Ras-related protein Rab10 facilitates TLR4 signaling by promoting replenishment of TLR4

Ras-related protein Rab10facilitates TLR4signaling by promoting replenishment of TLR4onto the plasma membrane

Di Wang a,1,Jun Lou b,1,Chuan Ouyang a,Weilin Chen a,Yiqi Liu a,Xinyuan Liu b,Xuetao Cao a,c,Jianli Wang a,

and Linrong Lu a,d,2

a Institute of Immunology and d Program in Molecular and Cellular Biology,Zhejiang University School of Medicine,Hangzhou310058,China;

b Xin Yuan Institute of Medicine and Biotechnology,Life Science College,Zhejiang Sci-Tech University,Hangzhou310018,China;and

c Institute of Immunology an

d National Key Laboratory of Medical Immunology,Second Military Medical University,Shanghai200433,China

Communicated by Harvey Cantor,Dana-Farber Cancer Institute,Boston,MA,June30,2010(received for review May7,2010)

The Toll-like receptor(TLR)4receptor complex,TLR4/MD-2,plays an important role in the in?ammatory response against lipopolysaccha-ride,a ubiquitous membrane component in Gram-negative bacteria. Ligand recognition by TLR4initiates multiple intracellular signaling pathways,leading to production of proin?ammatory mediators and type I IFN.Ligand interaction also leads to internalization of the surface receptor complex into lysosomes,leading to the degradation of TLR4and the termination of LPS response.However,surface level of TLR4receptor complex is maintained via continuous replenish-ment of TLR4from intracellular compartments like Golgi and endo-somes.Here we show that continuous replenishment of TLR4from Golgi to plasma membrane is regulated by the small GTPase Rab10, which is essential for optimal macrophage activation following LPS stimulation.Expression of Rab10is inducible by LPS.Blockade of Rab10function leads to decreased membrane TLR4expression and diminished production of in?ammatory cytokines and interferons upon LPS stimulation.These?ndings suggest that Rab10expression provides a mechanism to re?ne TLR4signaling by regulating the traf?cking rate of TLR4onto the plasma membrane.In addition, we show that altered Rab10expression in macrophages in?uences disease severity in an in vivo model of LPS-induced acute lung injury, suggesting Rab10as a possible therapeutic target for human acute respiratory distress syndrome(ARDS).

small GTPases|membrane traf?cking|LPS|acute respiratory distress syndrome

T oll-like receptors(TLRs)play important roles in the innate immune response to bacterial and viral pathogens(1).LPS, a common immunostimulatory bacterial membrane component constituting the outer membrane of Gram-negative bacteria,is recognized by TLR4(2).The TLR4receptor complex is comprised of TLR4and MD-2(TLR4/MD-2),a small extracellular glyco-protein that associates with the extracellular domain of TLR4. TLR/MD-2activates a signaling cascade through the Toll/IL-1R (TIR)domain of its cytoplasmic tail,which recruits the adaptor protein myeloid differentiation factor88(MyD88),allowing for subsequent activation of IL-1R-associated kinases(IRAKs)and tumor necrosis factor receptor-associated factor6(TRAF6), leading to NF-κB and MAPK pathways as well as induction of proin?ammatory cytokines.TLR4can also induce the expression of type I interferons using TIR domain-containing adapter inducing IFN-β(TRIF)as a critical MyD88-independent adaptor(3). Activation of TLR4is a tightly regulated process.In addition to direct regulation toward different signaling pathways,the amount of TLR4/MD-2present on the cell surface also controls the LPS response.Surface TLR4amount is determined by both receptor traf?cking from the Golgi apparatus to the cell membrane as well as internalization of the cell surface receptor into endosomal compartments(4).TLR4is synthesized and folded in the ER. MD-2also resides in the ER and physically associates with TLR4. The formation and surface expression of the TLR4receptor complex is regulated by ER-resident heat shock protein chaperone glycoprotein96(gp96)and protein associated with Toll-like re-ceptor4(PRAT4A)(5,6).Large ratios of TLR4localize in the subcellular compartments,such as Golgi apparatus,endosomes, and lysosomes,indicating that translocation of TLR4from Golgi to the cell surface is a regulated process,which may play a role in the temporal and spatial regulation of TLR4signaling(4).

Ras related in brain(Rab)proteins are small guanosine tri-phosphatases(GTPases)belonging to the Ras superfamily that regulate vesicular formation,movement,and fusion processes(7). Despite their function in membrane traf?cking,Rab may also be involved in signal transduction by regulating the membrane traf-?cking of cell surface receptors for hormone,cytokine,and che-mokine(8–10).In this study,we show that Rab10plays a critical role in TLR4signaling.We were able to show that Rab10ex-pression can up-regulate LPS-induced production of TNF-α,IL-6, and IFN-β,as well as potentiate LPS-induced activation of mul-tiple intracellular signaling pathways,including MAPK,NF-κB, and IFN regulatory factor3(IRF3)signaling pathways.Confocal analysis has revealed that Rab10primarily localizes in TGN58K and EEA-1positive subcellular compartments and colocalizes with TLR4.More importantly,cell surface levels of TLR4can be regulated by overexpression or RNAi knockdown of Rab10ex-pression.Taken together,these?ndings suggest that Rab10is a positive regulator of TLR4signaling,possibly by promoting transport of TLR4from the Golgi to plasma membrane. Finally,using an in vivo model of human acute respiratory distress syndrome(ARDS),we show that modi?cations in sur-face TLR4expression via overexpression of Rab10in macro-phages exaggerates LPS-induced lung injury,indicating Rab10as a potential therapeutic target for treatment of ARDS as well as other in?ammatory diseases in humans.

Results and Discussion

Rab10Expression Is Up-Regulated upon TLR4Activation by LPS.The Rab10gene was initially identi?ed from screening LPS-induced genes in bone marrow derived dendritic cells.Rab10was among the most highly-expressed genes induced by LPS stimulation in these cells;2μg/mL of LPS resulted in a20-fold increase in Rab10 mRNA expression,as judged from quantitative RT-PCR(Fig.1A and B).Because subsequent expression analysis revealed that Rab10production was higher in macrophages,we further veri?ed this using peritoneal macrophages and RAW264.7cells,a macro-phage-derived cell line.Similarly,both cells express Rab10and expression was also increased upon LPS stimulation at both the

Author contributions:D.W.,J.L.,C.O.,W.C.,X.L.,X.C.,J.W.,and L.L.designed research;D. W.,J.L.,C.O.,and Y.L.performed research;D.W.,J.L.,C.O.,W.C.,and L.L.analyzed data; and D.W.and L.L.wrote the paper.

The authors declare no con?ict of interest.

1D.W.and J.L.contributed equally to this work.

2To whom correspondence should be addressed.Email:lu_linrong@https://www.wendangku.net/doc/1115551184.html,.

This article contains supporting information online at https://www.wendangku.net/doc/1115551184.html,/lookup/suppl/doi:10. 1073/pnas.1009428107/-/DCSupplemental.

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mRNA and protein levels (Fig.1C and D ),suggesting the po-tential involvement of Rab10in regulation of TLR4signaling.

Silencing of Rab10Expression Reduces Production of LPS-Induced Proin ?ammatory Mediators in Macrophages.To determine whether

Rab10was involved in regulation of TLR4signaling,we silenced the expression of Rab10in RAW264.7cells by stable transfection of a vector expressing Rab10siRNA.Rab10expression was sig-

ni ?cantly down-regulated by Rab10siRNA,but not by corre-sponding scrambled controls (Fig.2A ).We found that production of TNF-α,IL-6,and IFN-βin RAW264.7cells upon LPS stimu-lation was signi ?cantly inhibited at the mRNA and protein level after Rab10silencing (Fig.2C and D ).

LPS-induced production of TNF-αand IL-6is related to LPS-initiated MyD88-dependent NF-κB or MAPK activation,whereas IFN-βproduction is primarily mediated by the TRIF-dependent activation of IRF3pathway (11).To con ?rm whether Rab10regulated TLR4-mediated signaling,we analyzed TLR4signaling pathways in RAW264.7cells stably silenced for Rab10expression.We found that activation of ERK1/2was substan-tially decreased in Rab10-silenced cells,compared with control siRNA-transfected Raw264.7cells (Fig.2E ).Activation of p38and JNK MAPK by LPS was also altered to a lesser extent (Fig.2E ).Analysis of AKT,IRF3,and I κB phosphorylation also showed that Rab10knockdown interrupted LPS-induced phos-phorylation of IRF3,Akt,and I κB (Fig.2F ).

To further de ?ne Rab10-mediated regulation of LPS-induced NF-κB and IRF3pathways in macrophages,RAW264.7cells were transiently transfected with a NF-κB –luciferase (NF-κB –luc)or IFN-β–luciferase (IFN-β–luc)construct.Analysis of LPS-treated and -untreated cells for luciferase reporter gene activity in Rab10knockdown cells showed decreased LPS-induced transcriptional activity of both NF-κB and IFN-βpromoters in RAW264.7macrophages (Fig.2B ).To exclude the possibility of any fundamental defect after Rab10knockdown in RAW264.7cells,we analyzed cell proliferation,cell cycle,and apoptosis in RAW264.7cells following Rab10knockdown.No obvious dif-ferences were observed compared with unmodi ?ed cells (Fig.S1A –C ).Furthermore,Rab10knockdown in RAW264.7cells did not signi ?cantly affect the production of proin ?ammatory mediators after poly I:C or CpG stimulation (Fig.S1D and E ).Taken together,these data demonstrate that Rab10regulates LPS-initiated activation of the MAPK,NF-κB,and IRF3path-ways and LPS-induced production of proin ?ammatory

mediators

Fig. 1.Activation of TLR4enhances Rab10expression in both DC and macrophages.(A and B )Dendritic cells and (C and D )RAW264.7cells were treated with 1μg/mL LPS for different time periods or with indicated doses of LPS for 12h;mRNA expression levels of Rab10were assayed by quanti-tative PCR.Results are presented as fold changes of Rab10mRNA levels compared with untreated controls.(D )Western blot analysis of Rab10and β-actin protein expression in RAW264.7cells treated with 1μg/mL of

LPS.

