Use of protein-protein interactions in affinity chromatography.

Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins.     

Probing the limits of aptamer affinity with a microfluidic SELEX platform

Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K_d) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K_d of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K_d.     

A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions

To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GalphaZ and RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40- to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.     

A miniaturized sandwich immunoassay platform for the detection of protein-protein interactions

Background  

Analysis of protein-protein interactions (PPIs) is a valuable approach for the characterization of huge networks of protein complexes or proteins of unknown function. Co-immunoprecipitation (coIP) using affinity resins coupled to protein A/G is the most widely used method for PPI detection. However, this traditional large scale resin-based coIP is too laborious and time consuming. To overcome this problem, we developed a miniaturized sandwich immunoassay platform (MSIP) by combining antibody array technology and coIP methods.     

Identification of novel protein-protein interactions using a versatile mammalian tandem affinity purification expression system

Identification of protein-protein interactions is essential for elucidating the biochemical mechanism of signal transduction. Purification and identification of individual proteins in mammalian cells have been difficult, however, due to the sheer complexity of protein mixtures obtained from cellular extracts. Recently, a tandem affinity purification (TAP) method has been developed as a tool that allows rapid purification of native protein complexes expressed at their natural level in engineered yeast cells. To adapt this method to mammalian cells, we have created a TAP tag retroviral expression vector to allow stable expression of the TAP-tagged protein at close to physiological levels. To demonstrate the utility of this vector, we have fused a TAP tag, consisting of a protein A tag, a cleavage site for the tobacco etch virus (TEV) protease, and the FLAG epitope, to the N terminus of human SMAD3 and SMAD4. We have stably expressed these proteins in mammalian cells at desirable levels by retroviral gene transfer and purified native SMAD3 protein complexes from cell lysates. The combination of two different affinity tags greatly reduced the number of nonspecific proteins in the mixture. We have identified HSP70 as a specific interacting protein of SMAD3. We demonstrated that SMAD3, but not SMAD1, binds HSP70 in vivo, validating the TAP purification approach. This method is applicable to virtually any protein and provides an efficient way to purify unknown proteins to homogeneity from the complex mixtures found in mammalian cell lysates in preparation for identification by mass spectrometry.     

Establishment of the platform for reverse chemical genetics targeting novel protein-protein interactions

In the "drug discovery" era, protein-protein interaction modules are becoming the most exciting group of targets for study. Although combinatorial libraries and active natural products are rapidly and systemically being equipped by both for-profit and not-for-profit organizations, complete drug-screening systems have not been achieved. There is a growing need for the establishment of drug discovery assays for highly effective utilization of the collected small molecules on a large scale. To generate drug-screening systems, we plan to identify novel protein-protein interactions that may participate in human diseases. The interactions have been identified by MS/MS analysis following immunoprecipitation using antibodies prepared from our cDNA projects. The intracellular pathway involving the identified interaction is computationally constructed, which then clarifies its relationship to the candidate disease. The development of reverse chemical genetics based on such information should help us to realize a significant increment in the number of drug discovery assays available for use. In this article, I describe our strategy for drug discovery and then introduce the applicability of fluorescence intensity distribution analysis (FIDA) and the expression-ready constructs called "ORF trap clones" to reverse chemical genetics.     

Non-additivity in protein-protein interactions

The energy of binding between proteins may be seen as the sum of the contributions of the individual amino acid residues. These contributions are additive when the binding energy, due to different amino acid residues, is independent of the interactions between amino acids in the same polypeptide chain. A measure of non-additivity is the coupling free energy. In this communication it is shown that: (1) the coupling free energy is the sum of intramolecular and intermolecular contributions; and (2), when additivity exists, experimentally determined values for the free energy of transfer of amino acids from water to the hydrophobic protein-protein interface are a very good approximation of their contribution to the energy of binding. Additivity cycles can be useful in determining the precise conditions where this approximation holds.     

