Biochemistry

The eukaryotic cytoskeleton is formed in part by microtubules, which are relatively rigid filaments with inherent structural polarity. One consequence of this polarity is that the two ends of a microtubule have different properties with important consequences for their cellular roles. These differences are often challenging to probe within the crowded environment of the cell. Fluorescence microscopy–based in vitro assays with purified proteins and stabilized microtubules have been used to characterize polarity-dependent and end-specific behaviors. These assays require ways to visualize the polarity of the microtubules, which has previously been achieved either by the addition of fluorescently tagged motor proteins with known directionality or by fluorescently polarity marking the microtubules themselves. However, classical polarity-marking protocols require a particular chemically modified tubulin and generate microtubules with chemically different plus and minus segments. These chemical differences in the segments may affect the behavior of interacting proteins of interest in an undesirable manner. We present here a new protocol that uses a previously characterized, reversibly binding microtubule plus-end capping protein, a designed ankyrin repeat protein (DARPin), to efficiently produce polarity-marked microtubules with different fluorescently labeled, but otherwise biochemically identical, plus- and minus-end segments.

Alpha-protein kinase 1 (ALPK1) is normally activated by bacterial ADP-heptose as part of the innate immune response, leading to the initiation of downstream signalling events that culminate in the activation of transcription factors such as NF-κB and AP-1. In contrast, disease-causing mutations in ALPK1 that cause ROSAH syndrome or spiradenoma allow ALPK1 to be activated in cells in the absence of bacterial infection (i.e., without ADP-heptose). This protocol describes a semi-quantitative reporter assay based on ALPK1 knockout HEK-Blue cells that measures the activity of transfected wildtype and disease-causing forms of ALPK1 by virtue of their ability to activate the transcription factors NF-κB and AP-1. These cells express a synthetic gene encoding alkaline phosphatase under the control of an NF-κB/AP-1-dependent promoter, and consequently, the activation of ALPK1 leads to the production of alkaline phosphatase, which is secreted into the culture media and can be measured colorimetrically at 645 nm after the addition of a detection reagent.

Bioorthogonal chemical reporters are non-native chemical handles introduced into biomolecules of living systems, typically through metabolic or protein engineering. These functionalities can undergo bioorthogonal reactions, such as copper-catalyzed alkyne-azide cycloaddition (CuAAC), with small-molecule probes, enabling the tagging and visualization of biomolecules. This approach has greatly enhanced our understanding of cellular dynamics, enzyme targeting, and protein post-translational modifications. Herein, we report a protocol for preparing protein lysates for click reaction and in-gel fluorescence analysis, exemplified using alk-16, a terminal alkyne-functionalized stearic acid analog, to investigate proteins with fatty acylation. This protocol provides methods for the fluorescent visualization of chemical reporter–labeled proteomes or individual proteins of interest (POIs).

The planar lipid bilayer (PLB) technique represents a highly effective method for the study of membrane protein properties in a controlled environment. The PLB method was employed to investigate the role of mitochondrial inner membrane protein 17 (MPV17), whose mutations are associated with a hepatocerebral form of mitochondrial DNA depletion syndrome (MDS). This protocol presents a comprehensive, step-by-step guide to the assembly and utilization of a PLB system. The procedure comprises the formation of a lipid bilayer over an aperture, the reconstitution of the target protein, and the utilization of electrophysiological recording techniques to monitor channel activity. Furthermore, recommendations are provided for optimizing experimental conditions and overcoming common challenges encountered in PLB experiments. Overall, this protocol highlights the versatility of the PLB technique in advancing our understanding of membrane protein function and its broad application in various fields of research.

ALPK1 is an atypical protein kinase that is activated during bacterial infection by ADP-heptose and phosphorylates TIFA to activate a cell signaling pathway. In contrast, specific mutations in ALPK1 allow it to also be activated by endogenous human nucleotide sugars such as UDP-mannose, leading to the phosphorylation of TIFA in the absence of infection. This protocol describes a quantitative, cell-free phosphorylation assay that can directly measure the catalytic activity of wildtype and disease-causing ALPK1 in the presence of different nucleotide sugars. In this method, overexpressed ALPK1 is first immunoprecipitated from the extracts of ALPK1 knockout HEK-Blue cells transfected with plasmids encoding either FLAG-tagged wildtype or mutant ALPK1, and then subjected to a radioactive phosphorylation assay in which the phosphorylation of purified GST-tagged TIFA by ALPK1 is quantified by measuring the incorporation of radioactivity derived from radiolabeled ATP.