Fig.2.Silencing of Rab10expression reduces LPS-induced production of proin ?ammatory mediators and LPS-initiated signaling pathways in macrophages.(A )RAW264.7cells were transfected with plasmids encoding siRNAs against Rab10(Rab10iA and KD251)and selected with 600μg/mL neomycin.The ef ?-ciency of silencing was evaluated by Western blot.(B )Control or Rab10silenced RAW264.7cells were cotransfected with pTK-RL and NF-κB –luc or IFN-β–luc reporter plasmids followed by 24-h incubation.Cells were then stimulated with 1μg/mL LPS for an additional 8h before luciferase activity was measured.(C and D )Production of proin ?ammatory mediators by Rab10silenced RAW264.7cells after LPS treatment was measured by (C )quantitative PCR and (D )enzyme-linked immunoassay.(E and F )Cell lysates of Rab10silenced RAW264.7cells were prepared and blotted with indicated anti-phospho Abs.Total Erk,Jnk,P38,Akt,IRF3,and I κB were probed as quantitative controls.Data shown represents three independent experiments.*P <0.05,**P <0.01.

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in macrophages,suggesting that Rab10expression is essential for optimal TLR4signaling initiated by LPS.

Rab10Overexpression Enhances TLR4Signaling and Effector Func-tions.To further support the role of Rab10in regulation of TLR4

signaling,we overexpressed Rab10in peritoneal macrophages.Overexpression of Rab10was con ?rmed by RT-PCR (Fig.3A ).We found that both LPS-induced MyD88-dependent production of proin ?ammatory cytokines and TRIF-dependent production of IFN-βwere signi ?cantly enhanced after Rab10overexpression,indicating that Rab10overexpression promoted LPS-initiated TLR4signaling in macrophages (Fig.3C and D ).

To con ?rm the effects of Rab10overexpression on LPS-initiated MAPK,NF-κB,and IRF3activation,we examined the activation status of ERK1/2,JNK1/2,p38,Akt,and IRF3in RAW264.7cells by Western blotting.We found that Rab10overexpression could enhance LPS-induced activation of ERK1/2and Akt (Fig.3E ).Although the effect of Rab10overexpression on phosphorylation of I κB and IRF3was not as obvious,the transactivation activity of both NF-κB and IFN-βpromoter induced by LPS were signi ?-cantly enhanced by Rab10overexpression (Fig.3B ),consistent with increased secretion of cytokines.

Mutation of the corresponding Thr23into Asn in Rab10can result in GTP-binding de ?ciency (12).Thus,Rab10T23N mutant failed to enhance LPS-induced signaling and cytokine production in macrophages (Fig.S2).Although Q68L mutant of Rab10can serve as an activated form of Rab10,this mutant also loses its ability to promote LPS-stimulated cytokine production,indicating that the GTPase activity or Rab10recycling is necessary for its function,as previously reported (13).These observations support the notion that Rab10regulation of TLR4signaling requires GTPase activity of Rab10.

Colocalization of Rab10and TLR4in Endomembrane Compartments.

Because most of the Rab proteins function via regulating protein transport between different subcellular compartments,we ex-

amined subcellular localization of Rab10and its mutants in RAW264.7cells by confocal microscopy.It showed that Rab10localized into vesicular and perinuclear membrane organelles (Fig.4A –C ).Additional staining revealed that Rab10localized into both Golgi (Fig.4A )and early endosomal compartments (Fig.4B )but not into late endosomes (Fig.4C)or lysosomes (Fig.4D ).These data are consistent with previous reports showing that Rab10was a Golgi-associated and early endosomal compartment-associated small GTPase (12).An inactive mutant of Rab10(Rab10T23N)localized to the Golgi and distributed in the cytosol,whereas the active mutant (Rab10Q68L)localized mainly in the early endo-somal compartment (Figs.S3A –D ).

To elucidate the mechanism of Rab10in regulating TLR4signaling,we investigated the dynamic localization of Rab10in RAW264.7cells after LPS ligation.We found that TLR4was par-tially localized in Golgi (Fig.4E )but not in lysosomes (Fig.S3E )before LPS treatment.It was partially transported into EEA-1-positive compartments (Fig.4F )10and 20min after LPS treatment.More importantly,TLR4was colocalized with Rab10(Fig.4G and H ).These data suggested a direct linkage between Rab10localization and TLR4transport.Moreover,Rab10was aligned along microtubules after LPS stimulation,suggesting that micro-tubules provide the transportation tracks for Rab10-containing vesicles (Fig.4I ).

Rab10Regulates Surface Expression of TLR4.Recent studies have

shown Rab10regulates membrane transport of proteins like GLUT4(12).We therefore determined whether Rab10could promote membrane transport of TLR4onto plasma membrane of macrophages.We examined TLR4surface expression in macrophages after Rab10overexpression or silencing.We found that overexpression of Rab10increased TLR4surface expression on bone marrow-derived macrophages and reduced the extent of LPS-induced transient down-regulation of surface TLR4(Fig.5A and B ).Similarly,cell surface expression of TLR4on RAW264.7cells was modulated via Rab10silencing (Fig.5C

).

Fig.3.Overexpression of Rab10promotes LPS-induced production of proin ?ammatory mediators and LPS-initiated signaling pathways in macrophages.(A )RAW264.7cells were transfected with plasmids encoding Rab10,Rab10T23N,and Rab10Q68L.Ef ?ciency of Rab10overexpression was evaluated by RT-PCR.(B )RAW264.7cells were cotransfected with pTK-RL with NF-κB –luc or IFN-β–luc reporter plasmids and the pcDNA-Rab10or empty vector plasmid.After 24h of culture,the cells were stimulated with 1μg/mL LPS for an additional 8h.Luciferase activity was measured.Similar results were obtained in three independent experiments.*P <0.05,**P <0.01.(C )Peritoneal macrophages were transfected with pcDNA-Rab10or empty vector.After 48h,cells were stimulated with 1μg/mL LPS for the indicated time periods.Relative mRNA expression of IL-6,TNF-α,and IFN-βwas measured by quantitative PCR.(D )Supernatants from the cultures above were collected and the concentration of IL-6,TNF-α,and IFN-βevaluated by enzyme-linked immunoassay.(E )Control and Rab10overexpressed RAW264.7cells were treated with 1μg/mL LPS for the indicated time periods.Cell lysates were prepared and blotted with the indicated anti-phospho Abs.Total Erk,Jnk,P38,Akt,I κB,IRF3,and β-actin were probed as quantitative controls.

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Because Rab10gene modi ?cation did not affect mRNA and total protein levels of TLR4expression in RAW264.7macro-phages,these results suggested that Rab10in ?uenced TLR4surface expression by promoting its translocation rather than regulating its transcription and translation.The dominant role of traf ?cking on TLR surface level were further con ?rmed by the ?ndings that surface TLR4expression was affected by BFA,a speci ?c inhibitor for Golgi-mediated protein secretion,but not protein synthesis inhibitor CHX or DRB,an inhibitor for RNA polymerase II,in a given period (Fig.5D ).These results sug-gested that replenishment of surface TLR4depends on trans-

portation of “ready-state ”TLR4molecules on the Golgi but not de novo protein synthesis (14).

Surface expression of F4/80,another macrophage molecule,showed no detectable changes after Rab10overexpression (Fig.S4A ).In addition,Rab10did not affect phagocytosis (Fig.S4B and C ),antigen-presentation capacity (Fig.S4D )or cell pro-liferation (Fig.S4E )in RAW264.7macrophages.

Elevated Expression of Rab10in Macrophages Enhances LPS-Induced Acute Lung Injury Because TLR4signaling is a key pathway during

the in ?ammatory process of acute lung injury (15),we

determined

Fig. 4.TLR4is localized to Rab10-positive Golgi and early endosomes compartments.(A –D )RAW264.7cells were trans-fected with GFP-Rab10plasmid;48h later,cells were labeled with LysoTracker Red (D ),or immunostained with primary Ab against TGN58K (A ),EEA-1(B ),or LAMP-1(C )as indicated and DyLight549conjugated goat anti-mouse IgG.(E –F )RAW264.7cells were transfected with TLR4-HA vector;48h later,cells were left untreated or treated with 1μg/mL LPS for the in-dicated time periods.Subcellular localization of TLR4was ex-amined by confocal microscopy after immunostaining for TLR4and the indicated compartment markers.(G –H )RAW264.7cells were cotransfected with TLR4-HA and Rab10plasmids and treated with 1μg/mL LPS for the indicated time periods.Cells were immunostained with anti-HA Ab and anti-Rab10Ab,manifested with DyLight549Conjugated goat anti-mouse IgG and DyLight488Conjugated goat anti-rabbit IgG before image analysis by confocal microscopy.(I )RAW264.7cells were trans-fected with Rab10and treated with 1μg/mL LPS for the in-dicated times.Cells were then immunostained with anti-tublin-αAb and anti-Rab10Ab,manifested with DyLight549Conju-gated goat anti-mouse IgG and DyLight488Conjugated goat anti-rabbit IgG before images analysis by confocal

microscopy.

