Loss of the KN Motif and AnKyrin Repeat Domain 1 (KANK1) Leads to Lymphoid Compartment Dysregulation in Murine Model.

The KN Motif and AnKyrin Repeat Domain 1 (KANK1) is proposed as a tumour suppressor gene, as its expression is reduced or absent in several types of tumour tissue, and over-expressing the protein inhibited the proliferation of tumour cells in solid cancer models. We report a novel germline loss of heterozygosity mutation encompassing the KANK1 gene in a young patient diagnosed with myelodysplastic neoplasm (MDS) with no additional disease-related genomic aberrations. To study the potential role of KANK1 in haematopoiesis, we generated a new transgenic mouse model with a confirmed loss of KANK1 expression. KANK1 knockout mice did not develop any haematological abnormalities; however, the loss of its expression led to alteration in the colony forming and proliferative potential of bone marrow (BM) cells and a decrease in hematopoietic stem and progenitor cells (HSPCs) population frequency. A comprehensive marker expression analysis of lineage cell populations indicated a role for Kank1 in lymphoid cell development, and total protein analysis suggests the involvement of Kank1 in BM cells' cytoskeleton formation and mobility.

Two single-point mutations in Ankyrin Repeat one drastically change the threshold temperature of TRPV1.

TRPV1 plays an important role in the thermosensory system; however, the mechanism controlling its heat activation property is not well understood. Here, we determine the heat activation properties of TRPV1 cloned from tailed amphibians, which prefer cooler environments, finding the threshold temperatures were approximately 10 °C lower compared with rat TRPV1 (rTRPV1). We find that two amino acid residues (Gln, Leu/Val) in the Ankyrin Repeat 1 (ANK1) region of the N-terminal domain are conserved among tailed amphibians and different from those (Arg, Lys) in rTRPV1. We observe the activation by heat in all urodelan TRPV1s is markedly elevated by substitution of these two amino acids. Conversely, reciprocal substitutions of rTRPV1 apparently lowers the high threshold temperature. Our studies demonstrate that tailed amphibians express TRPV1 with a reduced heat-activation threshold by substitution of two amino acid residues in the ANK1 region that likely contribute to cool-habitat selection.

Targeting Oncolytic Adenoviruses to Cancer Cells Using a Designed Ankyrin Repeat Protein Lipocalin-2 Fusion Protein.

Oncolytic viruses are a promising technology to attack cancer cells and to recruit immune cells to the tumor site. Since the Lipocalin-2 receptor (LCN2R) is expressed on most cancer cells, we used its ligand LCN2 to target oncolytic adenoviruses (Ads) to cancer cells. Therefore, we fused a Designed Ankyrin Repeat Protein (DARPin) adapter binding the knob of Ad type 5 (knob5) to LCN2 to retarget the virus toward LCN2R with the aim of analyzing the basic characteristics of this novel targeting approach. The adapter was tested in vitro with Chinese Hamster Ovary (CHO) cells stably expressing the LCN2R and on 20 cancer cell lines (CCLs) using an Ad5 vector encoding luciferase and green fluorescent protein. Luciferase assays with the LCN2 adapter (LA) showed 10-fold higher infection compared with blocking adapter (BA) in CHO cells expressing LCN2R and in cells not expressing the LCN2R. Most CCLs showed an increased viral uptake of LA-bound virus compared with BA-bound virus and for five CCLs viral uptake was comparable to unmodified Ad5. Flow cytometry and hexon immunostainings also revealed increased uptake of LA-bound Ads compared with BA-bound Ads in most tested CCLs. Virus spread was studied in 3D cell culture models and nine CCLs showed increased and earlier fluorescence signals for LA-bound virus compared with BA-bound virus. Mechanistically, we show that the LA increases viral uptake only in the absence of its ligand Enterobactin (Ent) and independently of iron. Altogether, we characterized a novel DARPin-based system resulting in enhanced uptake demonstrating potential for future oncolytic virotherapy.

Tiller Number1 encodes an ankyrin repeat protein that controls tillering in bread wheat.

