Affinity Protein-protein Interactions Research Articles

Protein repeats show clade-specific volatility in Aves

Protein repeats are a source of rapid evolutionary and functional novelty. Repeats are crucial in development, neurogenesis, immunity, and disease. Repeat length variability and purity can alter the outcome of a pathway by altering the protein structure and affecting the protein-protein interaction affinity. Such rampant alterations can facilitate species to rapidly adapt to new environments or acquire various morphological/physiological features. With more than 11.000 species, the avian clade is one of the most speciose vertebrate clades, with near-ubiquitous distribution globally. Explosive adaptive radiation and functional diversification facilitated the birds to occupy various habitats. High diversity in morphology, physiology, flight pattern, behavior, coloration, and life histories make birds ideal for studying protein repeats’ role in evolutionary novelty. Our results demonstrate a similar repeat diversity and proportion of repeats across all the avian orders considered, implying an essential role of repeats in necessary pathways. We detected positively selected sites in the polyQ repeat of RUNX2 in the avian clade; and considerable repeat length contraction in the Psittacopasserae. The repeats show a species-wide bias towards a contraction in Galloanseriformes. Interestingly, we detected the length contrast of polyS repeat in PCDH20 between Galliformes and Anseriformes. We speculate that length variability of serine repeat and its interaction with β-catenin in the Wnt/β-catenin signalling pathway could have facilitated fowls to adapt to their respective environmental conditions. We believe our study emphasizes the role of protein repeats in functional/morphological diversification in birds. We also provide an extensive list of genes with considerable repeat length contrast to further explore the role of length volatility in evolutionary novelty and rapid functional diversification.

Use of Open Surface Plasmon Resonance (OpenSPR) to Characterize the Binding Affinity of Protein-Protein Interactions.

Surface Plasmon Resonance(SPR) is a label-free optical technique to assess protein-protein interaction kinetics and affinities in a real-time setting. Traditionally, Biacore SPR employs a continuous film of gold to detect any change in the angle of re-emitted light when the refractive index of a ligand conjugated to the flat gold surface is altered by its interaction with a local analyte. In contrast, the Nicoya Lifesciences' OpenSPR technology uses gold nanoparticles to detect small changes in the absorbance peak wavelength of a conjugated ligand after its engagement by an analyte. Specifically, when broadband white light is shone onto the gold nanoparticles, it produces a strong resonance absorbance peak corresponding to the refractive index of a ligand conjugated to the surface of gold nanoparticles. Upon its interaction with an analyte, however, the absorbance wavelength peak of the conjugated ligand will be changed and timely recorded as sensorgrams of dynamic ligand-analyte interactions. Thus, the improvement in the detection method (from traditional detection of changes in the angle of re-emitted light to the contemporary detection of changes in the wavelength of the absorbance peak) features OpenSPR as a cost-effective and user-friendly technique for in-depth characterization of protein-protein interactions. Here, we describe the detailed method that we used to characterize procathepsin L (pCTS-L) interactions with two putative pattern recognition receptors (TLR4 and RAGE) using the 1st generation of Nicoya Lifesciences' OpenSPR instrument with a 1-channel detection. Key features • Nicoya OpenSPR is a benchtop small-size equipment that provides in-depth label-free binding kinetics and affinity measurement for protein-protein interactions in real-time fashion. • This technology is relatively intuitive and user-friendly for scientists at any skill level. • OpenSPR sensors employ nanotechnology to reduce the cost of manufacturing complex optical hardware and Sensor Chips, and similarly reduce the consumption of precious analyte samples. • The manufacturer provides online training for OpenSPR (Catalog: TRAIN-REMOTE) and TraceDrawer (Catalog: TRAIN-TD) to customer scientists.