Fig.5.Rab10expression regulates TLR4cell surface expression.(A )Bone marrow-derived macrophages were transfected with pcDNA-Rab10or empty vector and stimulated with LPS for indicated time periods.TLR4surface expression was analyzed by FACS analysis.Mean ?uorescence intensity (MFI)is shown.(B )Relative TLR4surface mean ?uorescence after LPS treatment for indicated time periods were graphed (MFI at time 0as 100%).(C )TLR4surface expression in Rab10silenced RAW264.7cells is shown.(D )RAW264.7cells were incubated with serum-free medium for up to 3h in the presence or absence of 100μM 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB),10μg/mL cycloheximide,and 5μM brefeldin A (BFA)before 1μg/mL of LPS treatment (30min);cell surface TLR4was assessed by ?ow cytometry and the mean ?uorescence intensity of surface TLR4staining were plotted .Similar results were obtained in three independent experiments.*P <0.05,**P <0.01.

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whether a change in surface expression of TLR4controlled by Rab10could in ?uence the in ?ammatory response using an in vivo model.Systemic administration of LPS to induce pulmonary in-?ammation mimics ARDS in humans and is characterized by a se-vere acute in ?ammatory response in lungs and neutrophilic alveolitis.LPS was administered to BALB/c mice infused via a jug-ular venous canula before i.v.injection of saline,bone marrow de-rived macrophages,or macrophages transfected with pCDNA4(null)or pCDNA4-Rab10plasmid.The total in ?ammatory cell count in bronchoalveolar lavage (BAL)?uid increased approxi-mately 20-fold at d 3following administration of LPS,and this was primarily attributed to an increase in neutrophils.Treatment of animals with macrophages transfected with pCDNA4-Rab10sig-ni ?cantly increased the total cell and neutrophil counts in BAL ?uid (Fig.6A and B )(P <0.05compared with LPS/saline group),whereas treatment with macrophages alone or macrophages transfected with pCDNA4also elevated the cell in ?ltration but to a lesser extent that was not statistically signi ?cant.

Histological assessment of lung sections 36h after LPS ad-ministration revealed marked in ?ammatory in ?ltrates,inter-alveolar septal thickening,and interstitial edema (Fig.S5).

Proin ?ammatory cytokines were measured in BAL ?uid col-lected from mice.We found that IL6,TNF-α,IL1β,and IFN-γwere all elevated in BAL ?uid in response to LPS challenge compared with na?ve controls that received saline (Fig.6C –F ).Treatment with macrophages alone or macrophages with pCDNA4showed variably elevated levels of proin ?ammatory cytokines,whereas treatment with BMMC-Rab10dramatically enhanced the levels of TNF-αand IL6.

In sum,these ?ndings indicate that continuous replenishment of TLR4receptor from the ER and Golgi is a limiting step for TLR4signaling and this process is mediated by a Golgi-associated small GTPase Rab10.Optimal TLR4signaling requires the ex-pression and activity of Rab10and continuous translocation of TLR4from inner membrane compartments onto the cell surface.Rab10silencing not only reduced surface expression of TLR4,but also diminished the activation of MAPK,NF-κB,and IRF3,coupled with decreased production of TNF-α,IL-6,and IFN-βin

macrophages after LPS treatment.They also suggest that Rab10-mediated translocation of TLR4may ?ne tune the in ?ammatory response after infection.These data provide initial evidence that Rab10is directly involved in regulating TLR signaling.

Bacterial and viral infections are important risk factors for ARDS.In 2003,the majority of patients who succumbed to severe acute respiratory syndrome (SARS)developed ARDS.Recently,H5N1avian in ?uenza infection in humans also resulted in high lethality due to ARDS.Recent studies of a murine model of ARDS have shown that innate immune signaling via TLR4in lung macrophages is a key pathway in determining ARDS susceptibility in vivo (15).Our mouse acute lung injury experiment suggests a role for Rab10as a potential target to modulate the intensity of LPS/TLR4-mediated in ?ammatory responses and treatment of in ?ammatory diseases like ARDS in humans.Materials and Methods

Mice and Reagents.C57BL/6and BALB/c mice (6-to 8-wk-old)were purchased from Shanghai Slac Animal Inc.and maintained in Experimental Animal Center of Zhejiang University.Experiments and animal care were performed in ac-cordance with the guidelines of Zhejiang University.LPS derived from Escherichia coli 0111:B4was obtained from Sigma.Rabbit polyclonal antibody against Rab10was from Proteintech Group.Antibodies (Abs)speci ?c for total and phosphorylated forms of ERK1/2(Thr202/Tyr204),JNK1/2(Thr183/Tyr185),p38(Thr180/Tyr182),Akt (Ser473),IFN regulatory factor (IRF3)(Ser396),I κB (Ser32/36),and Abs against TLR4and hemagglutinin epitope (HA)tag were obtained from Cell Signaling Technology.Abs against β-actin,early endosome antigen 1(EEA-1),LAMP-1,and trans Golgi network 58K (TGN58K)were from Santa Cruz Biotechnology.Ab against tublin-αwas from Sigma.DyLight549conjugated goat anti-mouse IgG (H+L),DyLight488conjugated goat anti-rabbit IgG (H+L)and ?uorescent dyes were from Thermo Fisher Scienti ?c.The pGL-3luciferase and pRL-TK-Renilla luciferase plasmids were from Promega.Molecular biology reagents were obtained from Takara.

Plasmid Constructs.Recombinant vector encoding mouse Rab10(mRab10,GenBank Accession number NM_016676.5)was constructed by PCR-based ampli ?cation and subcloning into the pcDNA3.1eukaryotic expression vec-tor (Invitrogen).Vectors encoding GFP-tagged Rab10and mutants were constructed by subcloning Rab10and mutants to pEGFP-N plasmid.

GFP-tags

Fig.6.Infusion of BMDMs overexpressing Rab10aggravated mice acute lung injury.BMDMs were transfected with plasmids encoding Rab10or empty vector.The ef ?ciency of transfection was evaluated by RT-PCR.Acute lung injury was induced as described in Methods and Materials .Total cells (A )and neu-trophils (B )from BAL ?uid were enumerated to evaluate lung airspace in ?ammation.(C –F )Levels of proin ?ammatory cytokines in BAL ?uid was measured by enzyme-linked immunoassay.Group comparisons were analyzed by one-way ANOVA with Dunnett post hoc test.*P <0.05,**P <0.01,LPS/saline versus each treated group (BMDMs,BMDMs-pcDNA3.1,or BMDMs-pcDNA3.1-Rab10).n =6,5,7,5,7,6,respectively.This result represents two independent experiments.

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were placed at the C-terminals of Rab10ORF to avoid interfering with its localization.The TLR4-HA plasmid,NF-κB reporter plasmid,and IFN-βre-porter plasmid were constructed and prepared as previously(16).All of the clones were con?rmed by DNA sequencing.Primer sequences used in clon-ing are available on request.

Cell Preparation,Culture,and Transfection.C57BL/6mice were used for the preparation of primary mouse peritoneal macrophages(17).BALB/c mice were used for the preparation of primary mouse bone marrow derived macrophages(18).Mouse macrophage cell line RAW264.7was obtained from American Type Culture Collection and cultured as described previously (18).Transfections using jetPEI-Macrophage transfection reagent(PolyPlus transfection)were performed according to manufacture’s instruction.Stable cell lines were selected in600μg/mL G418for3–4wk.

RNA Interference Assay.For stable knockdown of mRab10,an expression vector(psilencer-U6neo;Ambion)with insertion of the speci?c siRNA duplexes of mRab10or the scrambled siRNA duplexes were transfected into RAW264.7cells.Two mRab10siRNA target sequences were synthesized as follows:5′-GGGGTAATGCAGAAGTGAT-3′(Rab10iA)and5′-GCATCATGC-TAGTGTATGA-3′(KD251as described in ref.12).

RT-PCR and Quantitative PCR.Total cellular RNA was extracted using TRIzol reagent(Invitrogen).Reverse transcription and quantitative PCR was per-formed as described(16).The real time PCR primers were synthesized as follows:IL6(forward:AGT TGC CTT CTT GGG ACT GA;reverse:TCC ACG ATT TCC CAG AGA AC);TNF-α(forward:CTG GGA CAG TGA CCT GGA CT;reverse: GCA CCT CAG GGA AGA GTC TG);IFN-β(forward:CCC TAT GGA GAT GAC GGA GA;reverse:CTG TCT GCT GGT GGA GTT CA).Rab10(forward:CGA TGC CTT CAA TAC CAC CT;reverse:GCC ACT TGC TGA TGT TCT CA).

Measurement of Cytokines.IL-6,TNF-α,and IFN-βconcentrations in cell cul-ture supernatants or in bronchoalveolar lavage?uid(BALF)were measured using murine cytokine-speci?c Quantikine ELISA kits(eBioscience).

Luciferase Reporter Assay.The determinations of NF-κB and IFN-βreporter plasmid activity were performed as described previously(19).

Flow Cytometric Analysis.To detect cell surface expression of TLR4and other markers,cells were incubated with phycoerythrin-labeled antibodies against mouse TLR4or F4/80(eBioscience)for30min on ice,washed,and analyzed in a FACScalibur?ow cytometer(Becton Dickinson).

Western Blot.Total cell lysates were prepared as described previously(20)and protein concentration determined by the bicinchoninic acid(BCA)protein assay(Pierce).Cell extracts were subjected to SDS/PAGE,transferred onto nitrocellulose membrane,and blotted as described previously(21).Immuno?uorescence Staining and Confocal Microscopy.Cells were cultured on coverslips for48h before staining.For the colocalization analysis of GFP-Rab10with LysoTracker,cells were stained with100nM LysoTracker Red (Molecular Probes)for30min,and then cells were directly examined by confocal microscopy as described previously(16).For colocalization analysis of Rab10with organelle markers and TLR4-HA,transfected RAW264.7cells were sequentially immunostained?rst with Ab against TGN58K,EEA-1, LAMP-1,or HA,and then with proper DyLight549conjugated IgG(H+L)or DyLight488conjugated IgG(H+L)secondary Abs.The immunostaining pro-cess was performed as described(22).Slides were?nally examined under an Olympus FluoView FV1000confocal microscopy(Olympus).Images were acquired under40×/0.75NA oil objective and processed using Olympus Fluoview ver1.4a viewer(Olympus).

