1. 蛋白质相互作用 廖侃
生化与细胞所
2017,9,26

2. Protein interaction：two or more proteins (same or different) interact or form complex
Multiple protein interaction: e.g. F-actin, microtuble, proteins interact to form a functional structure
Enzyme complex: e.g. fatty acid synthase, multiple enzymes to catalyze a set of reactions
Ribosome: protein complex to execute a complicated reaction
Antibody-antigen interaction: to neutralize pathogen
Kinase-substrate interaction: to pass signal
Transcription factors: to induce gene expression
etc……

3. Protein: an important constituent of life
Protein interaction: make the difference of being live or dead
Protein interaction: from protein to system

4. Protein interaction:
1、protein interaction has its own biological functions and significance. It is part of the life. It is important for life itself.
We study it to understand life.
e.g. the mechanism of Gal 4 in regulating gene expression
2、protein interaction provides the technology useful for us to dissect and understand life. It is useful tool.
We use it to study life.
e.g. yeast two-hybridization: transcription factor Gal 4 interact with DNA and polymerase complex
Two-hybridization is not what yeast has in mind when it creates the Gal 4 transcription factor.

5. Protein interaction:
3、It is the protein interaction that is affected in case of defect protein or disease.
We study it to understand disease.
e.g. the sickle-cell anemia
Different protein interaction
Anemia or anti-malaria
A single AA mutation

6. From the system point of view to understand the functions and importance of protein interaction in biological process

7. Protein interactions are overly complicated.
Think about proteins and each capable of interacting with hundreds if not thousands of others. Plus all the modifications.
Then, where to get started：
1、starting from an important protein or a protein that can get you a publication
2、mining the database to identify some important interactions
3、Patterning and profiling

8. Analysis of important proteins: most important ones are picked.
You don’t know beforehand if the new protein you pick is an important one.
Data mining: try to figure out something useful from chaos
Data mining is tricky. You may fish out a diamond or you may step on a mine.
Work hard. Diamond or mine, it is your luck.

9. 1、Starting from an important protein or event
e.g. EGFR endocytosis
Receptor clustering and internalization
Co-immunoprecipitation, cellular co-localization, yeast two-hybridization, etc.

10. The order of science The order of reality
The purpose of the study is to understand
how EGFR is removed from cellular membrane,
how many proteins are involved in this process,
what is the order of interactions for these proteins and their regulatory roles,
how the receptor is recycled or degraded etc.
Know the basic knowledge of EGFR functions for understanding life.
Try to find a way to activate, inactivate or disrupt this process in order to treat some disease.
Earn enough to have a respectable life or to be famous
The order of reality
The order of science
At this stage, skill and ability are more important than knowledge

11. Yeast protein interactions Human protein interactions
2、Mining the database
Yeast protein interactions
Human protein interactions
Look at the big picture, you lose some details. Focus on details, you blur the big picture.
There is no “博大精深”. Either “博大” or “精深”, not both.

12. 2、Mining the database 1. Protein-Protein Interaction Databases
2. Predicted PPI Databases
3. Structural PPI Databases
4. Small Ligands & Peptides
5. Protein-Nucleic Acid Interaction
6. PPI Prediction
7. Interaction Network Visualization Software
8. Many more…….

13. Bioinformatics
Whole
Functional module
section
individual
System biology
Experimental analysis

14. Big picture or detail, precise or blur, data mining or mechanism analysis: make your choice
Bioinformatics and system biology
Functional analysis and experimentation
Your curiosity and the reality
All depend on what resource you have and how many.
Research is to gain the ability of “管中窥豹”, but some of you may not get that ability and end up with “盲人摸象” .
So, make your choice wisely.