Single bead affinity detection (SINBAD) for the analysis of protein-protein interactions

We present a miniaturized pull-down method for the detection of protein-protein interactions using standard affinity chromatography reagents. Binding events between different proteins, which are color-coded with quantum dots (QDs), are visualized on single affinity chromatography beads by fluorescence microscopy. The use of QDs for single molecule detection allows the simultaneous analysis of multiple protein-protein binding events and reduces the amount of time and material needed to perform a pull-down experiment.     

Diversity of protein-protein interactions

In this review, we discuss the structural and functional diversity of protein-protein interactions (PPIs) based primarily on protein families for which three-dimensional structural data are available. PPIs play diverse roles in biology and differ based on the composition, affinity and whether the association is permanent or transient. In vivo, the protomer's localization, concentration and local environment can affect the interaction between protomers and are vital to control the composition and oligomeric state of protein complexes. Since a change in quaternary state is often coupled with biological function or activity, transient PPIs are important biological regulators. Structural characteristics of different types of PPIs are discussed and related to their physiological function, specificity and evolution.     

Principles of protein-protein interactions

In the postgenomic era, one of the most interesting and important challenges is to understand protein interactions on a large scale. The physical interactions between protein domains are fundamental to the workings of a cell: in multi-domain polypeptide chains, in multi-subunit proteins and in transient complexes between proteins that also exist independently. Thus experimental investigation of protein-protein interactions has been extensive, including recent large-scale screens using mass spectrometry. The role of computational research on protein-protein interactions encompasses not only prediction, but also understanding the nature of the interactions and their three-dimensional structures. I will discuss properties such as sequence conservation and co-regulation of genes and proteins involved in different types of physical interactions. Given that all proteins consist of their evolutionary units, the domains, all interactions occur between these domains. The interactions between domains belonging to different protein families will be the second topic of my talk.     

Rapid functional analysis of protein-protein interactions by fluorescent C-terminal labeling and single-molecule imaging

Detection of protein-protein interactions is a fundamental step to understanding gene function. Here we report a sensitive and rapid method for assaying protein-protein interactions at the single-molecule level. Protein molecules were synthesized in a cell-free translation system in the presence of Cy5-puro, a fluorescent puromycin, using mRNA without a stop codon. The interaction of proteins thus prepared was visualized using a single-molecule imaging technique. As a demonstration of this method, a motor protein, kinesin, was labeled with Cy5-puro at an efficiency of about 90%, and the processive movement of kinesin along microtubules was observed by using total internal reflection microscopy. It took only 2 h from the synthesis of proteins to the functional analysis. This method is applicable to the functional analysis of various kinds of proteins.     

Motifs involved in protein-protein interactions

Summary Interactions between proteins are extremely variable. However, in the dimeric proteins comprised of regular motifs, interface interactions are similar to those that stabilize monomers. Additional stability is gained by converting loops within motifs or domains to linkers across interfaces. In multi-domain proteins, interactions can be greatly effected by the conformation of linkers between domains. Complex association of subunits, involving higher rotational symmetry or cubic symmetry, frequently involves motif sharing across interfaces.     

Molecular basis of a million-fold affinity maturation process in a protein-protein interaction

Protein engineering is becoming increasingly important for pharmaceutical applications where controlling the specificity and affinity of engineered proteins is required to create targeted protein therapeutics. Affinity increases of several thousand-fold are now routine for a variety of protein engineering approaches, and the structural and energetic bases of affinity maturation have been investigated in a number of such cases. Previously, a 3-million-fold affinity maturation process was achieved in a protein-protein interaction composed of a variant T-cell receptor fragment and a bacterial superantigen. Here, we present the molecular basis of this affinity increase. Using X-ray crystallography, shotgun reversion/replacement scanning mutagenesis, and computational analysis, we describe, in molecular detail, a process by which extrainterfacial regions of a protein complex can be rationally manipulated to significantly improve protein engineering outcomes.     

Electrostatic aspects of protein-protein interactions

Structural and mutational analyses reveal a central role for electrostatic interactions in protein-protein association. Experiment and theory both demonstrate that clusters of charged and polar residues that are located on protein-protein interfaces may enhance complex stability, although the total effect of electrostatics is generally net destabilizing. The past year also witnessed significant progress in our understanding of the effect of electrostatics on protein association kinetics, specifically in the characterization of a partially desolvated encounter complex.     