Murine Model of LPS-Induced Acute Lung Injury.Bone marrow derived mac-rophages(BMDMs)were isolated and cultured using a standard protocol as previously described(18).BMDMs were transfected with pcDNA3.1or pcDNA3. 1-mRab10plasmid using jetPEI-Macrophage transfection reagent(PolyPlus transfection),respectively.Female mice were divided into six groups(n=6,5, 7,5,7,6,respectively),Saline,BMDMs,BMDMs transfected with pcDNA3.1,or pcDNA3.1-mRab10plasmid(2×106cells,200μL total volume each)were given to each group via a jugular venous canula30min before LPS challenge.Mice were anesthetized and endotracheally intubated with a sterile plastic catheter and challenged with1.5mg/mL LPS(Escherichia coli0111:B4;Sigma)or normal saline.After24h,mice were killed and lungs were divided into two parts:the left lung lobes were lavaged three times with1mL of PBS with1%FCS and 5U/mL heparin;the right half were?xed by4%paraformaldehyde for his-tology.Cell counts were determined on BAL smear slides stained with Wight and Giemsa(Beyotime).Number of neutrophils was calculated as the per-centage of neutrophils multiplied by the total number of cells in the BAL?uid samples.BAL?uid collected was then centrifuged at800g,and supernatant was collected for analysis of cytokines levels.

Statistical Analysis.All experiments were independently performed three times in triplicate.Results are given as means plus or minus the https://www.wendangku.net/doc/1115551184.html,-parison between two groups were performed using Student t test,whereas differences between the treated mice groups versus the injured mice group (LPS/saline)were assessed using a one-way ANOVA(with post hoc compar-isons using Dunnett test)with GraphPad Prism version4.00statistic soft-ware.A value of P<0.05was considered statistically signi?cant. ACKNOWLEDGMENTS.We thank Drs Yuehai Ke,Wei Liu,Hu Hu,and Xue Zhang for their helpful discussion and Guifeng Xiao and Lan Xu for their excellent technical assistance.This work was supported by grants from the National Natural Science Foundation of China(30972724to L.L.and30901311 to D.W.),Zhejiang Provincial Natural Science Foundation of China(R2090202 to L.L.and Y2090401to D.W.),National Key Basic Research Program of China (2007CB512400to J.W),and the National High Technology Research and Development Program of China(2006AA02A239and2007AA021102to J.W.).

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蛋白质结构分析原理及工具-文献综述

蛋白质结构分析原理及工具 （南京农业大学生命科学学院生命基地111班） 摘要：本文主要从相似性检测、一级结构、二级结构、三维结构、跨膜域等方面从原理到方法再到工具，系统地介绍了蛋白质结构分析的常用方法。文章侧重于工具的列举，并没有对原理和方法做详细的介绍。文章还列举了蛋白质分析中常用的数据库。 关键词：蛋白质；结构预测；跨膜域；保守结构域 1 蛋白质相似性检测 蛋白质数据库。由一个物种分化而来的不同序列倾向于有相似的结构和功能。物种分化后形成的同源序列称直系同源，它们通常具有相似的功能；由基因复制而来的序列称为旁系同源，它们通常有不同的功能[1]。因此，推测全新蛋白质功能的第一步是将它的序列与进化上相关的已知结构和功能的蛋白质序列比较。表一列出了常用的蛋白质序列数据库和它们的特点。 表一常用蛋白质数据库 网址可能有更新 氨基酸替代模型。进化过程中，一种氨基酸残基会有向另一种氨基酸残基变化的倾向。氨基酸替代模型可用来估计氨基酸替换的速率。目前常用的替代模型有Point Accepted Mutation (PAM)矩阵、BLOck SUbstitution Matrix (BLOSUM)矩阵[2]、JTT模型[3]。 序列相似性搜索工具。序列相似性搜索又分为成对序列相似性搜索和多序列相似性搜索。成对序列相似性搜索通过搜索序列数据库从而找到与查询序列相似的序列。分为局部联配和全局联配。常用的局部联配工具有BLAST和SSEARCH，它们使用了Smith-Waterman 算法。全局联配工具有FASTA和GGSEARCH，基于Needleman-Wunsch算法。多序列相似性搜索常用于构建系统发育树，这里不阐述。表二列举了常用的成对序列相似性比对搜索工具

分子生物学 蛋白质的结构与功能

第二章蛋白质的结构与功能 一、单项选择题(A型题)，每题只有一个答案 1、各种蛋白质的含氮量接近于： A、6.25% B、16% C、84% D、12.5% E、61% 2、侧链上含有羧基的氨基酸是： A、Arg B、Gln C、Asn D、Glu E、Cys 3、维系蛋白质分子α-螺旋、β-折迭稳定的化学键是： A、肽键 B、二硫键 C、离子键 D、硫水键 E、氢键 4、无D－、L－构型之分的氨基酸是： A、Lys B、Ala C、Gly D、Glu E、Ser 5、哪种条件有利于β-折迭形成？ A、Gly或Ala连续出现 B、Pro存在处 C、酸性氨基酸连续排列 D、碱性氨基酸连续排列 E、Asn、Leu存在处 6、编码氨基酸不包括： A、半胱氨酸 B、亮氨酸 C、精氨酸 D、丙氨酸 E、鸟氨酸 7、使蛋白质变性的因素不包括： A、高温 B、高压 C、乙醇 D、蛋白酶催化水解 E、强酸 8、蛋白质变性后的改变不包括： A、肽链断裂 B、、水溶性下降 C、活性丧失 D、易沉淀 E、易被消化 9、Glu的pk1=2.6，pk2=4.6，pk3=9.6，其pI为： A、2.6 B、3.6 C、4.6 D、6.1 E、7.1 10、下列关于蛋白质的描述正确的是： A、变性大多可逆 B、变性后一定沉淀 C、沉淀后一定变性 D、变性后丧失紫外吸收特性 E、凝固是不可逆的变性 11、下列哪种氨基酸是中性氨基酸？ A、Glu B、Asp C、Trp D、His E、Arg 12、蛋白质二级结构的结构单元是： A、氨基酸残基 B、α-螺旋 C、β-折迭 D、肽单元 E、结构域13、蛋白质溶液对下列哪种波长的光有最大的吸收？ A、240nm B、260nm C、280nm D、340nm E、415nm 14、用哪种方法沉淀蛋白质，能较好地保存其活性？ A、强酸 B、丙酮 C、加热 D、Hg2+ E、(NH4)2SO4 15、肽键是指： A、–CO-NH- B、–CO-OH- C、–CO=NH D、–COH E、–CH=NH 16、关于多肽链的描述，不正确的是： A、N端含自由α-NH2 B、C端含自由α-COOH C、书写方向是N→C D、主链由肽键和Cα构成 E、侧链指的是氨基酸残基 17、关于蛋白质分子结构的描述，正确的是： A、模序是一种具有特殊功能的二级结构 B、二硫键参与各种空间结构的维系 C、天然蛋白质分子均具有一、二、三、四级结构 D、变性时蛋白质分子一、二、三、四级结构解体 E、分子伴侣不影响正确空间结构的形成 18、关于蛋白质分子中次级键的描述，不正确的是： A、键能虽小但数量多 B、二硫键是重要的次级键 C、二级结构主要靠氢键维系 D、次级键参与侧链结构的形成 E、次级键是非其价键 19、关于α-螺旋的描述，不正确的是： A、常见的二级结构形式 B、是一种右手螺旋结构 C、富有弹性，机械强度大 D、多以右双

生物信息学的主要研究内容

常用数据库 在DNA序列方面有GenBank、EMBL和等 在蛋白质一级结构方面有SWISS-PROT、PIR和MIPS等 在蛋白质和其它生物大分子的结构方面有PDB等 在蛋白质结构分类方面有SCOP和CATH等 生物信息学的主要研究内容 1、序列比对（Alignment） 基本问题是比较两个或两个以上符号序列的相似性或不相似性。序列比对是生物信息学的基础，非常重要。两个序列的比对有较成熟的动态规划算法，以及在此基础上编写的比对软件包BLAST和FASTA，可以免费下载使用。这些软件在数据库查询和搜索中有重要的应用。 2、结构比对 基本问题是比较两个或两个以上蛋白质分子空间结构的相似性或不相似性。已有一些算法。 3、蛋白质结构预测，包括2级和3级结构预测，是最重要的课题之一 从方法上来看有演绎法和归纳法两种途径。前者主要是从一些基本原理或假设出发来预测和研究蛋白质的结构和折叠过程。分子力学和分子动力学属这一范畴。后者主要是从观察和总结已知结构的蛋白质结构规律出发来预测未知蛋白质的结构。同源模建（Homology）和指认（Threading）方法属于这一范畴。虽然经过30余年的努力，蛋白结构预测研究现状远远不能满足实际需要。 4、计算机辅助基因识别(仅指蛋白质编码基因)。最重要的课题之一 基本问题是给定基因组序列后，正确识别基因的范围和在基因组序列中的精确位置.这是最重要的课题之一，而且越来越重要。经过20余年的努力，提出了数十种算法，有十种左右重要的算法和相应软件上网提供免费服务。原核生物计算机辅助基因识别相对容易些，结果好一些。从具有较多内含子的真核生物基因组序列中正确识别出起始密码子、剪切位点和终止密码子，是个相当困难的问题，研究现状不能令人满意，仍有大量的工作要做。 5、非编码区分析和DNA语言研究，是最重要的课题之一 在人类基因组中，编码部分进展总序列的3~5%，其它通常称为“垃圾”DNA，其实一点也不是垃圾，只是我们暂时还不知道其重要的功能。分析非编码区DNA 序列需要大胆的想象和崭新的研究思路和方法。DNA序列作为一种遗传语言，不仅体现在编码序列之中，而且隐含在非编码序列之中。 6、分子进化和比较基因组学，是最重要的课题之一 早期的工作主要是利用不同物种中同一种基因序列的异同来研究生物的进化，构建进化树。既可以用DNA序列也可以用其编码的氨基酸序列来做，甚至于可通过相关蛋白质的结构比对来研究分子进化。以上研究已经积累了大量的工作。近年来由于较多模式生物基因组测序任务的完成，为从整个基因组的角度来研究分子进化提供了条件。 7、序列重叠群（Contigs）装配 一般来说，根据现行的测序技术，每次反应只能测出500或更多一些碱基对的序列，这就有一个把大量的较短的序列全体构成了重叠群（Contigs）。逐步把它们拼接起来形成序列更长的重叠群，直至得到完整序列的过程称为重叠群装配。拼接EST数据以发现全长新基因也有类似的问题。已经证明，这是一个NP-完备