15. Protein interaction and biological functions: hand-in-hand
No function? then no interaction
target
Identify interacting proteins
Functions of interaction

16. From protein interaction to biological functions:
Identify interacting proteins, ask the biological question later.
Pitfalls: no biological functions for interacting proteins
From biological functions to interacting protein”
Functional disruption, analyze the change in interacting proteins
Pitfalls: the parallel changes of irrelevant proteins

17. One of the purpose to study protein interaction is to understand the function, mechanism, importance and significance of the interaction.
Most of the methods and tools used to study protein interaction are actually developed during the study of protein interaction.
Without good methods and tools, one can’t get very far and deep in studying protein interaction.
Without the biological questions of protein interaction, one can’t develop good methods or tools to study protein interaction.

18. Identify protein interactions and tools to study protein interaction

19. Transient interaction
Protein interaction
Stable interaction
（protein complex）
Transient interaction
（enzyme-substrate）
Stable and transient interaction require different tools and methods to study them.

20. Study of protein interaction: methods and tools
The key of each and every method or tool is its sensitivity and specificity.
Sensitivity vs specificity:
Sensitivity: most interactions in reality can be detected. However, the detected interactions may not be happen in real world, so called false positive. The more sensitive, the more false positive.
Specificity: the interactions detected are mostly real. However, not all the real interactions may be detected, so called false negative. The more specific, the more false negative.

21. Methods and their internal limitations (something you can’t overcome)
The average of a whole group: most biochemical analytical methods, the average of all cells or proteins.
The average height of 姚明 and 郭敬明 is 1.90 meter. It tells you nothing about their heights
The average of a representative group: genetic analysis and cell biology analysis
Single molecule analysis: not everyone looks the same
Quantitative and qualitative analysis:

22. Quantitation is very important
Input：10~20mg total proteins
IP sample：Immunoprecipitated from 1000mg of total proteins
Relative exposure level

23. Identify protein interaction:
Direct protein-protein interaction
Genetic analysis
Sequence alignment, homology and structure
Verify protein interaction:
Different methods
Multiple organisms

24. 1. Direct protein-protein interaction

25. Direct protein-protein interaction: affinity of interaction
Protein interaction Affinity: A+B=AB
Kd=[A][B]/[AB], Kd: dissociation constant
Interaction Kd
Streptavidin-biotin binding M
Antibody-antigen interaction (good) M
Antibody-antigen interaction (weak) M
Enzyme-substrate M

26. Direct protein-protein interaction
Co-immunoprecipitation and antigen-antibody interaction
Other affinity interactions
Yeast two-hybridization and phage display
Surface Plasmon Resonance
FRET
Proteomic analysis

27. Direct protein-protein interaction
Co-immunoprecipitation and antigen-antibody interaction
Other affinity interactions
Yeast two-hybridization and phage display
Surface Plasmon Resonance
FRET
Proteomic analysis

28. Co-immunoprecipitation and antigen-antibody interaction
Western blot: denatured protein target, protein detection.
Co-immunoprecipitation: native protein target, identify proteins interacted with the target.
Co-immunoprecipitation：Ab-Pr1-Pr2
Nonspecific interaction: Pr1-Ab-Pr2
Screening with antibody: cDNA expression library, antibody screens for target protein, pickup the positive clones. MS analysis and PCR are much easier.

29. Western blot
Electric transfer efficiency
The wetting of PVDF membrane
SDS concentration in transfer buffer
Damaged membrane
Loose contact between gel and membrane
Bad antibody
Inappropriate antibody concentration
Reducing reagent
Incomplete block

30. How to make a pretty Western blot:
Acrylamide concentration of SDS－PAGE
The amount of sample loaded
The amount of sample in the adjacent lane, ionic strength and sample volume
Membrane: PVDF vs nitrocellulose
Good antibody

31. Co-immunoprecipitation: Ab-Pr1-Pr2 not Pr1-Ab-Pr2

32. Co-immunoprecipitation: Bad antiobody Incomplete block
Loading not in proption
Too much sample
Good gel 1 2 3 5 4

33. Screening with antibody:
cDNA expression library,
antibody screens for target protein,
pickup the positive clones.
MS analysis and PCR are much easier.
No one is using this type of method anymore.