Importance of interface and surface areas in protein-protein binding affinity prediction: A machine learning analysis based on linear regression and artificial neural network

Protein-protein interaction plays an important role in all biological systems. The binding affinity between two protein binding partners reflects the strength of their association, which is crucial to the elucidation of the biological functions of these proteins and also to the design of protein-based therapeutic agents. In recent years, many studies have been conducted in an effort to improve the ability to predict the binding affinity of a protein-protein complex. Different sequence and structural features have been adopted in the prediction, but the surface or interface areas of the protein-protein complex were often not given adequate consideration. In the present study, different types of interface and surface areas in the protein-protein complex were used to construct or train linear, nonlinear or mixed models using linear regression and artificial neural network to predict the binding affinity of protein-protein interactions. The relative importance of the different types of areas in the selected models for affinity prediction was analyzed using variable-controlling approach. In terms of performance, the best area-based binding affinity predictors appeared to be superior or at least comparable to the widely-used predictors PRODIGY (a contacts-based predictor) and LISA (Local Interaction Signal Analysis). This work highlights the importance of interface and surface areas in protein-protein binding interactions. It also sheds light on the more suitable computational approaches that may aid in solving some of the scientific and technical issues associated with protein-protein binding affinity prediction. SignificanceProtein-protein interactions are ubiquitous in living systems. Protein-protein binding affinity is a metric that estimates the binding strength between two protein binding partners. Reliable information on their binding affinity is of great value in understanding complex biological processes as well as in designing protein-based therapeutics. In this work, the interface and surface areas in protein-protein interaction are explored with respect to their relative importance in better predicting the protein-protein binding affinity. The results from this study showed that different types of areas contribute importantly to protein-protein interactions and thus should be jointly considered in an explicit manner to improve affinity predictions. In addition, the effective application of interface and surface areas may also facilitate the simulation of the protein folding and binding processes.

Quantitative FRET (qFRET) Technology for the Determination of Protein-Protein Interaction Affinity in Solution.

Protein–protein interactions play pivotal roles in life, and the protein interaction affinity confers specific protein interaction events in physiology or pathology. Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research to detect molecular interactions in vitro and in vivo. The FRET assay provides very high sensitivity and efficiency. Several attempts have been made to develop the FRET assay into a quantitative measurement for protein–protein interaction affinity in the past. However, the progress has been slow due to complicated procedures or because of challenges in differentiating the FRET signal from other direct emission signals from donor and receptor. This review focuses on recent developments of the quantitative FRET analysis and its application in the determination of protein–protein interaction affinity (KD), either through FRET acceptor emission or donor quenching methods. This paper mainly reviews novel theatrical developments and experimental procedures rather than specific experimental results. The FRET-based approach for protein interaction affinity determination provides several advantages, including high sensitivity, high accuracy, low cost, and high-throughput assay. The FRET-based methodology holds excellent potential for those difficult-to-be expressed proteins and for protein interactions in living cells.

Mechanically transduced immunosorbent assay to measure protein-protein interactions.

Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. Yet, conventional methods rely upon the law of mass action and cannot measure many PPIs due to a scarcity of reagents and limitations in the measurable affinity ranges. Here, we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that correlates to binding potential. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. The METRIS assay uses as little as 20 pmols of reagents to measure a wide range of affinities while exhibiting a high resolution and sensitivity. We use METRIS to measure several PPIs that were previously inaccessible using traditional methods, providing new insights into epigenetic recognition.

High-Mass Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Absolute Quantitation of Noncovalent Protein-Protein Binding Interactions.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a robust and powerful tool for studying biomacromolecules and their interactions. However, quantitative detection of high-mass analytes (kDa to MDa range) remains challenging for MALDI-MS. Herein, we successfully used commercially available purified proteins (β-galactosidase and BSA) as internal standards for high-mass MALDI-MS analysis and achieved absolute quantification of several high-mass analytes. We systematically evaluated four sample deposition methods, and using the sandwich deposition method with saturated sinapinic acid as the top layer, we performed a robust quantitative analysis by high-mass MALDI-MS. Combined with chemical cross-linking, this quantitative strategy was further used to evaluate the affinity of protein-protein interactions (PPIs), specifically of two soluble protein receptors (interleukin 1 receptor and interleukin 2 receptor) and two membrane protein receptors (rhodopsin and angiotensin 2 receptor 1) with their interaction partners. The measured dissociation constants of the protein complexes formed were between 10 nM and 5 μM. We expect this high-throughput, rapid method, which does not require labeling or immobilization of any of the interaction partners, to become a viable alternative to traditional biophysical methods for studying PPIs.

