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Mar 1, 2010 (Vol. 30, No. 5)

Protein-Protein Interactions

In-Depth Investigation of These Complex Relationships Is Increasing Knowledge Base

  • Click Image To Enlarge +
    Using a model protein-protein interaction system, scientists from Boston Biomedical Research Institute have found that affinity changes result from changes in disorder-to-order transitions.

    If nature did not allow protein-protein interactions to occur inside living cells (and viruses), life as we know it would simply not exist. Molecular biologists have long struggled with developing methods to study protein-protein interactions.

    Recent data presented at the Gordon Research Conference on Protein Interaction Dynamics in Galveston, TX, suggest that they soon might be able to breathe a little easier.

    Eric Sundberg, Ph.D., principal scientist at Boston Biomedical Research Institute, discussed “some of the complex factors that regulate protein molecular recognition, including cooperation among amino acid residues within protein interfaces and disordered protein regions involved in protein binding.”

    Dr. Sundberg’s lab uses directed evolution techniques, including phage display and yeast display, to insert random mutations that evolve proteins for better binding, all in an effort to “create model systems to study how these complex factors regulate protein-protein interactions.”

    Dr. Sundberg explained that “directed evolution is just like Darwinian evolution in that you go through iterative rounds of mutation and selection. Our selection for fitness would be through tighter binding. And these directed evolutionary techniques are common in the field.”

    He and his team have used phage display to alter the affinity of protein-protein complexes by mutating one protein in the complex. Using the T-cell receptor and Staphylococcal superantigen, they found that mutating one protein in a model complex increases its affinity for its partner proteins in the complex.

    “The particular region of the protein that we targeted in this study is a disordered disulfide loop that is disordered in its unbound state. Also, in the wild type, it is largely disordered in the complex (bound) state.

    “When we apply our evolutionary techniques, we mutate a stretch of five contiguous residues within that loop. And so, when they mutate, binding affinity increases, and then we select for variants that have higher affinity.”

    By isolating and analyzing (by x-ray crystallography) various intermediate complexes along an affinity continuum, Dr. Sundberg’s team was able to conclude that the random mutations caused greater disorder-to-order transitions, which, in turn, was the reason for increased affinity in the binding region.

  • Studying Disorder

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    Distributions of the percentage of intrinsically disordered residues in transcription regulatory proteins from E. coli, yeast, and human genomes (University of Pittsburgh)

    “Disorder-to-order transitions are difficult to quantify by experimental means and what we presented was one way to quantify these transitions,” said Dr. Sundberg. “In the last ten years it has been discovered that many proteins, especially in the human genome, have large regions of intrinsic disorder, yet they are functional proteins; efforts to understand those transitions from disorder have intensified.”

    Carlos Camacho, Ph.D., associate professor of computational biology at the University of Pittsburgh, also discussed the role of disorder in protein function. “Over the past 10 years, it has become quite apparent that disorder plays an important role in function. We now know that 40% of genes in humans and mammals contain highly disordered regions, which means that crystallization efforts will fail because these proteins don’t have a unique structure.”

    Dr. Camacho and collaborators have attempted to meet these challenges by developing thermodynamic theories that explicitly allow for the possibility of proteins to be disordered. Using this formal treatment, they demonstrated what had been previously concluded through empirical methods only, that as disorder is increased in an enzyme, the catalytic rate drops significantly. In contrast, “when the function of a protein is only to bind another protein, order is only needed if the binding interaction is relatively weak, but if the binding is strong then optimal binding tolerates disorder.

    “The reason that disordered proteins have been in the dark for so long is that you cannot measure positive-folding free energy experimentally. You can only measure proteins that fold into a stable structure, i.e., they have a negative-folding free energy,” said Dr. Camacho. However, computational/bioinformatics tools can assess the amount of disorder over whole genomes.

    Dr. Camacho has analyzed disordered proteins in many complete genomes from bacteria to humans, validating the predictions of the theory. Moreover, he and his colleagues have found that prokaryotic proteins are mostly ordered when forming weak complexes, whereas, disorder is ubiquitous in regulatory proteins present in higher organisms such as humans, which, as predicted, also form tightly bound complexes.


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