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Protein Complex and Protein-protein Interaction

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Title: Protein Complex and Protein-protein Interaction


1
Protein Complex and Protein-protein Interaction
  • ???
  • ?????????????
  • Email pengkp_at_yahoo.com.cn

2
Central dogma the story of life
Protein is the final player in cell life
DNA
RNA
Protein
3
Proteins function in association with other
proteins or biomolecules, but not in isolation
4
Introduction to Proteomics
  • the analysis of genomic complements of proteins
  • dynamic
  • systematic
  • discovery-driven

5
Goals of Proteomics
to discover drug target
to understand cellular processes
to discover protein function
to identify biomarker
to understand disease states
6
Types of Proteomics
  • Expression Proteomics
  • Quantitative study of protein expression and
    their changes between samples that differs by
    some variable
  • Functional Proteomics
  • To study protein-protein interaction, 3-D
    structures, cellular localization and PTMs in
    order to understand the physiological function of
    the whole set of proteome.

7
Approaches
Genetic yeast two-hybrid phage display
Biophysical Mass Spectrometry SPR FRET
Biochemical Blue native PAGE Far Western
Pull-down Coimmunoprecipitation TAP Crosslinking
Bioinformatic Co-occurrence Neighborhood Surface
patch
8
Blue Native PAGE
  • separation of native proteins in complex.
  • Coomassie Blue G stable and negatively charge
    multiprotein complex.
  • 6-aminocaproic acid solubilize membrane protein
    complex instead of salts.
  • the resolution is not so high that the
    prepurification is needed.