分子生物学主要研究内容

分子生物学主要研究内容 1. 核酸的分子生物学。 核酸的分子生物学研究 核酸的结构及其功能。由于 核酸的主要作用是携带和传 递遗传信息，因此分子遗传 学是其主要组成部分。由于 50年代以来的迅速发展，该 领域已形成了比较完整的理 论体系和研究技术，是目前分子生物学内容最丰富的一个领域。研究内容包括核酸/基因组的结构、遗传信息的复制、转录与翻译，核酸存储的信息修复与突变，基因表达调控和基因工程技术的发展和应用等。遗传信息传递的中心法则是其理论体系的核心。 2. 蛋白质的分子生物学。 蛋白质的分子生物学研究执行各种生命功能的主要大分子──蛋白质的结构与功能。尽管人类对蛋白质的研究比对核酸研究的历史要长得多，但由于其研究难度较大，与核酸分子生物学相比发展较慢。近年来虽然在认识蛋白质的结构及其与功能关系方面取得了一些进展，但是对其基本规律的认识尚缺乏突破性的进展。 3.细胞信号转导的分子生物学。 细胞信号转导的分子生物学研究细胞内、细胞间信息传递的分子基础。构成生物体的每一个细胞的分裂与分化及其它各种功能的完成均依赖于外界环境所赋予的各种指示信号。在这些外源信号的刺激下，细胞可以将这些信号转变为一系列的生物化学变化，例如蛋白质构象的转变、蛋白质分子的磷酸化以及蛋白与蛋白相互作用的变化等，从而使其增殖、分化及分泌状态等发生改变以适应内外环境的需要。信号转导研究的目标是阐明这些变化的分子机理，明确每一种信号转导与传递的途径及参与该途径的所有分子的作用和调节方式以及认识各种途径间的网络控制系统。信号转导机理的研究在理论和技术方面与上述核酸及蛋白质分子有着紧密的联系，是当前分子生物学发展最迅速的领域之一。 4.癌基因与抑癌基因、肽类生长因子、细胞周期及其调控的分子机理等。 从基因调控的角度研究细胞癌变也已经取得不少进展。分子生物学将为人类最终征服癌症做出重要的贡献。

蛋白质组学生物信息学分析介绍

生物信息学分析FAQ CHAPTER ONE ABOUT GENE ONTOLOGY ANNOTATION (3) 什么是GO？ (3) GO和KEGG注释之前，为什么要先进行序列比对（BLAST）？ (3) GO注释的意义？ (3) GO和GOslim的区别 (4) 为什么有些蛋白没有GO注释信息？ (4) 为什么GO Level 2的统计饼图里蛋白数目和差异蛋白总数不一致？ (4) 什么是差异蛋白的功能富集分析&WHY？ (4) GO注释结果文件解析 (5) Sheet TopBlastHits (5) Sheet protein2GO/protein2GOslim (5) Sheet BP/MF/CC (6) Sheet Level2_BP/Level2_MF/Level2_CC (6) CHAPTER TWO ABOUT KEGG PATHWAY ANNOTATION (7) WHY KEGG pathway annotation? (7) KEGG通路注释的方法&流程？ (7) KEGG通路注释的意义？ (7) 为什么有些蛋白没有KEGG通路注释信息？ (8) 什么是差异蛋白的通路富集分析&WHY？ (8) KEGG注释结果文件解析 (8) Sheet query2map (8) Sheet map2query (9) Sheet TopMapStat (9) CHAPTER THREE ABOUT FEATURE SELECTION & CLUSTERING (10) WHY Feature Selection? (10)

聚类分析（Clustering） (10) 聚类结果文件解析 (10) CHAPTER FOUR ABOUT PROTEIN-PROTEIN INTERACTION NETWORK (12) 蛋白质相互作用网络分析的意义 (12) 蛋白质相互作用 VS生物学通路？ (12) 蛋白质相互作用网络分析结果文件解析 (12)

蛋白质数据库

生物芯片北京国家工程研究中心 湖南中药现代化药物筛选分中心 暨湖南涵春生物有限公司 常用数据库名录 1、蛋白质数据库 PPI - JCB 蛋白质与蛋白质相互作用网络 ?Swiss-Prot - 蛋白质序列注释数据库 ?Kabat - 免疫蛋白质序列数据库 ?PMD - 蛋白质突变数据库 ?InterPro - 蛋白质结构域和功能位点 ?PROSITE - 蛋白质位点和模型 ?BLOCKS - 生物序列分析数据库 ?Pfam - 蛋白质家族数据库 [镜像： St. Louis (USA), Sanger Institute, UK, Karolinska Institutet (Sweden)] ?PRINTS - 蛋白质 Motif 数据库 ?ProDom - 蛋白质结构域数据库 (自动产生) ?PROTOMAP - Swiss-Prot蛋白质自动分类系统 ?SBASE - SBASE 结构域预测数据库 ?SMART - 模式结构研究工具 ?STRING - 相互作用的蛋白质和基因的研究工具

?TIGRFAMs - TIGR 蛋白质家族数据库 ?BIND - 生物分子相互作用数据库 ?DIP - 蛋白质相互作用数据库 ?MINT - 分子相互作用数据库 ?HPRD - 人类蛋白质查询数据库 ?IntAct - EBI 蛋白质相互作用数据库 ?GRID - 相互作用综合数据库 ?PPI - JCB 蛋白质与蛋白质相互作用网络 2、蛋白质三级结构数据库 ?PDB - 蛋白质数据银行 ?BioMagResBank - 蛋白质、氨基酸和核苷酸的核磁共振数据库?SWISS-MODEL Repository - 自动产生蛋白质模型的数据库 ?ModBase - 蛋白质结构模型数据库 ?CATH - 蛋白质结构分类数据库 ?SCOP - 蛋白质结构分类 [镜像: USA | Israel | Singapore | Australia] ?Molecules To Go - PDB数据库查询 ?BMM Domain Server - 生物分子模型数据库 ?ReLiBase - 受体/配体复合物数据库 [镜像： USA] ?TOPS - 蛋白质拓扑图 ?CCDC - 剑桥晶体数据中心 (剑桥结构数据库 (CSD))

生物信息学名词解释

1.计算生物信息学（Computational Bioinformatics）是生命科学与计算机科学、数理科学、化学等领域相互交叉而形成的一门新兴学科，以生物数据作为研究对象，研究理论模型和计算方法，开发分析工具，进而达到揭示这些数据蕴含的生物学意义的目的。 2.油包水PCR (Emulsion PCR) : 1) DNA片段和捕获磁珠混合; 2) 矿物油和水相的剧烈震荡产生油包水环境; 3) DNA片段在油包水环境中扩增;4) 破油并富集有效扩增磁珠。 3.双碱基编码技术：在测序过程中对每个碱基判读两遍，从而减少原始数据错误，提供内在的校对功能。代表测序方法：solid 测序。 4.焦磷酸测序法：焦磷酸测序技术是由4种酶催化的同一反应体系中的酶级联化学发光反应,适于对已知的短序列的测序分析,其可重复性和精确性能与SangerDNA测序法相媲美,而速度却大大的提高。焦磷酸测序技术不需要凝胶电泳,也不需要对DNA样品进行任何特殊形式的标记和染色,具备同时对大量样品进行测序分析的能力。在单核苷酸多态性、病原微生物快速鉴定、病因学和法医鉴定研究等方面有着越来越广泛的应用。例如：454测序仪 ：用蛋白质序列查找核苷酸序列。 :STS是序列标记位点（sequence-tagged site）的缩写，是指染色体上位置已定的、核苷酸序列已知的、且在基因组中只有一份拷贝的DNA短片断，一般长200bp －500bp。它可用PCR方法加以验证。将不同的STS依照它们在染色体上的位置依次排列构建的图为STS图。在基因组作图和测序研究时，当各个实验室发表其DNA测序数据或构建成的物理图时，可用STS来加以鉴定和验证，并确定这些测序的DNA片段在染色体上的位置；还有利于汇集分析各实验室发表的数据和资料，保证作图和测序的准确性。 :表达序列标签技术（EST，Expressed Sequence Tags）EST技术直接起源于人类基因组计划。 ：生物信息学数据库。UniGene试图通过计算机程序对GeneBank中的序列数据进行适当处理，剔除冗余部分，将同一基因的序列，包括EST序列片段搜集到一起，以便研究基因的转录图谱。UniGene除了包括人的基因外，也包括小鼠、大鼠等其它模式生物的基因。 :开放阅读框（ORF，open reading frame )是基因序列的一部分，包含一段可以编码蛋白的碱基序列，不能被终止子打断。编码一个蛋白质的外显子连接成为一个连续的ORF。 10.分子钟检验：只有分子钟的，没听过分子钟检验。一种关于分子进化的假说，认为两个物种的同源基因之间的差异程度与它们的共同祖先的存在时间(即两者的分歧时间)有一定的数量关系