34. Direct protein-protein interaction
Co-immunoprecipitation and antigen-antibody interaction
Other affinity interactions
Yeast two-hybridization and phage display
Surface Plasmon Resonance
FRET
Proteomic analysis

35. Affinity interactions: Any strong specific interactions
GST pull down：GST-tagged bait protein, fish out interacting proteins from cellular of tissue lysate. Glutathione affinity resin binds GST fusion protein.
Proteins binds to glutathione.
Biotin-Avidin interaction：Biotin labeled bait protein. Avidin affinity resin binds all biotin-containing proteins.
Many cellular proteins containing biotin.
Other tags, e.g. His-tag and zinc ion.

36. GST pull down binding wash elution
Preparation of bait: GST fusion protein purification

37. GST pull down
The purity of GST fusion protein is crucial to the analysis of bound proteins.
Two or more rounds of purification of GST fusion protein is generally required.

38. GST pull down

39. GST pull down
binding
wash
elution

40. Biotin-Avidin interaction: one of the most stable interactions in biology
The blocking of avidin to prevent the binding of biotin-containing proteins.

41. Direct protein-protein interaction
Co-immunoprecipitation and antigen-antibody interaction
Other affinity interactions
Yeast two-hybrid and phage display
Surface Plasmon Resonance
FRET
Proteomic analysis

42. Yeast two-hybridization
Identify new interacting proteins
Verify protein interaction
Analyze the interacting domains

43. New interacting protein identification: DNA binding domain linked bait protein screens the activation domain fused library
lacZ
UAS AD DB
lacZ
UAS DB AD
lacZ
UAS AD Y DB B

44. Identify new interacting protein
Bait plasmid
Target library BD
B-Pr
Marker1 AD
library
Marker2
lacZ
UAS AD Y DB
B-pr
lacZ
UAS AD DB
B-pr Y

45. Verify protein interaction
Protein A Protein B BD
B-Pr
Marker1 AD
A-Pr
Marker2
Blue clony: protein A interacts with B
White clony: no interaction between A and B

46. 1 2 3 4 Analyze interacting domains white blue white white
Protein A interacts with protein B. There are 4 domains in protein B.
Protein A
Protein B 1 2 3 4 AD 1
Marker2 AD 3 AD 4 AD 2 BD
B-Pr
Marker1
white blue white white

47. The advantages and disadvantages of yeast two- hybridization:
In vivo interaction.
Most works are handling DNA and plasmids.
Detect weak interaction.
Disadvantages:
Yeast vs mammalian cell protein modification
Fusion proteins may cause new interaction
Auto-activation
Library: directional and in frame (1/3)
Yeast protein mediation
False positive

48. DB AD X Y Auto activation lacZ UASBD
Marker1
Bait protein has transcription activation ability AD Y
lacZ
UAS
Marker2
The target protein has DNA binding ability

49. Phage display

50. Phage display
The most important advantage of phage display is the scale of phage numbers.
Mammalian cells: 106~107 cells/10cm plate, 100 plates have 108~109 cells
Bacteria: easily 1010 bacterial cells
Phage：1012 phages or more
For a 7 amino acids peptide: 8X1016 combinations. Mammalian cells or bacteria can’t handle that many of peptides.

51. Direct protein-protein interaction
Co-immunoprecipitation and antigen-antibody interaction
Other affinity interactions
Yeast two-hybrid and phage display
Surface Plasmon Resonance
FRET
Proteomic analysis

52. Surface Plasmon Resonance
SPR is used to monitor interactions occurring in a biospecific surface on a metal layer by measuring changes in the solute concentration at this surface as a result of the interactions.

54. The advantages of SPR
Label-free detection
SRP does not require any labels or reporter groups to detect biomolecular interactions. It follows every step in a multi-step analysis procedure, in contrast to label-based methods that often only report the final step.
Real time measurement
The progress of interactions is displayed directly, as a plot of response (which is directly related to concentration changes at the surface) against time. Immediate feedback on the status of an interaction speeds up assay development and analysis. The results can be processed further after the run, for example to extract kinetic constants for the interaction.