Prediction of physical protein-protein interactions

Many essential cellular processes such as signal transduction, transport, cellular motion and most regulatory mechanisms are mediated by protein-protein interactions. In recent years, new experimental techniques have been developed to discover the protein-protein interaction networks of several organisms. However, the accuracy and coverage of these techniques have proven to be limited, and computational approaches remain essential both to assist in the design and validation of experimental studies and for the prediction of interaction partners and detailed structures of protein complexes. Here, we provide a critical overview of existing structure-independent and structure-based computational methods. Although these techniques have significantly advanced in the past few years, we find that most of them are still in their infancy. We also provide an overview of experimental techniques for the detection of protein-protein interactions. Although the developments are promising, false positive and false negative results are common, and reliable detection is possible only by taking a consensus of different experimental approaches. The shortcomings of experimental techniques affect both the further development and the fair evaluation of computational prediction methods. For an adequate comparative evaluation of prediction and high-throughput experimental methods, an appropriately large benchmark set of biophysically characterized protein complexes would be needed, but is sorely lacking.     

Predicting protein-protein interactions from sequences in a hybridization space

To understand the networks in living cells, it is indispensably important to identify protein-protein interactions on a genomic scale. Unfortunately, it is both time-consuming and expensive to do so solely based on experiments due to the nature of the problem whose complexity is obviously overwhelming, just like the fact that "life is complicated". Therefore, developing computational techniques for predicting protein-protein interactions would be of significant value in this regard. By fusing the approach based on the gene ontology and the approach of pseudo-amino acid composition, a predictor called "GO-PseAA" predictor was established to deal with this problem. As a showcase, prediction was performed on 6323 protein pairs from yeast. To avoid redundancy and homology bias, none of the protein pairs investigated has > or = 40% sequence identity with any other. The overall success rate obtained by jackknife cross-validation was 81.6%, indicating the GO-PseAA predictor is very promising for predicting protein-protein interactions from protein sequences, and might become a useful vehicle for studying the network biology in the postgenomic era.     

Mean-field analysis of protein-protein interactions

Calculations were performed on the D1.3-E5.2 antibody-antibody complex estimating the binding affinities of the wild-type and 16 alanine substitutions. Analyzed were structural models of the interfacial region containing a zinc ion and crystallographic waters. A continuum approach was used to evaluate the electrostatic free energies and the hydrophobic effect was calculated by employing a buried molecular surface area relationship. Estimates of the absolute binding affinity reproduced the experimental value within the uncertainty of assessing entropic and strain energy contributions. The best correlation for mutants with experimental data was achieved when the hydrophilicity of created cavities were considered, and yielded a correlation coefficient of 0.7 and an average error of +/-1.4 kcal/mol. Empirically fitting the free energy function produced a smaller error of +/-1.0 kcal/mol. Depending on the electrical potential and electrostatic reorganization, scaling the 'protein dielectric constant' to approximately 10 may improve the accuracy of continuum models for evaluating amino acid substitutions.     

Ontological visualization of protein-protein interactions

Background  

Cellular processes require the interaction of many proteins across several cellular compartments. Determining the collective network of such interactions is an important aspect of understanding the role and regulation of individual proteins. The Gene Ontology (GO) is used by model organism databases and other bioinformatics resources to provide functional annotation of proteins. The annotation process provides a mechanism to document the binding of one protein with another. We have constructed protein interaction networks for mouse proteins utilizing the information encoded in the GO annotations. The work reported here presents a methodology for integrating and visualizing information on protein-protein interactions.     

Kinetic studies of protein-protein interactions

The structure of a protein-protein interaction, its affinity and thermodynamic characteristics depict a 'frozen' state of a complex. This picture ignores the kinetic nature of complex formation and dissociation, which are of major biological and biophysical interest. This review highlights recent advances in deciphering the kinetic pathway of protein-protein complexation, the nature of the encounter complex, transition state and intermediate along the reaction, and the effects of mutation, viscosity, pH and salt on association.