SWISS-MODEL_蛋白质结构预测教程

SWISS-MODEL 蛋白质结构预测 SWISS-MODEL是一项预测蛋白质三级结构的服务，它利用同源建模的方法实现对一段未知序列的三级结构的预测。该服务创建于1993年,开创了自动建模的先河,并且它是讫今为止应用最广泛的免费服务之一。 同源建模法预测蛋白质三级结构一般由四步完成： 1. 从待测蛋白质序列出发，搜索蛋白质结构数据库（如PDB,SWISS-PROT等），得到许多相似序列 （同源序列），选定其中一个（或几个）作为待测蛋白质序列的模板； 2. 待测蛋白质序列与选定的模板进行再次比对，插入各种可能的空位使两者的保守位置尽量对齐； 3. 建模：调整待测蛋白序列中主链各个原子的位置，产生与模板相同或相似的空间结构——待测蛋白 质空间结构模型； 4. 利用能量最小化原理，使待测蛋白质侧链基团处于能量最小的位置。 最后提供给用户的是经过如上四步（或重复其中某几步）后得到的蛋白质三级结构。 SWISS-MODEL工作模式 SWISS-MODEL服务器是以用户输入信息的最小化为目的设计的，即在最简单的情况下，用户仅提供一条目标蛋白的氨基酸序列。由于比较建模程序可以具有不同的复杂性，用户输入一些额外信息对建模程序的运行有时是有必要的，比如，选择不同的模板或者调整目标模板序列比对。该服务主要有以下三种方式: ?First Approach mode(简捷模式）：这种模式提供一个简捷的用户介面：用户只需要输入一条氨基酸序列，服务器就会自动选择合适的模板。或者，用户也可以自己指定模板（最多5条），这些模板可以来自ExPDB 模板数据库（也可以是用户选择的含坐标参数的模板文件）。如果一条模板与提交的目标序列相似度大于25%，建模程序就会自动开始运行。但是，模板的可靠性会随着模板与目标序列之间的相似度的降低而降低，如果相似度不到50%往往就需要用手工来调整序列比对。这种模式只能进行大于25个残基的单链蛋白三维结构预测。 ?Alignment Interface（比对界面）：这种模式要求用户提供两条已经比对好的序列，并指定哪一条是目标序列，哪一条是模板序列（模板序列应该对应于ExPDB模板数据库中一条已经知道其空间结构的蛋白序列）。服务器会依据用户提供的信息进行建模预测。 ?Project mode(工程模式）：手工操作建模过程：该模式需要用户首先构建一个DeepView工程文件，这个工程文件包括模板的结构信息和目标序列与模板序列间的比对信息。这种模式让用户可以控制许多参数，例如：模板的选择，比对中的缺口位置等。此外，这个模式也可以用于“first approach mode简捷模式”输出结果的进一步加工完善。 此外，SWISS-MODEL还具有其他两种内容上的模式： ?Oligomer modeling(寡聚蛋白建模):对于具有四级结构的目标蛋白,SWISS-MODEL提供多聚模板的模式，用于多单体的蛋白质建模。这一模式弥补了简捷模式中只能提交单个目标序列,不能同时预测两条及以上目标序列的蛋白三维结构的不足。 ?GPCR mode(G蛋白偶联受体模式)：是专门对7次跨膜G蛋白偶联受体的结构预测。

蛋白质结构预测在线软件

蛋白质预测分析网址集锦? 物理性质预测：? Compute PI/MW?? ?? SAPS?? 基于组成的蛋白质识别预测? AACompIdent???PROPSEARCH?? 二级结构和折叠类预测? nnpredict?? Predictprotein??? SSPRED?? 特殊结构或结构预测? COILS?? MacStripe?? 与核酸序列一样，蛋白质序列的检索往往是进行相关分析的第一步，由于数据库和网络技校术的发展，蛋白序列的检索是十分方便，将蛋白质序列数据库下载到本地检索和通过国际互联网进行检索均是可行的。? 由NCBI检索蛋白质序列? 可联网到：“”进行检索。? 利用SRS系统从EMBL检索蛋白质序列? 联网到：”，可利用EMBL的SRS系统进行蛋白质序列的检索。? 通过EMAIL进行序列检索?

当网络不是很畅通时或并不急于得到较多数量的蛋白质序列时，可采用EMAIL方式进行序列检索。? 蛋白质基本性质分析? 蛋白质序列的基本性质分析是蛋白质序列分析的基本方面，一般包括蛋白质的氨基酸组成，分子质量，等电点，亲水性，和疏水性、信号肽，跨膜区及结构功能域的分析等到。蛋白质的很多功能特征可直接由分析其序列而获得。例如，疏水性图谱可通知来预测跨膜螺旋。同时，也有很多短片段被细胞用来将目的蛋白质向特定细胞器进行转移的靶标（其中最典型的例子是在羧基端含有KDEL序列特征的蛋白质将被引向内质网。WEB中有很多此类资源用于帮助预测蛋白质的功能。? 疏水性分析? 位于ExPASy的ProtScale程序（?）可被用来计算蛋白质的疏水性图谱。该网站充许用户计算蛋白质的50余种不同属性，并为每一种氨基酸输出相应的分值。输入的数据可为蛋白质序列或SWISSPROT数据库的序列接受号。需要调整的只是计算窗口的大小（n）该参数用于估计每种氨基酸残基的平均显示尺度。? 进行蛋白质的亲/疏水性分析时，也可用一些windows下的软件如，bioedit,dnamana等。? 跨膜区分析? 有多种预测跨膜螺旋的方法，最简单的是直接，观察以20个氨基酸为单位的疏水性氨基酸残基的分布区域，但同时还有多种更加复杂的、精确的算法能够预测跨膜螺旋的具体位置和它们的膜向性。这些技术主要是基于对已知

提高蛋白质的稳定性

提高蛋白质的稳定性 葡萄糖异构酶（GI）在工业上应用广泛，为提高其热稳定性，朱国萍等人在确定第138位甘氨酸(Gly138)为目标氨基酸后，用双引物法对GI基因进行体外定点诱变，以脯氨酸（Pro138）替代Gly138，含突变体的重组质粒在大肠杆菌中表达，结果突变型GI比野生型的热半衰期长一倍；最适反应温度提高10～12℃；酶比活相同。据分析，Pro替代Gly138后，可能由于引入了一个吡咯环，该侧链刚好能够填充于Gly138附近的空洞，使蛋白质空间结构更具刚性，从而提高了酶的热稳定性。 融合蛋白质 脑啡肽(Enk)N端5肽线形结构是与δ型受体结合的基本功能区域，干扰素（IFN）是一种广谱抗病毒抗肿瘤的细胞因子。黎孟枫等人化学合成了EnkN端5肽编码区，通过一连接3肽编码区与人α1型IFN基因连接，在大肠杆菌中表达了这一融合蛋白。以体外人结肠腺癌细胞和多形胶质瘤细胞为模型，采用3H－胸腺嘧啶核苷掺入法证明该融合蛋白抑制肿瘤细胞生长的活性显著高于单纯的IFN，通过Naloxone竞争阻断实验证明，抑制活性的增高确由Enk导向区介导。 蛋白质活性的改变 通常饭后30～60min，人血液中胰岛素的含量达到高峰，120～180min内恢复到基础水平。而目前临床上使用的胰岛素制剂注射后120min后才出现高峰且持续180～240min，与人生理状况不符。实验表明，胰岛素在高浓度（大于10－5mol/L）时以二聚体形式存在，低浓度时（小于10－9mol/L）时主要以单体形式存在。设计速效胰岛素原则就是避免胰岛素形成聚合体。类胰岛素生长因子－I(IGF－I)的结构和性质与胰岛素具有高度的同源性和三维结构的相似性，但IGF－I不形成二聚体。IGF－I的B结构域（与胰岛素B链相对应）中B28－B29氨基酸序列与胰岛素B链的B28－B29相比，发生颠倒。因此，将胰岛素B链改为B28Lys－B29Pro，获得单体速效胰岛素。该速效胰岛素已通过临床实验。 治癌酶的改造 癌症的基因治疗分二个方面：药物作用于癌细胞，特异性地抑制或杀死癌细胞；药物保护正常细胞免受化学药物的侵害，可以提高化学治疗的剂量。疱症病毒（HSV）胸腺嘧啶激酶(TK)可以催化胸腺嘧啶和其他结构类似物如GANCICLOVIR和ACYCLOVIR无环鸟苷磷酸化。GANCICLOVIR和ACYCLOVIR缺少3｀端羟基，就可以终止DNA的合成，从而杀死癌