Quantitative Yeast-Yeast Two Hybrid for the Discovery and Binding Affinity Estimation of Protein-Protein Interactions.

Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semiempirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of bait-prey yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.

N-Terminal Protein Labeling with N-Hydroxysuccinimide Esters and Microscale Thermophoresis Measurements of Protein-Protein Interactions Using Labeled Protein.

Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein-protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy-to-apply protocol for high-stoichiometric, site-specific labeling of a protein at its N-terminus with N-hydroxysuccinimide (NHS) esters as a means to measure protein-protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent-labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2-E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Labeling a protein of interest at its N-terminus with NHS esters through stepwise reaction Alternate Protocol: Labeling a protein of interest at its N-terminus with NHS esters through a one-pot reaction Basic Protocol 2: Purifying the N-terminal fluorescent-labeled protein and determining its concentration and labeling efficiency Basic Protocol 3: Using MST to determine the binding affinity of an N-terminal fluorescent-labeled protein to a binding partner. Basic Protocol 4: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay.

4094 Structural Determinants of Immunogenicity for Peptide-Based Immunotherapy

OBJECTIVES/GOALS: Neoantigen vaccine immunotherapies have shown promise in clinical trials, but identifying which peptides to include in a vaccine remains a challenge. We aim to establish that molecular structural features can help predict which neoantigens to target to achieve tumor regression. METHODS/STUDY POPULATION: Proteins were prepared by recombinant expression in E. coli followed by in vitro refolding. Correctly folded proteins were purified by chromatography. Affinities of protein-protein interactions were measured by surface plasmon resonance (SPR) and thermal stabilities of proteins were determined by differential scanning fluorimetry. All experiments were performed at least in triplicate. Protein crystals were obtained by hanging drop vapor diffusion. The protein crystal structures were solved by molecular replacement and underwent several rounds of automated refinement. Molecular dynamics simulations were performed using the AMBER molecular dynamics package. RESULTS/ANTICIPATED RESULTS: A T cell receptor (TCR) expressed by tumor-infiltrating T cells exhibited a 20-fold stronger binding affinity to the neoantigen peptide compared to the self-peptide. X-ray crystal structures of the peptides with the major histocompatibility complex (MHC) protein demonstrated that a non-mutated residue in the peptide samples different positions with the mutation. The difference in conformations of the non-mutated residue was supported by molecular dynamics simulations. Crystal structures of the TCR engaging both peptide/MHCs suggested that the conformation favored by the mutant peptide was crucial for TCR binding. The TCR bound the neoantigen/MHC with faster binding kinetics. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that the mutation impacts the conformation of another residue in the peptide, and this alteration allows for more favorable T cell receptor binding to the neoantigen. This highlights the potential of non-mutated residues in contributing to neoantigen recognition.

A Quantitative Tri-fluorescent Yeast Two-hybrid System: From Flow Cytometry to In cellula Affinities

We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pm to 17 μm After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry

Measurements of biomolecular interactions are crucial to understand the mechanisms of the biological processes they facilitate. Bulk-based methods such as ITC and SPR provide important information on binding affinities, stoichiometry, and kinetics of interactions. However, the ensemble averaging approaches are not able to probe the intrinsic heterogeneity often displayed by biological systems. Interactions that involve cooperativity or result in the formation of multicomponent complexes pose additional experimental challenges. Single-molecule techniques have previously been applied to solve these problems. However, single-molecule experiments are often technically demanding and require labeling or immobilization of the molecules under study. A recently developed single-molecule method, mass photometry (MP), overcomes these limitations. Here we applied MP to measure the affinities of biomolecular interactions. We have demonstrated how MP allows the user to study multivalent complexes and quantify the affinities of different binding sites in a single measurement. Results obtained from this single-molecule technique have been validated by ITC and BLI. The quality and information content of the MP data, combined with simple and fast measurements and low sample consumption makes MP a new preferred method for measuring strong protein-protein interactions.

Proteomic navigation using proximity-labeling.

