Sirtuins are unique posttranslational modification enzymes that utilize NAD+ as the co-substrate to remove acyl groups from lysine residues. The deacylation events result in profound biological consequences, from transcription silencing to metabolism regulation. This article focuses on a newly developed technology using activity-based chemical probes to report sirtuin functional state in various settings. These chemical probes, thioacyllysine peptides carrying photo-cross-linker as well as bioorthogonal functionality, target the active site of sirtuins to form stalled reaction intermediate. Subsequently, the probe forms covalent adduct with the protein through photocrosslinking. Ultimately, the active sirtuin can be visualized via "click" chemistry-mediated conjugation to a fluorescent tag. Here, we describe the labeling protocols on recombinant protein, whole cell lysate, and in situ labeling. © 2020 Elsevier Inc. All rights reserved.Intrinsic protein properties that may not be apparent by only examining three-dimensional structures can be revealed by careful analysis of mutant protein variants. Deep mutational scanning is a technique that allows the functional analysis of millions of protein variants in a single experiment. To enable this high-throughput technique, the mutant genotype of protein variants must be coupled to a selectable function. This chapter outlines how artificial genetic circuits in the yeast Saccharomyces cerevisiae can maintain the genotype-phenotype link, thus enabling the general application of this approach. To do this, we describe how to engineer genetic selections in yeast, methods to construct mutant libraries, and how to analyze sequencing data. We investigate the structure-function relationships of the antimicrobial resistance protein TetX to illustrate this process. In doing so, we demonstrate that deep mutational scanning is a powerful method to dissect the importance of individual residues for the inactivation of antibiotic analogues, with consequences for the rational design of new drugs to combat antimicrobial resistance. © 2020 Elsevier Inc. All rights reserved.Chicken avidin and bacterial streptavidin are workhorses in biotechnology. We have used avidin as a scaffold protein to develop avidin variants with novel ligand-binding affinity, so-called antidins. This article covers the strategy applied in the development of antidins. Using a phage display developed for avidin, immobilized ligands were used to select binders from a phage pool displaying avidin variants with randomized sequence in the protein loops. Antidins binding various ligands with nanomolar affinity were obtained. Antidins have already been demonstrated to be suitable for a diagnostic assay measuring serum progesterone levels and they offer a promising alternative to antibodies for the recognition of small molecules. © 2020 Elsevier Inc. All rights reserved.It is now clear that some cysteines on some proteins are highly tuned to react with electrophiles. Based on numerous studies, it is also established that electrophile sensing underpins rewiring of several critical signaling processes. These electrophile-sensing proteins, or privileged first responders (PFRs), are likely critically relevant for drug design. However, identifying PFRs remains a challenging and unsolved problem, despite the development of several high-throughput methods to ID proteins that react with electrophiles. More importantly, we remain unable to rank how different PFRs identified under different conditions relate to one another, in terms of sensing or signaling capacity. Here we evaluate different methods to assay sensing functions of proteins and discuss these methods in the context of developing a "ranking scheme." Based on theoretical and experimental evidence, we propose that T-REX-the only targeted-electrophile delivery tool presently available-is a reliable method to rank PFRs. Finally, we address to what extent electrophile sensing and downstream signaling are correlated. Based on our current data, we observe that such behaviors are indeed correlated.  https://www.selleckchem.com/products/tegatrabetan.html  It is our hope that through this manuscript researchers from various arms of the stress signaling fields will focus on developing a quantitative understanding of precision electrophile labeling. © 2020 Elsevier Inc. All rights reserved.Phenotypic screening is a powerful approach to discover small molecules targeting pathways or disease biology with complex genetic causes. Following the initial discovery of these small molecules is their target identification, which is at the cornerstone in addressing their biological and clinical utility. Yet, finding the needle in the haystack remains a challenge. Nuclear lamins are type V intermediate filament proteins that form a filamentous structure underneath the inner nuclear envelope to support the mechanical stability of the mammalian cell nucleus. They also participate a myriad of other cellular signaling processes with incompletely understood molecular mechanisms. Small molecules that can directly bind to nuclear lamins will be incredible tools to address lamins' roles in different aspects of biology. However, these small molecules did not exist until recently. We previously discovered an acylpyrroloquinazoline called LBL1 that selectively killed breast cancer cells without harming normal human cells. To help understand the mechanism of action of LBL1, we recently took an unbiased chemical proteomics approach to identify its direct binding targets from the entire human cellular proteome. In this chapter, we describe our detailed methods to identify and validate lamins as the direct targets of LBL1. In this approach, we developed a clickable photoaffinity probe called LBL1-P that contains acylpyrroloquinazoline, trifluoromethyldiazirine and alkyne groups. Furthermore, we described a fluorescence microscopic method to validate that LBL1 directly targets lamin A in living cells. When properly designed, this approach should be broadly applicable to other bioactive small molecules. © 2020 Elsevier Inc. All rights reserved.The cyclic-AMP response element binding protein (CREB) is an important nuclear transcription factor and has been shown to be overexpressed and/or over-activated in many different cancer types, suggesting that targeting CREB is a novel approach for developing cancer therapies. Our lab discovered the first cell-permeable small molecule inhibitor of CREB, from which we further developed a potent CREB inhibitor with in vivo anti-cancer activity. In this article, we detailed our biochemical and cell-based bioassays to assess different small molecule CREB inhibitors. © 2020 Elsevier Inc. All rights reserved.