蛋白常用数据库

搞蛋白质的童鞋们，甭要只查NCBI了~蛋白质相关数据库启蒙~ ★ 小木虫(金币+1):奖励一下，谢谢提供资源 qinhy:恭喜，您的帖子被版主审核为资源贴了，别人回复您的帖子对资源进行评价后，您就可以获得金币了理由:资源贴2011-11-26 16:56 本来是带图的，可是弄过来就变成米图了，附件里面一个是PDF版、一个是WORD版均是带图的，童鞋们看带图的可能比较方便点哦~ 基于蛋白质序列的蛋白质相互作用位点预测（闲谈版） 这个不是论文不是论文啊~~这个是应某某的要求帮他找的，所以都是用现成的免费的网站数据库做的预测分析。无论文为依托，无原理为根据，纯粹就是流连各大网站作个的闲谈。 1、用这些网站先查查你要研究的蛋白质的底细。 这些网站的数据库大多数是实验或者一些相关文献报道的数据的组成。 ★String http://string.embl.de/ 输入你要搜寻的蛋白，它就把这个蛋白相关的数据反映给你，分confidence、evidence的数据可信度参考，同时还具有actions选项，反应它们之间可能是激活/抑制的关系。按按+、-号可以扩大缩小关联蛋白的数量范围。 往下拉一点点就是数据,哈哈，我们都要看数据吃饭啊~~ 分析的数据源自Neighborhood、Fusion、Occurrence、Coexpression、Experiments Database、Textminin及Homology，表示点得证明有数据，根据各项数据给出综合评分。评分越高相互存在关系可能性越高。点击下方各项图标等详细看到各项数据内容。 设条件确定筛选范围。 ★DIP https://www.wendangku.net/doc/1115551184.html,/dip/Main.cgi 跟上面的大同小异的功能，装上它附带的软件可能操作性会好一点，不过我米有试过哦。倒是跟它有链接的几个数据库都很强大，大家可以点击看看。 ★BIND http://www.bind.ca 文献有介绍的网站，不过我不能理解为什么我注册就注不了……. 2、继续查，用这些网站将要研究的蛋白质的家庭背景，月收入也大起底。 这里的网站可能跟相互作用方面的关系不大，但是如果知道这些，可以对研究的蛋白有更深的了解。 ★PDB https://www.wendangku.net/doc/1115551184.html,/pdb/home/home.do 要查3D结构就往这里查~通常说的PDB号为文献号末4位。 ★PIR https://www.wendangku.net/doc/1115551184.html,/pirwww/index.shtml 在蛋白质方面如NCBI般强大的网站，去上面晃荡下吧，会有收获滴。 ★KEGG http://www.genome.jp/kegg/ 粉强大的一个网站，我只说说它的KEGG PA THW AY子项，能迅速掌握一个蛋白质的功能通路，对于小白的偶们来说，很有用，有木有。 3、正题正题，做完上面那些后，接着就是纯预测的成分。也因为如此，要找着这些网站是很悲催的一件事。就算你找着了，你不懂语言，不懂算法，到底结果的可靠性怎样，见人见智。 需要PDB号作分析： promate http://bioinfo.weizmann.ac.il/promate/

生物信息学常用核酸蛋白数据库

（1）GenBank https://www.wendangku.net/doc/1115551184.html,/ （2）dbEST (Database of Expressed Sequence Tags) https://www.wendangku.net/doc/1115551184.html,/dbEST/index.html （3）UniGene 数据库 https://www.wendangku.net/doc/1115551184.html,/UniGene/ （4）dbSTS (Database of Sequence Tagged Sites) https://www.wendangku.net/doc/1115551184.html,/dbSTS/index.html （5）dbGSS (Database of Genome Survey Sequences) https://www.wendangku.net/doc/1115551184.html,/dbGSS/index.html （6）HTG (High-Throughput Genomic Sequences) https://www.wendangku.net/doc/1115551184.html,/HTGS/ （7）基因组数据库 https://www.wendangku.net/doc/1115551184.html,/sites/entrez?db=genome （8）dbSNP (Database of Single Nucleotide Polymorphisms) 单核苷酸多态性数据库https://www.wendangku.net/doc/1115551184.html,/sites/entrez?db=snp （9）EMBL (European Molecular Biology Laboratory) https://www.wendangku.net/doc/1115551184.html,/embl （10）DDBJ (DNA Data Bank of Japan) http://www.ddbj.nig.ac.jp/Welcome-e.html 启动子（11）EPD (Eukaryotic Promoter Database) http://www.epd.isb-sib.ch/ 2、蛋白质数据库 https://www.wendangku.net/doc/1115551184.html,/swissprot （2）TrEMBL (Translation of EMBL) https://www.wendangku.net/doc/1115551184.html,/swissprot/ （3）PIR (Protein Information Resource) https://www.wendangku.net/doc/1115551184.html, （4）PRF (Protein Research Foundation) http://www.prf.or.jp/en/os.html （5）PDBSTR (Re-Organized Protein Data Bank) http://www.genome.ad.jp （6）Prosite https://www.wendangku.net/doc/1115551184.html,/prosite 3、结构数据库 （1）PDB (Protein Data Bank) https://www.wendangku.net/doc/1115551184.html, （2）NDB（Nucleic Acid Database） https://www.wendangku.net/doc/1115551184.html,/ （3）DNA-Binding Protein Database https://www.wendangku.net/doc/1115551184.html,/ （4）SWISS-3D IMAGE http://www.expasy.ch/sw3d/

整理(蛋白质序列数据库)

蛋白质序列数据库 我们可以根据基因组序列预测新基因，预测编码区域，并推测其产物（即蛋白质）的序列。因此，随着基因组序列的不断增长，蛋白质序列也在不断增加。 PIR 历史上，蛋白质数据库的出现先于核酸数据库。在1960年左右，Dayhoff和其同事们搜集了当时所有已知的氨基酸序列，编著了《蛋白质序列与结构图册》。从这本图册中的数据，演化为后来的蛋白质信息资源数据库PIR（Protein Information Resource）。 PIR是由美国生物医学基金会NBRF（National Biomedical Research Foundation）于1984年建立的，其目的是帮助研究者鉴别和解释蛋白质序列信息，研究分子进化、功能基因组，进行生物信息学分析。它是一个全面的、经过注释的、非冗余的蛋白质序列数据库。所有序列数据都经过整理，超过99%的序列已按蛋白质家族分类，一半以上还按蛋白质超家族进行了分类。PIR提供一个蛋白质序列数据库、相关数据库和辅助工具的集成系统，用户可以迅速查找、比较蛋白质序列，得到与蛋白质相关的众多信息。目前，PIR已经成为一个集成的生物信息数据源，支持基因组研究和蛋白质组研究。至2004年，PIR 有近30万个蛋白质的登录数据项，包括来自不同生物体的蛋白质序列。 除了蛋白质序列数据之外，PIR还包含以下信息： （1）蛋白质名称、蛋白质的分类、蛋白质的来源； （2）关于原始数据的参考文献； （3）蛋白质功能和蛋白质的一般特征，包括基因表达、翻译后处理、活化等； （4）序列中相关的位点、功能区域。 对于数据库中的每一个登录项，有与其它数据库的交叉索引，包括到GenBank、EMBL、DDBJ、GDB、MELINE等数据库的索引。PIR中一个具体的登录项如图4.4所示。

蛋白质的功能域、结构及其药物设计----6

第六章 蛋白质的功能域、结构及其药物设计 随着人类基因组全序列测定的完成，预示着基因组研究从结构基因组(Structural Genomics)进入了功能基因组(Functional Genomics)研究时代。研究基因组功能当然首先要研究基因表达的模式。当前研究这一问题可以基于核酸技术，也可以基于蛋白质技术，即直接研究基因的表达产物。测定一个有机体的基因组所表达的全部蛋白质的设想是由Williams于1994年正式提出的，而“蛋白质组”(proteome)一词是Wilkins于1995年首次提出。蛋白质组是指由一个细胞或组织的基因组所表达的全部相应的蛋白质。蛋白质组与基因组相对应，均是一个整体概念，但是两者又有根本的不同：一个有机体只有一个确定的基因组，组成该有机体的所有不同细胞都共享有一个基因组；但是，基因组内各个基因表达的条件、时间和部位等不同，因而它们的表达产物(蛋白质)也随条件、时间和部位的不同而有所不同。因此，蛋白质组又是一个动态的概念。由于以上原因，再加上由于基因剪接，蛋白质翻译后修饰和蛋白质剪接，基因遗传信息的表达规律更趋复杂，不再是经典的一个基因一个蛋白的对应关系，而是一个基因可以表达的蛋白质数目大于一。由此可见，蛋白质组研究是一项复杂而艰巨的任务。 蛋白质结构与功能的研究已有相当长的历史，由于其复杂性，对其结构与功能的预测不论是方法论还是基础理论方面均较复杂。统计学方法曾被成功地应用于蛋白质二级结构预测中，如Chou和Fasman提出的经验参数法便是最突出的例子。 该方法统计分析了各种氨基酸的二级结构分布特征，得出相应参数(P а,P β 和P t )并 用于预测。本章将简要介绍蛋白质结构与功能预测的生物信息学途径。 第一节 蛋白质功能预测 一、根据序列预测功能的一般过程 如果序列重叠群(contig)包含有蛋白质编码区，则接下来的分析任务是确定表达产物——蛋白质的功能。蛋白质的许多特性可直接从序列上分析获得，如疏水性，它可以用于预测序列是否跨膜螺旋(transmenbrane helix)或是前导序列(leader sequence)。但是，总的来说，我们根据序列预测蛋白质功能的唯一方法是通过数据库搜寻，比较该蛋白是否与已知功能的蛋白质相似。有2条主要途径可以进行上述的比较分析： ①比较未知蛋白序列与已知蛋白质序列的相似性； ②查找未知蛋白中是否包含与特定蛋白质家族或功能域有关的亚序列或保守区段。 图6.1给出了根据序列预测蛋白质功能的大致过程。由于涉及数条技术路线，所得出的分析结果并不会总是相一致。一般来说，数据库相似性搜索获得的结果最为可靠，而来自PROSITE的结果相对不可靠。

蛋白质结构预测网址

蛋白质结构预测网址 物理性质预测： Compute PI/MW Peptidemass TGREASE SAPS 基于组成的蛋白质识别预测 AACompIdent PROPSEARCH 二级结构和折叠类预测 nnpredict Predictprotein SSPRED 特殊结构或结构预测 COILS MacStripe 与核酸序列一样，蛋白质序列的检索往往是进行相关分析的第一步，由于数据库和网络技校术的发展，蛋白序列的检索是十分方便，将蛋白质序列数据库下载到本地检索和通过国际互联网进行检索均是可行的。 由NCBI检索蛋白质序列 可联网到：“”进行检索。 利用SRS系统从EMBL检索蛋白质序列 联网到：”，可利用EMBL的SRS系统进行蛋白质序列的检索。 通过EMAIL进行序列检索 当网络不是很畅通时或并不急于得到较多数量的蛋白质序列时，可采用EMAIL方式进行序列检索。 蛋白质基本性质分析 蛋白质序列的基本性质分析是蛋白质序列分析的基本方面，一般包括蛋白质的氨基酸组成，分子质量，等电点，亲水性，和疏水性、信号肽，跨膜区及结构功能域的分析等到。蛋白质的很多功能特征可直接由分析其序列而获得。例如，疏水性图谱可通知来预测跨膜螺旋。同时，也有很多短片段被细胞用来将目的蛋白质向特定细胞器进行转移的靶标（其中最典型的例子是在羧基端含有KDEL序列特征的蛋白质将被引向内质网。WEB中有很多此类资源用于帮助预测蛋白质的功能。 疏水性分析 位于ExPASy的ProtScale程序（）可被用来计算蛋白质的疏水性图谱。该网站充许用户计算蛋白质的50余种不同属性，并为每一种氨基酸输出相应的分值。输入的数据可为蛋白质序列或SWISSPROT数据库的序列接受号。需要调整的只是计算窗口的大小（n）该参数用于估计每种氨基酸残基的平均显示尺度。 进行蛋白质的亲/疏水性分析时，也可用一些windows下的软件如， bioedit,dnamana等。 跨膜区分析 有多种预测跨膜螺旋的方法，最简单的是直接，观察以20个氨基酸为单位的疏水性氨基酸残基的分布区域，但同时还有多种更加复杂的、精确的算法能够预测跨膜螺旋的具体位置和它们的膜向性。这些技术主要是基于对已知跨膜螺旋的研究而得到的。自然存在的跨膜螺旋Tmbase 数据库，可通过匿名FTP获得()，参见表一

生物信息学论文完结版

生物信息学论文 学院：生命科学技术学院 专业：生物科学 班级：2013级 老师：高亚梅 学生：蔡欣月 学号：20134083003

链孢霉GH5-1及GH6-3基因生物信息学分析蔡欣月（黑龙江八一农垦大学，生命科学技术学院，2013级生物科学专业，黑龙江省，大庆市） 【摘要】目的：分析和预测链孢霉菌GH5-1和GH6-3基因及其编码蛋白质的结构和特征。方法：利用NCBI、CBS和ExPASy网站中的各种信息分析工具，并结合VectorNTIsuite8.0生物信息分析软件包，分析预测链孢霉菌GH5-1和GH6-3基因并预测该基因编码蛋白结构的特征和功能。结果：GH5-1基因全长2006bp,编码区具有390个氨基酸，在GenBank同源序列中，其与endoglucanase 3 [Neurospora crassa OR74A]基因氨基酸序列一致性达到100%，且有GH5-1保守域。GH5-1蛋白相对分子量预测为41907.4，理论等电点为5.14。预测GH5-1编码蛋白α螺旋(H ) 、β折叠(E )、无规则卷(L )的比例分别是16.92％、33.85％、49.23％，2个GTPase结构域。GH5-1蛋白为亲水蛋白，无跨膜区，有信号肽。GH6-3基因全长1914bp,编码区具有419个氨基酸，在GenBank同源序列中，其与exoglucanase 3 [Neurospora crassa OR74A]基因氨基酸序列一致性达到100%，且有GH6-3保守域。GH6-3蛋白相对分子量预测为44839.3，理论等电点为6.51。预测GH6-3编码蛋白α螺旋(H ) 、β折叠(E )、无规则卷(L )的比例分别是29.59％、16.71％、53.75％，1个GTPase结构域。GH6-3蛋白为亲水蛋白，有跨膜区，无信号肽。结论：成功预测GH5-1和GH6-3基因及其编码蛋白生化及其结构特征，为下一步对其进行克隆和表达奠定基础。 【关键词】链孢霉菌；糖基水解酶家族5（GH5-1）；糖基水解酶家族6（GH6-3）生物信息学 链孢霉菌又称脉孢菌、串珠菌、红色面包菌，俗称红霉菌，是食用菌生产中重要的竞争性杂菌之一。其广泛分布在自然界土壤中和和禾本科植物上，尤其在玉米芯上极易发生[1]。通过空气、土壤、腐烂植物、谷物等进行传播、在食用菌生产中，链孢菌和绿菌是生产中最常见的病原菌。链孢霉在高温高湿条件下最易发生，是夏季食用菌生产中危害严重的病原菌，该病原菌生活力强、生长迅速、繁殖快、分生孢子多、易传播，几乎会感染所有熟料栽培的食用菌，并且一旦感染很难彻底消灭，给生产造成较大的经济损失，严重危害所有食用菌的母种、原种、栽培种，以及香菇、木耳、银耳、银耳、灵芝等熟料菌简[2]。目前链孢霉菌的全基因组序列已经获得，但有关其蛋白和基因的各类研究仍为数较少，本文通过对链孢霉GH5-1和GH6-3基因及编码蛋白质进行生物信息学分析，分析其基本生化及结构特征，为下一步对其进行克隆表达和应用奠定基础。 一、材料与方法 1.1材料 通过ExPASy 数据库的UniProtKB（https://www.wendangku.net/doc/1115551184.html,或https://www.wendangku.net/doc/1115551184.html,/uniprot）获得链孢霉菌的GH5-1与GH6-3基因序列。GH5-1基因编号为NCU00762，NCBI的登录号为XM_959066.2，其他物种的GH5-1的氨基酸序列均来自Genbank，登录号见表1。GH6-3基因编号为NCU09680，NCBI的登录号为XM_952322.2，其他物种的GH6-3的氨基酸序列均来自Genbank，登录号见表2。 1.2方法 利用美国国家生物技术信息中心（NCBI,https://www.wendangku.net/doc/1115551184.html,）的基本局部比对搜索工具（BLAST,https://www.wendangku.net/doc/1115551184.html,/blast/），运用Blastx完成基因同源性分析。 应用ORF finder（https://www.wendangku.net/doc/1115551184.html,/gorf/orfig.cgi）寻找其开放读码框，并推导出可编码蛋白序列。 利用保守结构域（https://www.wendangku.net/doc/1115551184.html,/Structure/cdd/wrpsb.cgi）分析预测其保守域。 通过瑞士生物信息学研究所的蛋白分析专家系统（ExPASy，https://www.wendangku.net/doc/1115551184.html,）所提供的蛋白组学和分析工具：Protparam、Proscale程序分析GH5-1及GH6-3蛋白氨基酸组成、相对分子质量、等电点等基本理化性质；TMHMM程序预测GH5-1及GH6-3的跨膜区；SignalP程序预测GH5-1及GH6-3蛋白的信号肽，

稳定蛋白结构的相互作用

稳定蛋白质三维结构的作用力 稳定蛋白质三维结构的作用力主要是一些所谓弱的相互作用或称非共价键或次级键，包括氢键，范德华力，疏水作用和盐键（离子键）。此外共价二硫键在稳定某些蛋白质的构象方面也起着重要作用。 氢键(hydrogen bond)在稳定蛋白质的结构中起着极其重要的作用。多肽主链上的羰基氧和酰胺氢之间形成的氢键是稳定蛋白质二级结构的主要作用力。此外，还可在侧链与侧链，侧链与介质水，主链肽基与侧链或主链肽基与水之间形成。

由电负性原子与氢形成的基团如N-H和O-H具有很大的偶极矩，成键电子云分布偏向负电性大的原子，因此氢原子核周围的电子分布就少，正电荷的氢核（质子）就在外侧裸露。这一正电荷氢核遇到另一个电负性强的原子时，就产生静电吸引，即所谓氢键。 范德华力(van der waals force）广义上的范德华力包括3种较弱的作用力：定向效应，诱导效应，分散效应。 分散效应（dispersion effect)是在多数情况下主要作用的范德华力，它是非极性分子或基团间仅有的一种范德华力即狭义的范德华力，也称london分散力。这是瞬时偶极间的相互作用，偶极方向是瞬时变化的。 范德华力包括吸引力和斥力。吸引力只有当两个非键合原子处于接触距离（contact distance)或称范德华距离即两个原子的范德华半径之和时才能达到最大。就个别来说范德华力是很弱的，但其相互作用数量大且有加和效应和位相效应，因此成为一种不可忽视的作用力。 疏水作用（hydrophobic interaction)介质中球状蛋白质的折叠总是倾向与把疏水残基埋藏在分子的内部，这一现象称为疏水作用，它在稳定蛋白质的三维结构方面占有突出地位。疏水作用其实并不是疏水基团之间有什么吸引力的缘故，而是疏水基团或疏水侧链出自避开水的需要而被迫接近。 蛋白质溶液系统的熵增加是疏水作用的主要动力。当疏水化合物或基团进入水中时，它周围的水分子将排列成刚性的有序结构即所谓笼形结构(clathrate structure)。与此相反的过程（疏水作用），排列有序的水分子（笼形结构）将