The identification of bona fide protein-protein interactions and the mapping of proteomes was greatly enhanced by protein tagging for generic affinity purification methods and analysis by mass spectrometry (AP-MS). The high quality of AP-MS data permitted the development of proteomic navigation by sequential tagging of identified interactions. However AP-MS is laborious and limited to relatively high affinity protein-protein interactions. Proximity labeling, first with the biotin ligase BirA, termed BioID, and then with ascorbate peroxidase, termed APEX, permits a greater reach into the proteome than AP-MS enabling both the identification of a wider field and weaker protein-protein interactions. This additional reach comes with the need for stringent controls. Proximity labeling also permits experiments in living cells allowing spatiotemporal investigations of the proteome. Here we discuss proximity labeling with accompanying methodological descriptions for E. coli and mammalian cells.

Protein\u2013Protein Affinity Determination by Quantitative FRET Quenching

The molecular dissociation constant, Kd, is a well-established parameter to quantitate the affinity of protein-protein or other molecular interactions. Recently, we reported the theoretical basis and experimental procedure for Kd determination using a quantitative FRET method. Here we report a new development of Kd determination by measuring the reduction in donor fluorescence due to acceptor quenching in FRET. A new method of Kd determination was developed from the quantitative measurement of donor fluorescence quenching. The estimated Kd values of SUMO1-Ubc9 interaction based on this method are in good agreement with those determined by other technologies, including FRET acceptor emission. Thus, the acceptor-quenched approach can be used as a complement to the previously developed acceptor excitation method. The new methodology has more general applications regardless whether the acceptor is an excitable fluorophore or a quencher. Thus, these developments provide a complete methodology for protein or other molecule interaction affinity determinations in solution.

Isolation and Characterization of S100 Protein-Protein Complexes.

S100 proteins are small, mostly dimeric, EF-hand Ca2+-binding proteins. Upon Ca2+ binding, a conformational change occurs resulting in the exposure of a shallow hydrophobic binding groove in each subunit. Interestingly, S100 proteins can interact with their partners in two ways: symmetrically, when the two partners identically bind into each groove, or asymmetrically, when only one partner binds to the S100 dimer occupying both binding pockets. Here we present a heterologous expression and purification protocol for all known human S100 proteins as well as for their partner peptides. Moreover, we provide a detailed description of three in vitro methods to determine the affinity, stoichiometry, and kinetics of S100 protein-protein interactions.

Computational affinity maturation of camelid single-domain intrabodies against the nonamyloid component of alpha-synuclein

Improving the affinity of protein-protein interactions is a challenging problem that is particularly important in the development of antibodies for diagnostic and clinical use. Here, we used structure-based computational methods to optimize the binding affinity of VHNAC1, a single-domain intracellular antibody (intrabody) from the camelid family that was selected for its specific binding to the nonamyloid component (NAC) of human α-synuclein (α-syn), a natively disordered protein, implicated in the pathogenesis of Parkinson’s disease (PD) and related neurological disorders. Specifically, we performed ab initio modeling that revealed several possible modes of VHNAC1 binding to the NAC region of α-syn as well as mutations that potentially enhance the affinity between these interacting proteins. While our initial design strategy did not lead to improved affinity, it ultimately guided us towards a model that aligned more closely with experimental observations, revealing a key residue on the paratope and the participation of H4 loop residues in binding, as well as confirming the importance of electrostatic interactions. The binding activity of the best intrabody mutant, which involved just a single amino acid mutation compared to parental VHNAC1, was significantly enhanced primarily through a large increase in association rate. Our results indicate that structure-based computational design can be used to successfully improve the affinity of antibodies against natively disordered and weakly immunogenic antigens such as α-syn, even in cases such as ours where crystal structures are unavailable.