Anal Biochem 1991, 199223-231
9
Blue Native PAGE
_
detergent
CBB
6-ACA

10
Blue Native PAGE
Solubilization with nonionic detergent
(laurylmaltoside, TX-100, CHAPS, Mega 9,
octylglucoside, Brij 35, etc), supplemented with
6-aminocaproic acid
Sample Preparation
Blue Native PAGE
Separation gel 6-13 gradient Cathode buffer
contains 0.02 Coomassie blue G250
SDS-PAGE
Separation of members of multiprotein complex
11
Blue Native PAGE of chloroplast thylakoid
membranes
BN-PAGE of solubilized chloroplast thylakoid
membranes (a) followed by SDSPAGE in the second
dimension (b). CF0F1 ATP synthase was indicated.
BBRC 1999, 259569-575
12
Blue Native PAGE of chloroplast thylakoid
membranes
lane 1 LMW marker lane 2 CF0F1 ATP synthase,
purified by density gradient centrifugation lane
3 electroeluted protein from the intense band
(Rf 0.38) in BN-PAGE (a).
BBRC 1999, 259569-575
13
Blue Native PAGE of multiprotein complex from
whole cellular lysate
Dialysis permits the analysis of multiprotein
complexes of whole cellular lysates by BN-PAGE.
MCP 3176-182, 2004
14
Identification and analysis of distinct
proteasomes by WCL 2D BN/SDS-PAGE
Blue Native PAGE
A, WCL of HEK293 cells was separated by 2D
BN/SDS-PAGE (5.514 and 10, respectively), and
immunoblotting was performed with specific
antibodies recognizing either subunits of the 20S
core complex (Mcp21 and 2), or a subunit of the
19S cap of the 26S proteasome (S4 ATPase), or a
subunit of the PA28 regulatory subunit (PA28).
B, An identical sample was boiled in 1 SDS,
resolved by 2D BN/SDS-PAGE, and immunoblotted as
described in A.
MCP 3176-182, 2004
15
Visualization of MPCs on a 2D WCL BN/SDS gel
Blue Native PAGE
B, WCL of HEK293 cells was boiled with 1 SDS
before separation and staining.
A, WCL of HEK293 cells was prepared using Triton
X-100 and separated by 2D BN/SDS-PAGE (5.517 and
10, respectively).
MCP 3176-182, 2004
16
Far Western
17
Max functional cloning of a Myc-binding protein
Far Western
A. CKII, casein kinase II phosphorylation site
BR, basic region HLH, helix-loop-helix LZ,
leucine zipper. B. Plaques that express
beta-galactosidase fusion prteins were screened
for their ability to react with 125I-labeld
GST-MycC92. Top left, secondary plating of five
putative positive demonstrates the reactivity of
two of the primary plaques, Max11 and Max14. Top
right, as a negative control, GST was labeled to
a similar specific activity and compared with
GST-MycC92 for bidning to Max14 plaques. Bottom,
binding of GST-MycC92 to Mzx14 plaques was
assayed with or without affinity purified
carboxyl terminal-specific anti-Myc (Ab) or
peptide immunogen (peptide).
MycC92
Science 2511211-7, 1991
18
Association of Rb with HIP1
Far Western
HeLa nulear extract (100 ug) (lane 1, 2) and
HIP1 (200 ng) purified from HeLa (lane 3, 4)
were electrophoresed, blotted, and renatured in
situ. Adjacent strips were cut from the filters
and probed with 32P-GST-RB(379-928) (lane 1, 3)
or 32P-GST-RB(379-928706F) (lane 2, 4)
Cell 70351-364, 1992
19
GST Pulldown
20
Interactions of Cellular Polypeptides with the
Cytoplasmic Domain of the Mouse Fas Antigen
GST Pulldown
Fas 45-kilodalton transmembrane receptor that
initiates apoptosis The biochemical mechanisms
responsible for Fas action are incompletely
understood the cytoplasmic domain is clearly
necessary for Fas to function as a receptor The
cytoplasmic domain does not display any known
enzymatic activities but is capable of
interacting with a number of proteins.
JBC 2718627-32, 1996
21
GST-mFas fusion proteins
GST Pulldown
22
GST-mFas-associated polypeptides from 32S-labeled
HeLa, L929, and Jurkat cell lysates
GST Pulldown
Preclearation 25 ug GST/50 ul GSH-Seph. Incubatio
n 10 ug GST/GST-mFas-(194-306) Wash 0.5 NP-40,
20 mM Tris, pH 8.0, 200 mM NaCl Elution 50 ul 20
mM GSH in 50 mM Tris
23
GST-mFas-associated polypeptides are stable to
high salt concentrations
GST Pulldown
HeLa cell lysates were screened with either GST
or GST-mFas-(194306) as described above except
that the Sepharose-protein complexes were washed
with Lysis Buffer containing different salt
concentrations (as indicated). The eluted
material was subjected to 12 SDS-PAGE and
fluorography.
24
Association is blocked by preincubation with a
polyclonal antibody against GST-mFas
GST Pulldown
A. the antibody recognized the Fas intracellular
domain B. association of proteins from HeLa
lysate with GST-mFas was blocked by anti-GST-mFas
IgG C. anti-GST antibody had no effect up to 100
ug of IgG.
25
Differential association with mutant forms of
GST-mFas
GST Pulldown
26
Schematic representation of the mouse Fas antigen
and its binding proteins
GST Pulldown
27
Epitope tagging
GST Pulldown
28
Co-Immunoprecipitation
In the intact cell, protein X is present in a
complex with protein Y. This complex is preserved
after cell lysis and allows protein Y to be
coimmunoprecipitated with protein X (complex 1).
However, the disruption of subcellular
compartmentalization could allow artifactual
interactions to occur between some proteins, for
example, protein X and protein B (complex 2).
Furthermore, the antibody that is used for the
immunoprecipitation may cross-react
nonspecifically with other proteins, for example,
