Protein-protein interaction 蛋白间的相互作用

Protein-Protein Interactions

Protein Structure

•The fundamental unit of

protein structure is a

domain.

•This region of a polypeptide

chain folds into a distinct

structure and mediates the

protein’s biological

functionality.

Abstract View of Protein

Structure

•Proteins are represented

as shaded areas and the

domains as geometrical

figures.

•We assume that protein

domain (or domain

combination) interactions

are responsible for

protein interactions.

Gen ome: 30.000 genes

Transcript ome: 40-100.000 mRNAs

Prot eome:

100-400.000 proteins >1.000.000 interactions

Dimensions of Information

Complexity

Genomics vs. Post-Genomics

Human Genome Human Proteome Transcripts

Protein Interaction

105

106

A proteomics circuit showing the

interaction of RNA splicing proteins

Source: Campbell/Heyer. Discovering genomics, proteomics, and bioinformatics

Terminology

Protein Complexes are groups of proteins that interact with each other at the same time and place, forming a single multi-molecular machine.

E.g., anaphase-promoting complex, RNA-splicing and

protein export and transport complexes, etc. Functional modules consist of proteins that participate in a particular cellular process while binding each other at a different time and place (different conditions or phases of the cell cycle, in different cellular compartments, etc).

E.g., yeast pheromone response pathway, MAP

signaling cascades, etc.

How can we identify Protein-Protein Interactions ?

1. In vitro interactions.

2. In vivo interactions.

3. How does this fit together?

Identification of Protein-Protein

Interactions by

Co-Immunoprecipitation

Co-Immunoprecipitation - Principle

A variation of

Co-Immunoprecipitation:

Reverse Co-Immunopreciptiation combined with cross-linking Spatial organisation of P-P-interactions

Cross-linkers

e.g. bifunctional sulfhydril-reactive crosslinkers:

e.g. Maleimide-ester

Homobifunctional

Periodate

cleavable!

Identification of Protein-Protein Interactions by Pull-Down methods

prepare protein

The GST fusion protein might have different origin: Bacterial and yeast expression systems

Higher eukaryotic expression systems (insect cells/ baculovirus)

Various Affinity tags commonly used:

GST

Poly-Histidine (IMAC)

Calmoduline binding protein (CBP)

………

Multi-Tags e.g. TAP-tag: