Protein Interactions

Why study protein interaction ?
• Gene function can ultimately be broken down into a series of molecular interactions that take place among proteins and between proteins and other molecules • We discuss the techniques that are used to study protein interactions, and how these have recently been adapted for high throughput analysis on a proteomic scale • For example, if protein X is uncharacterized but interacts with proteins Y and Z, both of which are part of the RNA splicing machinery, it is likely that protein X is involved in this process also. • Every protein in the cell can eventually be linked into a functional network

Genetic approaches to detect protein interaction
Suppressor mutations :

• Screening for suppressor mutations, i.e. mutations in one gene that partially or fully compensate for a mutation in another. Have been widely employed in Drosophila and yeast

Limitations : • One potential problem is that the suppressor mutation may map to the same gene as the primary mutation, since second mutations in the same gene can suppress the primary mutant phenotype by introducing a compensatory conformational change within the same protein • Even if the suppressor maps to a different gene, the two gene products might not actually interact. For example, a mutation that abolishes the activity of an enzyme required for amino acid biosynthesis could be suppressed by a gain of function mutation in a transport protein that increases the uptake of that amino acid from the environment. • Furthermore, the mutations do not necessarily have to change the structures of proteins X and Y.

where individual mutations in the genes for proteins X and Y do not prevent interaction and are therefore viable.Enhancer mutations.e. those that worsen the phenotype generated by a primary mutation • One example of this strategy is the synthetic lethal screen. i. but simultaneous mutations in both genes prevent the interaction and result in a lethal phenotype .

• Investigators established a synthetic genetic array (SGA) system in which a mutation in one yeast gene can be crossed to a set of 5000 viable deletion mutants. the order of protein function can often be established by epistasis. In this type of experiment. This can be used to identify all the proteins involved in the same pathway or complex as a particular query protein • Mutations in different genes that generate similar phenotypes often indicate that the protein products are part of the same complex or the same biochemical or signaling pathway • For pathways. . If a loss-of-function mutation in gene X overrides a gain-offunction mutation in gene Y. allowing synthetic interactions to be mapped in a systematic fashion. it suggests that protein X acts downstream of protein Y in the pathway. loss-offunction and gain-of-function mutations (with opposite phenotypes) are combined in the same cell or organism.


Comparative Genomics Methods to detect protein interaction Three methods have been developed to infer protein interactions directly from genomic data These work best in bacteria because more bacterial genome sequences are available for comparison & bacterial genomes are often organized into functional units called operons where the encoded proteins tend to have related functions .

. which has two domains. • A well-known example is the S. • Multi-domain proteins in one species may be represented by two or more interacting subunits in another.<1> Domain fusion method • domain fusion or Rosetta stone methodis based on the principle that protein domains are structurally and functionally independent units that can operate either as discrete polypeptides or as part of the same polypeptide chain. coli. cerevisiae topoisomerase II protein. and which is represented by the two separate subunits GyrA and GyrB in E.

it suggests they are functionally related and that their products may interact.<2>Conservation of gene position method • Second method is based on the knowledge that bacterial genes are often organized into operons. • Therefore. if two genes are neighbors in a series of bacterial genomes. Limitations: • Genes whose functions are apparently unrelated may also be organized into operons. .

<3> Phylogenetic Profiling • Evolutionary conservation of genes involved in the same function. • The conservation of three or four uncharacterized genes in 20 aerobic bacteria and their absence in 20 anaerobes might indicate that the products are required for aerobic metabolism. • Since proteins usually function as complexes. . and would tend to lead to the loss of the other components over evolutionary time since mutations in the corresponding genes would have no further detrimental effect. the loss of one component would render the entire complex non-functional.



Co .immunoprecipitation The two interacting proteins precipitate as a complex .

Affinity Chromatography Interacting proteins will be retained on the column as a conjoined complex .

. and can be detected by the change in the emission wavelength of the acceptor fluorophore. FRET occurs only when the two fluorophores are up to 10 nm apart.Flouresence Resonance Energy Transfer (FRET) Energy is transferred from an excited donor fluorophore to a nearby acceptor fluorophore.

a co-immunoprecipitation.• Traditional protein interaction analysisTechniques s. • These methods can’t be used for high throughput analysis . • X-ray crystallography and NMR spectroscopy that can be used to characterize protein interactions at the atomic level. .and cross-linking used previously to characterize the interactions of individual proteins. hence development of functional genomics techniques. affinity chromatography.