SPR—Based study of affinity of cytochrome P450s / redox partners interactions modulated by steroidal substrates

The goal of this work was to test the hypothesis that the affinity of protein-protein interactions in the cytochrome P450-dependent monooxygenase system is modulated by the low-molecular-weight compounds (substrates or inhibitors). The surface plasmon resonance (SPR) based study was carried out using the recombinant protein preparations of three microsomal cytochromes P450 (CYP17A1, CYP21A2, and CYP2C19) and their redox partners: cytochrome b5 (CYB5A), NADPH - cytochrome P450 reductase (CPR), and also iron-sulfur protein adrenodoxin (Adx). As a result, we have revealed some specificity of the influence of the steroid substrates on the binding affinity of CYPs with their redox partners, namely: the lack of effect on CPR/CYPs and Adx/CYP complex formation, and a significant effect on interactions between CYB5A and steroidogenic CYPs. The equilibrium dissociation constant (Kd) value of the CYB5A/CYP17A1 complex decreased by 5 times in the presence of progesterone (P4), which was due to a 10 times increase in the association rate constant (kon). In this case, a twofold increase in the dissociation rate constant (koff) value of CYB5A/CYP17A1 complex formation was observed. It was also demonstrated that the affinity of CYB5A/CYP17A1 interaction increased in the presence of two other steroidal substrates 17α-hydroxyprogesterone and pregnenolone and that effect was comparable with P4. In contrast, only the twofold decrease in the affinity of CYB5A/CYP21A2 interaction in the presence of P4 was caused by a slight increase in the koff value (the kon value of the complex did not change). This indicates a different format of the steroidal substrates effects expressed in a change in the stability of the CYB5A/CYPs complexes. Thus, it was found that P4 modulated the both kinetic and equilibrium constants of CYB5A/CYP17A1 and CYB5/CYP21A2 complex formation and complexes, while not affecting the CYB5A/CYP2C19 interaction (2C19 is the cytochrome P450 isoenzyme possessing broad substrate specificity), thereby indicating a specific influence of steroidal substrates on interactions involving steroidogenic CYPs. Our results are consistent with current understanding of the role of CYB5A as a regulator of cytochrome P450 activity in P450-dependent monooxygenase system.

FRETting about the affinity of bimolecular protein-protein interactions.

Fluorescence resonance energy transfer (FRET) is a powerful tool to study macromolecular interactions such as protein-protein interactions (PPIs). Fluorescent protein (FP) fusions enable FRET-based PPI analysis of signaling pathways and molecular structure in living cells. Despite FRET's importance in PPI studies, FRET has seen limited use in quantifying the affinities of PPIs in living cells. Here, we have explored the relationship between FRET efficiency and PPI affinity over a wide range when expressed from a single plasmid system in Escherichia coli. Using live-cell microscopy and a set of 20 pairs of small interacting proteins, belonging to different structural folds and interaction affinities, we demonstrate that FRET efficiency can reliably measure the dissociation constant (KD ) over a range of mM to nM. A 10-fold increase in the interaction affinity results in 0.05 unit increase in FRET efficiency, providing sufficient resolution to quantify large affinity differences (> 10-fold) using live-cell FRET. This approach provides a rapid and simple strategy for assessment of PPI affinities over a wide range and will have utility for high-throughput analysis of protein interactions.

Structural motifs in eIF4G and 4E-BPs modulate their binding to eIF4E to regulate translation initiation in yeast.

The interaction of the eukaryotic initiation factor 4G (eIF4G) with the cap-binding protein eIF4E initiates cap-dependent translation and is regulated by the 4E-binding proteins (4E-BPs), which compete with eIF4G to repress translation. Metazoan eIF4G and 4E-BPs interact with eIF4E via canonical and non-canonical motifs that bind to the dorsal and lateral surface of eIF4E in a bipartite recognition mode. However, previous studies pointed to mechanistic differences in how fungi and metazoans regulate protein synthesis. We present crystal structures of the yeast eIF4E bound to two yeast 4E-BPs, p20 and Eap1p, as well as crystal structures of a fungal eIF4E–eIF4G complex. We demonstrate that the core principles of molecular recognition of eIF4E are in fact highly conserved among translational activators and repressors in eukaryotes. Finally, we reveal that highly specialized structural motifs do exist and serve to modulate the affinity of protein-protein interactions that regulate cap-dependent translation initiation in fungi.

Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif

Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs.

Engineering High Affinity Protein-Protein Interactions Using a High-Throughput Microcapillary Array Platform.

Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the μSCALE platform for affinity maturation of a protein-protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of μSCALE as a new tool for directed evolution.