距离变化。 其不足是扫描时需要通过复杂的控制系统来保持 适当的样品与探针之间的距离,并且其近场激发

生物发光共振能量转移

BRET 生物发光共振能量转移 技术简介: 生物发光共振能量转移(BRET)技术是近10年来出现的一种新的检测蛋白质-蛋白质相互作用的技术.它的最大优势是能在活细胞中实时进行检测,因此能够进行相互作用动力学的研究。在应用这个系统研究感兴趣的蛋白前,首先需要将Rluc或者EYFP 融合到每个蛋白或者他们的辅蛋白上。在细胞中,融合蛋白共表达,然后和荧光素酶和其底物腔肠素反应,当能量供体Rluc,能量受体EYFP 之间距离比较近的时候 (10-100?),那么光将从Rluc(峰值480nm发射)传递到EYFP,形成它的激发,并且发射一个波长在530nm 的发射光,BRET 量化的程度以发射光530nm 和480nm 的比率表示。 实验流程 1、用于BRET的表达质粒构建; 2、融合蛋白表达检测; 3、BRET实验; 4、实验结果分析及提交实验报告 客户提供 1、目的基因cDNA(也可由我公司提供基因克隆) 2、用于实验细胞(如果我公司细胞库有可不需提供) 公司提供 详细实验步骤、使用仪器、及结果分析等详细实验报告。 服务周期和价格 具体咨询本公司 荧光共振能量转移(FRET) 荧光共振能量转移-FRET(Fluorescent Resonance Energy Transfer) 指两个荧光发色基团在足够靠近时,当供体分子吸收一定频率的光子后被激发到更高的电子能态,在该电子回到基态前,通过偶极子相互作用,实现了能量向邻近的受体分子转移。

(1)FRET发生条件: 能量匹配:供体分子的发射光谱可以被受体分子吸收并产生荧光信号。供体分子的发射光谱应与受体分子的激发光谱必须有显著重叠(>30%); 作用距离:供体分子与受体分子的作用距离为1-10nm; FRET效率公式:用于计算分子/基团间作用距离R 偶极-偶极作用:供体分子与受体分子作用时的向量必须满足一定条件。 (2)FRET的应用: 通过FRET技术可以获得有关两个蛋白分子之间相互作用的空间信息(作用距离、作用方向、能量传递效率)。常用于解决如下问题:蛋白分子的共定位;蛋白分子聚合体;转录机制;转化途径;分子运动;蛋白折叠等生物学问题。 (3)FRET常见的供体-受体荧光分子对: 荧光蛋白 类: 染料类:

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