55. Direct protein-protein interaction
Co-immunoprecipitation and antigen-antibody interaction
Other affinity interactions
Yeast two-hybrid and phage display
Surface Plasmon Resonance
FRET
Proteomic analysis

56. Emission frequency = Excitation frequency
Fluorescence Resonance Energy Transfer (FRET)
Emission frequency
Excitation frequency
Acceptor
Donor
(2-10nm)
Emission frequency = Excitation frequency

57. Donor is excited at the maximum absorbance wavelength (380 nanometers).
Acceptor signal is increased at the acceptor emission maximum (510 nanometers)

58. Sensitivity to photobleaching
Wavelength of the Fluorescent Proteins
Color defining GFP
mutation (source)
Blue (BFP)
Y66H
Green (GFP)
Y66W
Cyan (CFP)
S65T
Yellowish (YFP)
S65G, T203Y
Red (D.s. RFP)
D. striata
Excitation (max)
382 nm
434 nm
488 nm
514 nm
558 nm
Emission (max)
446 nm
476 nm
509 nm
527 nm
583 nm
Sensitivity to photobleaching
High
Low
Moderate

62. Advantages of FRET
Measure interactions between two proteins in vivo
Very sensitive
Excellent resolution
Helpful in characterizing major conformational changes in macromolecules
Determine the interaction qualitatively and quantitatively
Limitations
FRET only occurs when the two fluorophores are within 2-10nm of each other, which means that the fluorophores must be brought together via very close protein-protein interaction.
Require expensive equipment.
Require labeling of proteins using fluorophore or GFP fusion

63. Direct protein-protein interaction
Co-immunoprecipitation and antigen-antibody interaction
Other affinity interactions
Yeast two-hybrid and phage display
Surface Plasmon Resonance
FRET
Proteomic analysis

64. 2. Analysis of protein interaction at single molecule level

65. Atomic force interaction

66. 3. Genetic analysis of functional interaction
Lose of function and functional complimentary analysis
Functional compliment is not a proof of direct protein interaction, but it is the indication of possible interaction

67. Genetic protein interactionB C A A B C X + - B C A X ++ + - A B C
+++

68. By expressing A, B and C in cell, we found that only A activated the gene expression.
By expressing two of these three proteins, A and B combination are more active than other two combinations.
By expressing all three proteins, we got the most active gene expression.
In these cases, it is most likely that A, B and C form a complex to up-regulate gene expression. A is most likely the DNA binding protein and B mediates the association of A and C.

69. Direct protein interaction: there is a physical protein-protein interaction.
Genetic protein interaction: the functional compliment or enhancement may indicate a possible physical interaction.

70. Identification of interacting proteins
Verifying the interaction
Biological functions

71. Functional mechanism study: Lose of function and gain of function
Experimental
Analysis
(methods and tools)
Biological
question
Understanding
of the question
Lead to new biological questions

72. Gain of functions: gene transfection. the cell obtains a new function.
A protein, which is not normally expressed in a certain type of cell, may change the phenotype of the cell.
Transgenic animals

73. Lose of functions: mutation or gene knockdown or knockout
Lose of functions: mutation or gene knockdown or knockout. Cell loses function.
Nullifing gene, inhibiting protein expression, dominant negative mutation, knockout, RNAi, protein fragment, etc.
Knockout animals

74. Phenotype to functional change

75. Functional change to protein interaction and mechanism
Interaction and function

76. From protein to its function,
from protein to cell, from protein function to cell function,
from cell to animal, from cell function to animal phenotype

77. Other important methods in protein studies:
Protein isoelectric point and buffer pH
Protein concentration determination, not melamine
Fluorescence and ECL
Chromatography: many types
Ultracentrifugation
Electrophoresis: SDS, native, isoelectric focusing

78. Thank you. That’s all, folks.
Be creative in studying protein-protein interaction.
Thank you. That’s all, folks.