Wheat (Triticum aestivum L.) is a major staple food for more than one-third of the world's population. Tiller number is an important agronomic trait in wheat, but only few related genes have been cloned. Here, we isolate a wheat mutant, tiller number1 (tn1), with much fewer tillers. We clone the TN1 gene via map-based cloning: TN1 encodes an ankyrin repeat protein with a transmembrane domain (ANK-TM). We show that a single amino acid substitution in the third conserved ankyrin repeat domain causes the decreased tiller number of tn1 mutant plants. Resequencing and haplotype analysis indicate that TN1 is conserved in wheat landraces and modern cultivars. Further, we reveal that the expression level of the abscisic acid (ABA) biosynthetic gene TaNCED3 and ABA content are significantly increased in the shoot base and tiller bud of the tn1 mutants; TN1 but not tn1 could inhibit the binding of TaPYL to TaPP2C via direct interaction with TaPYL. Taken together, we clone a key wheat tiller number regulatory gene TN1, which promotes tiller bud outgrowth probably through inhibiting ABA biosynthesis and signaling.

Single amino acids set apparent temperature thresholds for heat-evoked activation of mosquito transient receptor potential channel TRPA1.

Animals detect heat using thermosensitive transient receptor potential (TRP) channels. In insects, these include TRP ankyrin 1 (TRPA1), which in mosquitoes is crucial for noxious heat avoidance and thus is an appealing pest control target. However, the molecular basis for heat-evoked activation has not been fully elucidated, impeding both studies of the molecular evolution of temperature sensitivity and rational design of inhibitors. In TRPA1 and other thermosensitive TRPs, the N-terminal cytoplasmic ankyrin repeat (AR) domain has been suggested to participate in heat-evoked activation, but the lack of a structure containing the full AR domain has hindered our mechanistic understanding of its role. Here, we focused on elucidating the structural basis of apparent temperature threshold determination by taking advantage of two closely related mosquito TRPA1s from Aedes aegypti and Culex pipiens pallens with 86.9% protein sequence identity but a 10 °C difference in apparent temperature threshold. We identified two positions in the N-terminal cytoplasmic AR domain of these proteins, E417 (A. aegypti)/Q414 (C. pipiens) and R459 (A. aegypti)/Q456 (C. pipiens), at which a single exchange of amino acid identity was sufficient to change apparent thresholds by 5 to 7 °C. We further found that the role of these positions is conserved in TRPA1 of a third related species, Anopheles stephensi. Our results suggest a structural basis for temperature threshold determination as well as for the evolutionary adaptation of mosquito TRPA1 to the wide range of climates inhabited by mosquitoes.

Crystal structures of HER3 extracellular domain 4 in complex with the designed ankyrin-repeat protein D5.

The members of the human epidermal growth factor receptor (HER) family are among the most intensely studied oncological targets. HER3 (ErbB3), which had long been neglected, has emerged as a key oncogene, regulating the activity of other receptors and being involved in progression and tumor escape in multiple types of cancer. Designed ankyrin-repeat proteins (DARPins) serve as antibody mimetics that have proven to be useful in the clinic, in diagnostics and in research. DARPins have previously been selected against EGFR (HER1), HER2 and HER4. In particular, their combination into bivalent binders that separate or lock receptors in their inactive conformation has proved to be a promising strategy for the design of potent anticancer therapeutics. Here, the selection of DARPins targeting extracellular domain 4 of HER3 (HER3d4) is described. One of the selected DARPins, D5, in complex with HER3d4 crystallized in two closely related crystal forms that diffracted to 2.3 and 2.0 Šresolution, respectively. The DARPin D5 epitope comprises HER3d4 residues 568-577. These residues also contribute to interactions within the tethered (inactive) and extended (active) conformations of the extracellular domain of HER3.

A membrane-bound ankyrin repeat protein confers race-specific leaf rust disease resistance in wheat.

Plasma membrane-associated and intracellular proteins and protein complexes play a pivotal role in pathogen recognition and disease resistance signaling in plants and animals. The two predominant protein families perceiving plant pathogens are receptor-like kinases and nucleotide binding-leucine-rich repeat receptors (NLR), which often confer race-specific resistance. Leaf rust is one of the most prevalent and most devastating wheat diseases. Here, we clone the race-specific leaf rust resistance gene Lr14a from hexaploid wheat. The cloning of Lr14a is aided by the recently published genome assembly of ArinaLrFor, an Lr14a-containing wheat line. Lr14a encodes a membrane-localized protein containing twelve ankyrin (ANK) repeats and structural similarities to Ca2+-permeable non-selective cation channels. Transcriptome analyses reveal an induction of genes associated with calcium ion binding in the presence of Lr14a. Haplotype analyses indicate that Lr14a-containing chromosome segments were introgressed multiple times into the bread wheat gene pool, but we find no variation in the Lr14a coding sequence itself. Our work demonstrates the involvement of an ANK-transmembrane (TM)-like type of gene family in race-specific disease resistance in wheat. This forms the basis to explore ANK-TM-like genes in disease resistance breeding.