Membrane protein structures offer a more accurate basis for understanding their functional correlates when derived from full-length proteins in their native lipid environment. Producing such samples has been a primary challenge in the field. Here, we present robust, step-by-step biochemical and biophysical protocols for generating monodisperse assemblies of full-length transmembrane proteins within lipidic environments. These protocols are particularly tailored for cases where the size and molecular weight of the proteins align closely with those of the lipid islands (nanodiscs). While designed for single-span bitopic membrane proteins, these protocols can be easily extended to proteins with multiple transmembrane domains. The insights presented have broad implications across diverse fields, including biophysics, structural biology, and cryogenic electron microscopy (cryo-EM) studies.

MreB is a prokaryotic actin homolog. It is essential for cell shape in the majority of rod-shaped cell-walled bacteria. Structural and functional characterization of MreB protein is important to understand the mechanism of ATP-dependent filament dynamics and membrane interaction. In vitro studies on MreBs have been limited due to the difficulty in purifying the homogenous monomeric protein. We have purified MreB from the cell-wall-less bacteria Spiroplasma citri, ScMreB5, using heterologous expression in Escherichia coli. This protocol provides a detailed description of purification condition optimization that led us to obtain high concentrations of stable ScMreB5. Additionally, we have provided a protocol for detecting the presence of monovalent ions in the ScMreB5 AMP-PNP-bound crystal structure. This protocol can be used to obtain a high yield of ScMreB5 for carrying out biochemical and reconstitution studies. The strategies used for ScMreB5 show how optimizing buffer components can enhance the yield and stability of purified protein.

Single-stranded RNA bacteriophages (ssRNA phages) infect their hosts by binding to the host receptor pili. Purification of pili usually involves mechanical shearing of pili from cells followed by precipitation. However, previous methods often result in low efficiency or unstable results due to pili retraction. This protocol presents an optimized method for purifying receptor type IV pili from Acinetobacter genomospecies 16 (A. gp16), incorporating enhancements in shearing and collection steps to achieve high yields. We found that repeated passage through syringe needles increases yield, and temperature control is crucial during purification. Additionally, the CsCl density gradient was optimized specifically for this specific strain. The purified type IV pili are suitable for cryogenic electron microscopy (cryo-EM) and various biochemical experiments.

Chromogranin B and other members of the granin protein family form condensates that recruit clients like proinsulin. The condensation in the lumen of trans-Golgi network (TGN) is critical for the biogenesis of secretory granules. Here, we describe a protocol to purify the tagged version of chromogranin B close to its native form at the TGN, which can then be utilized for microscopy-based assays to monitor condensate formation in vitro and client partitioning depending on the material properties of chromogranin B assemblies.

Progress in bioinformatics has facilitated the identification of a large number of putative glycosyltransferases (GTs) associated with many physiological processes. However, many of these GTs remain with unknown biochemical function due to numerous technical limitations. One of these limitations is the lack of innovative tools for large-scale screening of enzyme activity in vitro and testing protein–protein interactions (PPIs) between GT partners. Currently, testing the enzyme activity of a protein requires its production in a heterologous expression system and purification before enzyme assays, a process that is time-consuming and not amenable to high-throughput screening. To overcome this, we developed a platform called in vitro GT-array (i-GT-ray). In this platform, 96-well microplates are coated with plasmid DNA encoding for tagged GTs and a capture antibody. Tagged GTs are produced from plasmid DNA via a cell-free in vitro transcription/translation (IVTT) system and captured through the anti-tag capture antibody directly on microplates. After washing to remove IVTT components, the captured enzymes can be considered purified, and their activity can be tested directly on microplates. The whole process can be performed in less than two days, compared to several weeks for currently available screening methods. The i-GT-ray platform has also been adapted to investigate PPIs between GTs. Here, we provide a practical user guide for the preparation of GT-arrays coated with plasmid DNA and a capture antibody that can be used for monitoring enzyme activity and PPIs of GTs in a high-throughput manner.