蛋白质相互作用 廖侃 生化与细胞所 2017,9,26

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……

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

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.

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

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

Protein interactions are overly complicated. Think about 10000 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

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.

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.

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

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.

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…….

Bioinformatics Whole Functional module section individual System biology Experimental analysis

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.

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

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

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.

Identify protein interactions and tools to study protein interaction

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

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.

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:

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

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

1. Direct protein-protein interaction

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 10-14M Antibody-antigen interaction (good) 10-8-10-10M Antibody-antigen interaction (weak) 10-6M Enzyme-substrate 10-6-10-10M

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

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

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.

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

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

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

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

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.

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

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.

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

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.

GST pull down

GST pull down binding wash elution

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

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

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

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

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

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

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

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

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

Phage display

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.

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

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.

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.

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

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

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

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

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

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

2. Analysis of protein interaction at single molecule level

Atomic force interaction

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

Genetic protein interaction B C A A B C X + - B C A X ++ + - A B C +++

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.

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

Identification of interacting proteins Verifying the interaction Biological functions

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

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

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

Phenotype to functional change

Functional change to protein interaction and mechanism Interaction and function

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

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

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