ASB16-AS1 up-regulated and phosphorylated TRIM37 to activate NF-κB pathway and promote proliferation, stemness, and cisplatin resistance of gastric cancer.

BACKGROUND: Long non-coding RNA (lncRNA) ASB16 antisense RNA 1 (ASB16-AS1) is recognized as an oncogene in several cancer types, but its relation to GC is unknown. Tripartite motif containing 37 (TRIM37) has been proven to accelerate the development of gastric cancer (GC), whereas the molecular mechanism assisted ASB16-AS1 and TRIM37 in regulating GC progression remains unclear. METHODS: Differentially expressed lncRNAs in GC samples were analyzed based on Gene Expression Omnibus (GEO) data. CCK-8 and colony formation assays were applied to determine the proliferative ability of GC cells. Stem cell-like phenotype of GC cells was assessed by sphere formation assay and flow cytometry analysis. Luciferase reporter assay, RNA immunoprecipitation (RIP), pulldown, and co-immunoprecipitation (Co-IP) were performed to verify the interplay of RNA molecules. RESULTS: ASB16-AS1 was upregulated in GC samples according to GEO data and qRT-PCR analysis. ASB16-AS1 strengthened the proliferative ability and stem cell-like characteristics in GC cells. More importantly, ASB16-AS1 encouraged GC cell growth in vivo. Mechanistically, ASB16-AS1 strengthened TRIM37 expression by sequestering miR-3918 and miR-4676-3p. ASB16-AS1 activated NF-kappa B (NF-κB) pathway by cooperating with ATM serine/threonine kinase (ATM) to induce TRIM37 phosphorylation. CONCLUSION: In summary, ASB16-AS1 exerted oncogenic functions in GC through modulating TRIM37 expression at both mRNA and protein levels.

Epistatic effect of Ankyrin repeat and kinase domain containing 1 - Dopamine receptor D2 and catechol-o-methyltransferase single nucleotide polymorphisms on the risk for hazardous use of alcohol in Lithuanian population.

AIM: The Lithuanian population has outstanding rates of alcohol consumption and alcohol related mortality. Alteration of brain dopaminergic system play a role in the risk for addiction disorders. We evaluated the association of one single nucleotide polymorphism rs1800497 in the Ankyrin Repeat and Kinase Domain Containing 1 - Dopamine Receptor D2 complex (ANKK1-DRD2) and a catechol-o-methyltransferase (COMT) rs4680 single nucleotide polymorphism with the risk for alcohol use disorder and impulsiveness in Lithuanian population. Both genetic polymorphisms are known to alter brain dopaminergic activity, thus we also investigated the possible interaction effect of these polymorphisms. METHODS: The study included 329 participants recruited from the local community. Hazardous alcohol use was evaluated using the Alcohol Use Disorder Identification Test (AUDIT). Impulsiveness was measured using the Barratt Impulsiveness Scale - 11 (BIS-11). Between group differences of AUDIT and BIS-11 scores were examined stratified by genetic polymorphisms and their combinations. The independent effect of each polymorphism and their interaction for hazardous alcohol use were evaluated using adjusted logistic regression analyses. RESULTS: The ANKK1-DRD2 rs1800497 polymorphism was associated with total AUDIT score, but not with the hazardous use of alcohol, as indicated by the AUDIT test cut-off of 8. The COMT rs4680 GG genotype was associated with the hazardous use of alcohol (adjusted OR = 2.094, p = 0.029), but this association was not statistically significant after adjustment for multiple comparisons. Presence of both COMT rs4680 and ANKK1-DRD2 rs1800497 GGxCT/TT polymorphisms was associated with significantly increased risk for hazardous use of alcohol (adjusted OR = 5.016, p = 0.005). The COMT rs4680 and ANKK1-DRD2 rs1800497 genetic polymorphisms, and their combination were not associated with impulsiveness. CONCLUSIONS: Our study demonstrated that the interaction of COMT rs4680 and ANKK1-DRD2 rs1800497 genetic polymorphisms is associated with a hazardous use of alcohol.

