Research Methodology of Biotechnology: Protein-Protein Interactions Yao-Te Huang Aug 16, 2011.

Research Research Methodology of Methodology of Biotechnology: Biotechnology: Protein-ProteinProtein-Protein

Interactions Interactions

Yao-Te HuangYao-Te Huang

Aug 16, 2011Aug 16, 2011

IntroductionIntroductionProtein interactions and functions are intimately related.The structure of a protein influences its function by determining the other molecules with which it can interact and the consequences of those interactions.

Introduction (contd.)Introduction (contd.)Experimental methods available to detect protein interactions vary in their level of resolution.These observations can be classified into four levels: (a) atomic scale, (b) binary interactions, (c) complex interactions, and (d) cellular scale.

Introduction (contd.)Introduction (contd.)Atomic-scale methods:

showing the precise structural relationships between interacting atoms and residues The highest resolution methods: e.g., X-ray crystallography and NMRNot yet applied to study protein interactions in a high-throughput manner.

Introduction (contd.)Introduction (contd.)Binary-interaction methods:

Methods to detect interactions between pairs of proteinsDo not reveal the precise chemical nature of the interactions but simply report such interactions take placeThe major high-throughput technology: the yeast two-hybrid system

Introduction (contd.)Introduction (contd.)Complex-interaction methods:

Methods to detect interactions between multiple proteins that form complexes.Do not reveal the precise chemical nature of the interactions but simply report that such interactions take place.The major high-throughput technology: systematic affinity purification followed by mass spectrometry

Introduction (contd.)Introduction (contd.)Cellular-scale methods:

Methods to determine where proteins are localized (e.g., immunofluorescence).It may be possible to determine the function of a protein directly from its localization.

COIB (2001), 12:334-339

Principles of protein-Principles of protein-protein interaction analysisprotein interaction analysis

These small-scale analysis methods are also useful in proteomics because the large-scale methods tend to produce a significant number of false positives.They include (a) genetic methods, (b) bioinformatic methods, (c) Affinity-based biochemical methods, and (d) Physical methods.

Genetic methodsGenetic methodsClassical genetics can be used to investigate protein interactions by combining different mutations in the same cell or organism and observing the resulting phenotype.Suppressor mutation: A secondary mutation that can correct the phenotype of a primary mutation.

Suppressor mutationSuppressor mutation

Synthetic lethal effectSynthetic lethal effect

Bioinformatic methodsBioinformatic methods(A) The domain fusion method (or

Rosetta stone method):The sequence of protein X (a single-domain protein from genome 1) is used as a similarity search query on genome 2. This identifies any single-domain proteins related to protein X and also any multi-domain proteins, which we can define as protein X-Y.As part of the same protein, domain X and Y are likely to be functionally related.

The domain fusion method The domain fusion method (or Rosetta stone method)(or Rosetta stone method)The sequence of domain Y can then be used to identify single-domain orthologs in genome 1.Thus, Gene Y, formerly an orphan with no known function, becomes annotated due to its association with Gene X. The two proteins are also likely to interact. The sequence of protein X-Y may also identify further domain fusions, such as protein Y-Z. This links three proteins into a functional group and possibly identifies an interacting complex.

The domain fusion method The domain fusion method (or Rosetta stone method)(or Rosetta stone method)

Bioinformatic methodsBioinformatic methods(B) The phylogenetic profile:

It describes the pattern of presence or absence of a particular protein across a set of organisms whose genomes have been sequenced. If two proteins have the same phylogenetic profile (that is, the same pattern of presence or absence) in all surveyed genomes, it is inferred that the two proteins have a functional link. A protein’s phylogenetic profile is a nearly unique characterization of its pattern of distribution among genomes. Hence any two proteins having identical or similar phylogenetic profiles are likely to be engaged in a common pathway or complex.

When homology is present, the elements are shaped on a gradient from light red (low level of identity) to dark red (high level of identity)

YPL207W clusters with the ribosomal proteinsand can be assigned a function in protein synthesis.

Affinity-based Affinity-based biochemical methodsbiochemical methods

Affinity chromatography can be used to trap interacting proteins. If protein X is immobilized on Sepharose beads (e.g., using specific antibodies), then proteins (and other molecules) interacting with protein X can be captured from a cell lysate passed through the column. After washing away unbound proteins, the bound proteins can be eluted, separated by SDS-PAGE and analyzed by mass spectrometry.

(A) Affinity chromatography (A) Affinity chromatography followed by SDS-PAGE & Mass followed by SDS-PAGE & Mass

spectrometryspectrometry

(B) Immunoprecipitation(B) ImmunoprecipitationThe addition of antibodies specific for protein X to a cell lysate will result in the precipitation of the antibody-antigen complex.The technique is usually carried out with polyclonal antisera.The precipitated complexes are separated from the cell lysate by centrifugation, washed and then fractionated by SDS-PAGE, and the bound proteins can be identified by mass spectrometry.

ImmunoprecipitationImmunoprecipitation

(C) GST pulldown(C) GST pulldownThe protein X is expressed as a fusion to GST. After mixing the fusion protein with a cell lysate and allowing complexes to form, glutathione-coated beads are added to capture the GST part of the fusion. The beads are recovered by centrifugation, washed and the recovered proteins fractionated and identified by mass spectrometry.

GST pulldownGST pulldown