protein A (complex 3). The key to identification
of proteinprotein interactions by
coimmunoprecipitation is to perform the proper
controls so as to identify protein Y but not
protein A and B.
29
Co-Immunoprecipitation
Antibody Identification
The protein against which the antibody was raised
should be precipitated from cell lysate. (1)
Independent antibodies raised against the same
protein recognize the same polypeptide (2)
Target protein should not be identified with
antibodies from cell lines without target protein
30
False positive and control
Co-Immunoprecipitation
1. Antibody control Monoclonal Ab another MoAb
against similar protein Antiserum serum before
immunization from the same animal Polyclonal Ab
purified PoAb against another protein 2. Multiple
antibodies different Abs against different
epitopes the epitope may be the site for
association with other proteins 3. Cell lines
depleted of target protein Control experiment
should be practised in depleted cell lines 4.
Inactive biological mutant 5. Interaction
verification before and after cell
lysis unphysiological interaction
31
Reduction of nonspecific protein background
Co-Immunoprecipitation
1. to increase ionic strength in wash buffer 2.
to reduce the amount of primary Ab 3. to
preclear cell lysate with control Ab.
32
Binding of pVHL to Elongin B and C
Co-Immunoprecipitation
1. von Hippel-Lindau disease is a hereditary
cancer syndrome characterized by the development
of multiple tumors 2. VHL susceptibility gene,
mutated in the majority of VHL kindreds, is a
tumor suppressor 3. to elucidate the
biochemical mechanisms underlying tumor
suppression by pVHL, search for cellular proteins
that bound to wt pVHL, but not to tumor-derived
pVHL mutants.
Science 2691444-6, 1995
33
Identification of VHL-associated proteins
Co-Immunoprecipitation
Lysates from 786-O renal carcinoma cells,
transfected with the indicated pVHL constructs,
were immunoprecipitated with anti-HA (A and B) or
with anti-VHL (C). Detection by autoradiography
(A, C) or by immunoblotting (B). open arrows exo
pVHL closed arrows VHL-AP
pVHL(1-115) without residues frequently altered
by naturally occurring VHL mutations and, unlike
pVHL(wt), does not suppress tumor formation in
vivo. pVHL(167W) the predicted product of a
mutant VHL allele that is common in VHL families.
34
Mapping the p14 and p18 binding site on pVHL
Co-Immunoprecipitation
A. 786-O cells producing HA-VHL(wt) or
HA-VHL(1-115) were labeled with 35S-methione,
lysed, and immunoprecipitated with anti-HA.
Parental 786-O cells were similarly labeled,
lysed, and incubated with GSH Sepharose preloaded
with GST-VHL(117-213) or GST alone. B and C.
786-O cells were labeled, lysed, and incubated
with GSH Sephorase preloaded with the indicated
GST-VHL fusion protein. In (C), the indicated
peptides (final conc. 0.1, 1, or 10 uM) were
added to the GST-VHL fusion protein before
incubation with the radiolabeled extract. The wt
peptide is TLKERCLQWRSLVKP (underlined residues
are sites of germ-line missense mutations). The
mutant peptide is TLKERFLQWRSLVKP.
35
the binding site for Elongin B and C in pVHL
Co-Immunoprecipitation
Distribution of germ-line VHL mutations. The
shaded region represents the bidning site for
Elongin B and C.
36
Binding of pVHL to Elongin B and Elongin C in vivo
Co-Immunoprecipitation
A. ACHN (VHL /), CAKI-1 (VHL /), 786-O (VHL
-/-), and 293 (VHL /) cells were labeled with
35S-methione, lysed, and immunoprecipitated with
anti-VHL or a control antibody. The
immunoprecipitaes were washed under high-salt
conditions. The identification of pVHL(wt) (open
arrow) was confirmed by anti-pVHL immunoblot
analysis. The 19 kD protein immediately above
p18 () in the ACHN, CAKI-1, and 293 cell
anti-VHL immunoprecipitates reacts with a
polyclonal antibody to VHL. B. Comparison of
peptides generated by partial proteolysis of
Elongin B and C, translated in vitro, with p18
and p14.
37
TAP tandem affinity purification
38
Sequence and structure of the TAP tag
TAP
39
Overview of the TAP procedure
TAP
40
Schematic representation of the split TAP tag
strategy
TAP
41
Schematic representation of the substraction
strategy
TAP
42
Protein composition of TAP-purified U1 snRNP
TAP
43
Step-by-step analysis of the TAP strategy
TAP
Proteins present in the final TAP fraction (lanes
7 and 8), or present after each of the single
affinity purification steps (lanes 14), were
analyzed. Snu71-TAP (lanes 1, 3, and 7) or
wild-type extracts (lanes 2, 4, and 8) were used.
Lane 5 molecular weight marker. Lane 6 an
amount of TEV protease identical to the amount
used to elute proteins bound to IgG beads (lanes
2, 3, 7, and 8). Right arrows indicate the U1
snRNP-specific proteins including the tagged
Snu71p after TEV cleavage the arrow on the left
indicates the Snu71p protein fused to the TAP tag
before TEV cleavage.
44
TAP in higher eucaryotes
TAP
Questions overexpression endogenous
expression Solutions RNA interference Knockin
technique
45
Strengths and weaknesses of commonly used
affinity approaches for the retrieval of protein
complexes
46
FRET fluorescence resonance energy transfer
47
FRET
E energy transfer efficiency R0 intermolecular
distance when half of energy is transfered r