The ankyrin repeat-containing protein MdANK2B regulates salt tolerance and ABA sensitivity in Malus domestica.

KEY MESSAGE: The ankyrin repeat-containing protein MdANK2B was identified to contribute to increasing resistance to salt stress and decreasing sensitivity to ABA in Malus domestica. Ankyrin (ANK) repeat-containing proteins occur widely in prokaryotes, eukaryotes, and even in some viruses and play a critical role in plant growth and development, as well as the response to biotic and abiotic stress. However, the function of ANK repeat-containing proteins in apple (Malus domestica) has not yet been investigated. Here, we identified apple MdANK2B based on homology analysis with the Arabidopsis ANK repeat-containing proteins AtAKR2A and AtAKR2B. MdANK2B was found to be localized in the cytoplasm, and its encoding gene was highly expressed in both apple leaves and fruits. In addition, MdANK2B gene expression was highly induced by salt stresses and abscisic acid (ABA). Overexpression of MdANK2B increased resistance to salt stress and decreased sensitivity to ABA in both transgenic apple calli and seedlings. In addition, overexpression of MdANK2B reduced the accumulation of reactive oxygen species (ROS) by enhancing the activity of antioxidant enzymes in response to salt stress. Our data revealed the role of MdANK2B in response to salt stress and ABA treatment in apple, which widens the known functions of ANK repeat-containing proteins in response to abiotic stress.

An ankyrin-repeat and WRKY-domain-containing immune receptor confers stripe rust resistance in wheat.

Perception of pathogenic effectors in plants often relies on nucleotide-binding domain (NBS) and leucine-rich-repeat-containing (NLR) proteins. Some NLRs contain additional domains that function as integrated decoys for pathogen effector targets and activation of immune signalling. Wheat stripe rust is one of the most devastating diseases of crop plants. Here, we report the cloning of YrU1, a stripe rust resistance gene from the diploid wheat Triticum urartu, the progenitor of the A genome of hexaploid wheat. YrU1 encodes a coiled-coil-NBS-leucine-rich repeat protein with N-terminal ankyrin-repeat and C-terminal WRKY domains, representing a unique NLR structure in plants. Database searches identify similar architecture only in wheat relatives. Transient expression of YrU1 in Nicotiana benthamiana does not induce cell death in the absence of pathogens. The ankyrin-repeat and coiled-coil domains of YrU1 self-associate, suggesting that homodimerisation is critical for YrU1 function. The identification and cloning of this disease resistance gene sheds light on NLR protein function and may facilitate breeding to control the devastating wheat stripe rust disease.

Design of Protein Logic Gate System Operating on Lipid Membranes.

Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein-membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein-membrane logic gate with an OR-AND logic. The system consists of an engineered pH-dependent pore-forming protein listeriolysin O and its DARPin-based inhibitor, conjugated to a lipid vesicle membrane. The gate responds to low pH and removal of the inhibitor from the membrane either by switching to a reducing environment, protease cleavage, or any other signal depending on the conjugation chemistry used for inhibitor attachment to the membrane. This unique protein logic gate vesicle system advances generic sensing and actuator platforms used in synthetic biology and could be utilized in drug delivery.

Silencing circular RNA circANKRD36 remits lipopolysaccharide-induced inflammatory damage by regulating microRNA-15/MyD88.

Bedsore is a familiar disease, which fearfully harms the health of the patients. We investigated the efficacy and mechanism of circular RNA circANKRD36 on HaCaT cell in inflammatory damage. CCK-8 and flow cytometry were respectively used to investigate the efficacies of lipopolysaccharide (LPS), circANKRD36, and microRNA (miR)-15 on cell viability and apoptosis. Moreover, circANKRD36 and miR-15 expression were changed by cell transfection and investigated by reverse transcription-quantitative polymerase chain reaction. Furthermore, the levels of Bax, pro caspase-3, cleaved caspase-3, interleukin (IL)-1ß, IL-6, and proteins of the pathway were investigated by Western blot. Otherwise, the levels of IL-1ß and IL-6 were investigated by enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) was investigated by ROS assay. The relation between myeloid differentiation factor 88 (MyD88) and miR-15 was investigated by luciferase assay. LPS caused inflammatory damage and upregulated circANKRD36. circANKRD36 was silenced in cells and si-circANKRD36 remitted inflammatory damage. Furthermore, si-circANKRD36 negatively regulated miR-15 and miR-15 inhibitor could reverse the efficacies of si-circANKRD36. Besides, si-circANKRD36 restrained the NF-κB pathway by upregulating miR-15. Finally, MyD88 was authenticated as a target of miR-15. circANKRD36 remitted cell inflammatory damage upregulating miR-15/MyD88 via the NF-κB pathway in HaCaT cells.