highthroughput strategies that link genes and proteins into functional networks are essential.• The aim of functional genomics is to determine functions for the many anonymous genes and cDNAs amassing in databases. .

which provide a platform for organizing and querying the increasing amount of interaction data.Library-based interaction mapping. 2 . 3 .Bioinformatics tools and databases of interacting proteins.Three areas of Functional Genomics : 1 . .High-throughput protein analysis and annotation.

within the environment of a cell (in vivo) . in vitro and 2.Library-based screening methods allow the large-scale analysis of binary interactions • Allow hundreds or thousands of proteins to be screened in parallel • all experimentally identified proteins are linked to the genes or cDNAs that encode them. the corresponding clones can be rapidly isolated and used to interrogate DNA sequence databases • Two broad classes of library: those in which protein interactions are assayed 1. • Therefore. once interacting proteins have been detected.

Library-based methods for the global analysis of binary interactions Standard cDNA expression libraries Phage display method The yeast two-hybrid system .

Standard cDNA expression libraries Expression libraries are usually screened with labeled antibodies. labeled calmodulin has been used to screen for calmodulin-binding proteins. Low throughput Does not provide the native conditions for the folding of all proteins. so a significant number of interactions would not be detected. In place of antibodies. other proteins can be used as probes. For example. .

Phage display method (1) M13 (a filamentous phage containing ss-DNA encased in a protein coat): contains five coat proteins. two of which are gVIIIp (gene VIII protein) and gIIIp (gene III protein). .

Phage display method (2) .

.Phage display method (2): contd.

The phage display method .

These do not have to be covalently joined together. . When the two strains are mated. This principle is exploited to identify protein interactions. but can be assembled to form a dimeric protein. Bait proteins are expressed in one yeast strain as a fusion with a DNA-binding domain and candidate prey proteins are expressed in another strain as fusions with a transactivation domain. functional transcription factors are assembled only if the bait and prey interact.The yeast two-hybrid system Transcription factors generally comprise two functionally independent domains. This can be detected by including a reporter gene activated by the hybrid transcription factor. one for DNA binding and one for transcriptional activation.

The yeast two-hybrid yeast .

large-scale studies. the degree of overlap in the reported interactions is very low (10-15%). This suggest either that the screens were not comprehensive or that even minor differences in experimental conditions could influence the types of interactions that are detected. where independent groups have carried out similar.Limitations of the yeast two-hybrid system First. .

suggesting there is also high level of false positives. suggesting there is a high level of false negatives.Limitations of the yeast two-hybrid system Secondly. a significant number of interactions that are detected in large-scale screens appear spurious when investigated in more detail. a significant number of well-characterized interactions are not detected in the large-scale screens. . Thirdly.

A variant of the yeast two-hybrid system .

defined clones are generated for each bait and prey matrix approach .


random library method Bait and/or prey are represented by random clones from a highly complex expression library .



After several rounds of such “panning” the remaining. • Phage display is a more suitable alternative  the expression of fusion proteins in such a way that a foreign peptide sequence is “displayed” on the bacteriophage surface. coli. • Traditional clone-based library not an ideal platform for proteomewide interaction screening (Drawbacks-labor-intensive and technically demanding screening procedures). in which non-interacting phages are discarded and bound phages are eluted and used to reinfect E.In vitro expression libraries are of limited use for interaction screening • Immunological screening (the use of antibodies as probes-specialized form of interaction analysis. Screening is basically a reiterative affinity-purification process. tightly-bound phage are isolated and the inserts sequenced to identify the interacting peptides . Libraries of phage can be produced and screened to identify peptides that interact with a given probe (such as an antibody) which is immobilized on a membrane or in the well of a microtiter plate.

• .• The major disadvantage of all in vitro library systems is that interactions occur in an unnatural environment where the protein may be incorrectly folded or partially unfolded.

In vivo interaction screening method The yeast two-hybrid system • Is the prototype of a range of related techniques in which protein interactions are assayed in vivo. • The principle of the system is that proteins often comprise several functionally independent domains. which can function not only when they are covalently linked in the same polypeptide chain. but also when they are brought together through noncovalent interactions. .

. • A library of “prey” is then generated in which each clone is expressed as a fusion protein with the transcription factor’s transactivation domain.• Transcription factors generally contain independent DNA-binding and transactivation domains. • The general strategy is as follows: protein X is expressed as a fusion (a hybrid) with the DNA-binding domain of a transcription factor to generate a “bait”. and this means that a functional transcription factor can be created if separately expressed DNA-binding and transactivation domains can be persuaded to interact.

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