distance between fluorophores
E R06/(R06 r6) when r 2R0, E 1/65
48
Imaging protein phosphorylation by FRET
FRET
49
Detection of protein interaction by FRET
FRET
in vitro
phosphorylation
in vivo
50
FRET reveals interleukin (IL)-1-dependent
aggregation of IL-1 type I receptors that
correlates with receptor activation
FRET
JBC 27027562-8, 1995
51
Abbreviation
FRET
IL-1 interleukin 1 IL-1 RI IL-1 type I
receptor IL-1ra IL-1 receptor antagnist CHO-mu1c
CHO-K1 cells stably transfected with wild- type
IL-1 receptor CHO-extn CHO-K1 cells stably
transfected with cytoplasmic tail-truncated
IL-1 receptor M5 noncompetitive anti-IL1 RI
monoclonal antibody FITC-M5 M5 labeled with a
donor probe, FITC Cy3-M5 M5 labeled with a
acceptor probe, Cy3
52
IL-1a-dependent FRET between donor FITC-M5 and
acceptor Cy3-M5 bound to IL-1 RI on the surface
of CHO-mu1c cells
FRET
A, a mixture of 5 nM FITC-M5 and 5 nM Cy3-M5 was
incubated with CHO-mu1c cells (3 X 106 cells/ml)
containing wild-type transfected receptors for 50
min at 22 C. IL-1a or IL-1ra was added at a
final concentration of 30 nM immediately after
the time point at t 0 min (arrow), and changes
in the ratio of Cy3-M5 fluorescence to FITC-M5
fluorescence were monitored over time. Changes in
this ratio were also monitored for the control
sample to which no ligand was added. B,
normalized fluorescence ratio for cells with
added IL-1a or IL-1ra calculated from data in A.
53
IL-1a but not IL-1ra causes aggregation between
IL-1 RI-labeled with FITC and Cy3 Fab fragments
of M5 as detected by FRET
FRET
A mixture of 20 nM FITC-M5-Fab and 20 nM
Cy3-M5-Fab was added to CHO-mu1c cells
transfected with wild-type receptors and
incubated at 22 C for 50 min. IL-1a or IL-1ra
was added to a final concentration of 10 nM
immediately after the time point at 0 min.
Changes in the normalized ratio of Cy3-M5 Fab
fluorescence to FITC-M5 Fab fluorescence were
monitored over time at 22 C.
54
IL-1-dependent energy transfer between IL-1 RI is
temperature
FRET
A mixture of 20 nM FITC-M5 Fab and 12 nM Cy3-M5
Fab was added to CHO-mu1c cells (3 X 106
cells/ml) with transfected wild-type IL-1 RI and
preincubated at either 4 C (A) or 22 C (B) for
50 min. Immediately after the base-line data
point at t 0 min, IL-1a was added (arrow) at a
final concentration of 10 nM to both samples.
Changes in the normalized ratio of Cy3-M5 Fab
fluorescence to FITC-M5 Fab fluorescence was
monitored over time at the corresponding
preincubation temperature. At t 85 min, the
temperature for sample (A) was changed from 4 to
22 C, and the temperature for sample (B) was
changed from 22 to 4 C. Changes in the
normalized fluorescence ratio continued to be
monitored until t 180 min.
55
IL-1a-dependent FRET can be detected between
FITC-M5 Fab and Cy3-M5 Fab bound to the
cytoplasmic tail deleted mutant IL-1 RI on
CHO-extn cells
FRET
A mixture of 20 nM FITC-M5 Fab and 12 nM Cy3-M5
Fab was added to wild-type transfected receptors
on CHO-mu1c cells and incubated at 22 C for 50
min (A). A mixture of 20 nM FITC-M5 Fab and 12 nM
Cy3-M5 Fab was added to CHO-extn cells
(cytoplasmic tail deleted mutant IL-1 RI) and
incubated at 22 C for 50 min (B). IL-1a was
added to a final concentration of 20 nM at the
arrow, and changes in the normalized ratio of
Cy3-M5 Fab fluorescence to FITC-M5 Fab
fluorescence were monitored over time at 22 C.
A
B
56
SPR Surface Plasma Resonance
57
Diagram of BIAcore
SPR
58
Interactions betweenlectins and immobilized
glycoproteins
SPR
59
Interactions betweenlectins and immobilized
glycoproteins
SPR
An overlay plot of binding curves showing the
interaction between lectins and immobilized
thyroglobulin. Lectin solutions (50 µg/ml in 10
mM HEPES, 0.5 mM MnCl2 , 0.5 M CaCl2 and 0.05
surfactant, pH 7.4) were injected. Bound lectin
was dissociated by 100 mM HCl (15 µl, 5 µl/min).
60
Summary of the interaction of seven lectins of
different nominal specificities with immobilized
glycoproteins
SPR
Binding of lectin to the glycoprotein is
indicated by and lack of binding by - in
the above table. As control experiments, the
lectins were injected over (i) an immobilized
non-glycosylated protein (recombinant HIV-1
reverse transcriptase expressed in E. coli) and
(ii) a blank surface which was subjected to
immobilizationchemistry in absence of a protein.
The lectins did not show any binding in the
control experiments.
61
SPR-MS Ligand Fishing with Biacore 3000
SPR
Selective binding, recovery and identification by
MALDI MS of a specific interaction partner
62
Other important techniques in protein interaction
research
Mass Spectrometry Cross-linking Ultracentrifuge Ch
IP (Chromatin immunoprecipitation)
63
Mass spectrometry is indispensable for protein
identification and will be in the center of
proteomics research.
Mass Spectrometry
High sensitivityHigh resolutionHigh throughput
64
Reference data bases
  • Interactions
  • MIPS
  • DIP
  • YPD
  • Intact (EBI)
  • BIND/ Blueprint
  • GRID
  • MINT
  • Prediction server
  • Predictome (Boston U)
  • Plex (UTexas)
  • STRING (EMBL)
  • Protein complexes
  • MIPS
  • YPD

65
From defining the proteome to understanding
function
Thanks!
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