Potential Genes Associated with the Survival of Lung Adenocarcinoma Were Identified by Methylation.

BACKGROUND: Lung adenocarcinoma (LUAD) is the most common pathological type of lung cancer. The purpose of this study is to search for genes related to the prognosis of LUAD through methylation based on a linear mixed model (LMM). METHODS: Gene expression, methylation, and survival data of LUAD patients were downloaded from the TCGA database. Based on the LMM model, the GEMMA algorithm was used to screen the predictive genes related to LUAD survival. The Cox model was used to further screen the predicted genes, and then, protein-protein interaction (PPI) network was constructed. Through the software plugin Cytoscape MCODE 3.8.0, the most closely related genes in the PPI network module were selected for in-depth biological function analysis to further explore the interaction and correlation between genes. RESULTS: We screened out 97 predictive genes from 18,834 genes and eliminated one gene associated with lung squamous cell carcinoma from previous studies, leaving 96 genes. The MCODE and the Kaplan-Meier curve analysis were used to finally identify two genes ASB16 and NEDD4 that are related to the prognosis of LUAD. CONCLUSIONS: The newly identified two genes associated with the prognosis of LUAD may provide a basis for the treatment of patients.

Structure of the thermo-sensitive TRP channel TRP1 from the alga Chlamydomonas reinhardtii.

Algae produce the largest amount of oxygen on earth and are invaluable for human nutrition and biomedicine, as well as for the chemical industry, energy production and agriculture. The mechanisms by which algae can detect and respond to changes in their environments can rely on membrane receptors, including TRP ion channels. Here we present a 3.5-Å resolution cryo-EM structure of the transient receptor potential (TRP) channel crTRP1 from the alga Chlamydomonas reinhardtii that opens in response to increased temperature and is positively regulated by the membrane lipid PIP2. The structure of crTRP1 significantly deviates from the structures of other TRP channels and has a unique 2-fold symmetrical rose-shape architecture with elbow domains and ankyrin repeat domains submerged and dipping into the membrane, respectively. Our study provides a structure of a TRP channel from a micro-organism and a structural framework for better understanding algae biology and TRP channel evolution.

Ankyrin repeat-containing N-Ank proteins shape cellular membranes.

Cells of multicellular organisms need to adopt specific morphologies. However, the molecular mechanisms bringing about membrane topology changes are far from understood-mainly because knowledge of membrane-shaping proteins that can promote local membrane curvatures is still limited. Our analyses unveiled that several members of a large, previously unrecognised protein family, which we termed N-Ank proteins, use a combination of their ankyrin repeat array and an amino (N)-terminal amphipathic helix to bind and shape membranes. Consistently, functional analyses revealed that the N-Ank protein ankycorbin (NORPEG/RAI14), which was exemplarily characterised further, plays an important, ankyrin repeat-based and N-terminal amphipathic helix-dependent role in early morphogenesis of neurons. This function furthermore required coiled coil-mediated self-assembly and manifested as ankycorbin nanodomains marked by protrusive membrane topologies. In summary, here, we unveil a class of powerful membrane shapers and thereby assign mechanistic and cell biological functions to the N-Ank protein superfamily.

Spatiotemporal expression profile of embryonic and adult ankyrin repeat and EF-hand domain containing protein 1-encoding genes ankef1a and ankef1b in zebrafish.

