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.

Enhancer mutations, i.e. those that worsen the phenotype generated by a primary mutation
One example of this strategy is the synthetic lethal screen, where individual mutations in the genes for proteins X and Y do not prevent interaction and are therefore viable, 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, allowing synthetic interactions to be mapped in a systematic fashion. 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, the order of protein function can often be established by epistasis. In this type of experiment, loss-offunction and gain-of-function mutations (with opposite phenotypes) are combined in the same cell or organism. If a loss-of-function mutation in gene X overrides a gain-offunction mutation in gene Y, it suggests that protein X acts downstream of protein Y in the pathway.

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

<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. Multi-domain proteins in one species may be represented by two or more interacting subunits in another. A well-known example is the S. cerevisiae topoisomerase II protein, which has two domains, and which is represented by the two separate subunits GyrA and GyrB in E. coli.

<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, it suggests they are functionally related and that their products may interact. 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, the loss of one component would render the entire complex non-functional, 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.

Co - immunoprecipitation

The two interacting proteins precipitate as a complex

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

Flouresence Resonance Energy Transfer (FRET)

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

 Traditional protein interaction analysisTechniques s.a co-immunoprecipitation, affinity chromatography,and cross-linking used previously to characterize the interactions of individual proteins. X-ray crystallography and NMR spectroscopy that can be used to characterize protein interactions at the atomic level. These methods cant be used for high throughput analysis , hence development of functional genomics techniques.

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

Three areas of Functional Genomics :

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

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. Therefore, once interacting proteins have been detected, 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. in vitro and 2. within the environment of a cell (in vivo)

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. In place of antibodies, other proteins can be used as probes. For example, 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.

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

The yeast two-hybrid system

Transcription factors generally comprise two functionally independent domains, one for DNA binding and one for transcriptional activation. These do not have to be covalently joined together, but can be assembled to form a dimeric protein. This principle is exploited to identify protein interactions. 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. When the two strains are mated, functional transcription factors are assembled only if the bait and prey interact. This can be detected by including a reporter gene activated by the hybrid transcription factor.

The yeast two-hybrid yeast

Limitations of the yeast two-hybrid system

First, where independent groups have carried out similar, 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.

Limitations of the yeast two-hybrid system

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

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

In vitro expression libraries are of limited use for interaction screening

Immunological screening (the use of antibodies as probes-specialized form of
interaction analysis.

Traditional clone-based library not an ideal platform for proteomewide interaction screening (Drawbacks-labor-intensive and technically demanding screening procedures). 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. 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. Screening is basically a reiterative affinity-purification process, in which non-interacting phages are discarded and bound phages are eluted and used to reinfect E. coli. After several rounds of such panning the remaining, tightly-bound phage are isolated and the inserts sequenced to identify the interacting peptides

 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.

 Transcription factors generally contain independent DNA-binding and transactivation domains, and this means that a functional transcription factor can be created if separately expressed DNA-binding and transactivation domains can be persuaded to interact. 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. A library of prey is then generated in which each clone is expressed as a fusion protein with the transcription factors transactivation domain.