Recent human next-generation sequencing (NGS) studies indicate a correlation between ANKEF1 (ankyrin repeat and EF-hand domain containing protein 1) expression and cilia formation or function. Additionally, a single study conducted in the African clawed frog (Xenopus laevis) showed ankef1 is down-regulated after pharmacological fibroblast growth factor (FGF) inhibition and plays a role in protocadherin-mediated cell protrusion and adhesion. That study also revealed a critical role for ankef1 in the embryonic development of the frog, with morphants exhibiting phenotypes including spina bifida and a shortened body axis. Interestingly, while little is known about ANKEF1 function in other vertebrate systems, recent proteomic data has shown ANKEF1 enriched in ciliated cells. Likewise, publicly available EST profile databases imply ANKEF1 expression in multiple human tissues, including high levels in the testes. Together, these previous studies suggest an important role for ANKEF1 in ciliated tissues and during embryonic development. Here, we report cloning of zebrafish (Danio rerio) ankef1a, as well as its paralog, ankef1b, and expression analyses by whole-mount in situ hybridization (WISH) and quantitative polymerase chain reaction (qPCR) during embryonic development and in adult tissues. WISH shows both forms are ubiquitously expressed early in development, with more discrete expression of both transcripts in embryonic tissues known to precede or possess motile cilia, including dorsal forerunner cells (DFC) and the otic vesicles, respectively. Additionally, both transcripts are enriched in the developing pharynx and swim bladder. Our qPCR results indicate enhanced expression in the testes, along with increased expression in brain. Certainly, our experiments in the zebrafish model system with ankef1a and ankef1b provide a solid foundation for future studies to uncover the molecular pathways through which Ankef1 acts in both healthy and disease states.

Structural and functional analysis of parameters governing tankyrase-1 interaction with telomeric repeat-binding factor 1 and GDP-mannose 4,6-dehydratase.

Human tankyrase-1 (TNKS) is a member of the poly(ADP-ribose) polymerase (PARP) superfamily of proteins that posttranslationally modify themselves and target proteins with ADP-ribose (termed PARylation). The TNKS ankyrin repeat domain mediates interactions with a growing number of structurally and functionally diverse binding partners, linking TNKS activity to multiple critical cell processes, including Wnt signaling, Golgi trafficking, and telomere maintenance. However, some binding partners can engage TNKS without being modified, suggesting that separate parameters influence TNKS interaction and PARylation. Here, we present an analysis of the sequence and structural features governing TNKS interactions with two model binding partners: the PARylated partner telomeric repeat-binding factor 1 (TRF1) and the non-PARylated partner GDP-mannose 4,6-dehydratase (GMD). Using a combination of TNKS-binding assays, PARP activity assays, and analytical ultracentrifugation sedimentation analysis, we found that both the specific sequence of a given TNKS-binding peptide motif and the quaternary structure of individual binding partners play important roles in TNKS interactions. We demonstrate that GMD forms stable 1:1 complexes with the TNKS ankyrin repeat domain; yet, consistent with results from previous studies, we were unable to detect GMD modification. We also report in vitro evidence that TNKS primarily directs PAR modification to glutamate/aspartate residues. Our results suggest that TNKS-binding partners possess unique sequence and structural features that control binding and PARylation. Ultimately, our findings highlight the binding partner:ankyrin repeat domain interface as a viable target for inhibition of TNKS activity.

Site-Selective Enzymatic Labeling of Designed Ankyrin Repeat Proteins Using Protein Farnesyltransferase.

Affinity agents coupled to a functional moiety play an ever-increasing role in modern medicine, ranging from radiolabeled selective binders in diagnosis to antibody-drug conjugates in targeted therapies. In biomedical research, protein coupling to fluorophores, surfaces and nanoparticles has become an integral part of many procedures. In addition to antibodies, small scaffold proteins with similar target binding properties are being widely explored as alternative targeting moieties. To label these binders of interest with different functional moieties, conventional chemical coupling methods can be employed, but often result in heterogeneously modified protein products. In contrast, enzymatic labeling methods are highly site-specific and efficient. Protein farnesyltransferase (PFTase) catalyzes the transfer of an isoprenoid moiety from farnesyl diphosphate (FPP) to a cysteine residue in a C-terminal CaaX motif at the C-terminus of a protein substrate. The addition of only four amino acid residues minimizes the influence on the native protein structure. In addition, a variety of isoprenoid analogs containing different bioorthogonal functional groups, including azides, alkynes, and aldehydes, have been developed to enable conjugation to various cargos after being incorporated onto the target protein by PFTase. In this protocol, we present a detailed procedure for labeling Designed Ankyrin Repeat Proteins (DARPins) engineered with a C-terminal CVIA sequence using an azide-containing FPP analog by yeast PFTase (yPFTase). In addition, procedures to subsequently conjugate the labeled DARPins to a TAMRA fluorophore using strained-promoted alkyne-azide cycloaddition (SPAAC) reactions as well as the sample preparation to evaluate the target binding ability of the conjugates by flow cytometry are described.