Funding for this work has been provided by the National Science Foundation:
Improving Undergraduate STEM Education Grant (Award #1712268)
Research Coordination Networks in Undergraduate in Undergraduate Biology Education (Award # 1920270)
We are grateful to Karsten Theis, PhD, Westfield University, for helpful discussions about Jmol.

The authors declare that they have no relevant or material financial interests that relate to the research described in this paper.

This protocol outlines a ten-step process for the modeling of an enzyme active site, applied to four popular programs for biomolecular modeling. The critical steps of the protocol are: identifying the ligands in the active site, selecting residues within 5 &#197; to define an active site, and showing the interactions of the enzyme with the active site ligands. Distinguishing the ligands relevant to the biological function is paramount, as this allows the user to define the amino acid residues within 5 &#197; that can play a role in binding the ligands. Finally, using the program to display molecular interactions allows the user to develop the skills necessary to understand the molecular interactions that promote binding.
A limitation of computer-based molecular modeling protocols is the dependence on specific commands and syntax. While biochemical protocols may be tolerant of small changes in procedure, computer-based investigations may yield wildly different final products if the procedure is not closely adhered to. This is particularly important when using command-line interfaces where program-specific syntax is required to achieve a certain output, and a seemingly insignificant change in punctuation or capitalization can cause a command to fail. There are various Wikis and manuals for each program, where a user can find and troubleshoot command-line inputs; the user should pay careful attention to the details of command syntax. Although most molecular visualization programs include undo commands, due to the complexity of the interfaces, the undo command doesn&#39;t always faithfully reverse the last executed step. Therefore, saving the current working state often is encouraged, especially for new users.
Further limitations can arise from the data used to create the model itself. While the standards inherent in the Protein Data Bank ensure a certain level of consistency, users of molecular visualization programs will often encounter unexpected effects in a protein rendering. First, most structures are determined using X-ray crystallography, which provides a single model of the protein; however, NMR structures are often composed of multiple models that can be visualized one at a time. Second, structures determined from crystallography or cryogenic electron microscopy experiments may contain atoms whose position can&#39;t be elucidated and appear as gaps in certain representations of the protein. Protein structures may have alternate conformations of side chains, which, when displayed in stick rendering, appear as two groups protruding off of the same amino acid backbone. Even short sections of backbone may have such alternative conformations, and sometimes ligands are superimposed in the active site in more than one binding conformation.
For a crystal structure, the deposited 3D coordinates include all components of the asymmetric unit, which provides enough information to reproduce the repeating unit of a protein crystal. Sometimes, this structure will contain additional protein chains compared to the biologically active form of the protein (e.g., fetal hemoglobin mutant, PDB ID: 4MQK). Conversely, some programs may not automatically load all chains of the biologically active unit. For example, the SARS-CoV2 main protease (PDB ID: 6Y2E) loads half of the biologically active dimer (made up of two protein chains) when fetched using the commands described in this protocol in ChimeraX, PyMOL, and Jmol. Though slight modification of the command will load the biologically active dimer, this consideration may not be straightforward for the novice modeling program user. A different issue that can arise is in the identification of the active site or substrate itself. Crystallographic experiments are carried out using a variety of molecules, which may be modeled into the final structure. For example, sulfate molecules may bind phosphate binding sites in the active site, or they may bind other regions that are not relevant to the mechanism. These molecules may obscure the correct identification of the active site itself and may even suggest to the student that they are part of the mechanism.
Presumably, the user will wish to apply this procedure to other active/binding sites. To apply this protocol in the future work involving the analysis of new protein active sites, the user will need to identify which of the bound ligands are relevant to function. Some ligands are not associated with protein function, and instead are a result of the solvent or crystallization conditions used to conduct the experiment (e.g., the potassium ion present in the 3FGU model). The key ligands should be identified by consulting the original manuscript. With practice and, where applicable, an understanding of the line command syntax, a user will be able to apply the protocol for the desired modeling program to any enzyme active site, and to model other macromolecules of their choice.
Identifying and analyzing bound substrates and ligands is central to the elucidation of molecular mechanisms and structure-based drug design efforts, which have directly led to improvements in treatments for disease, including acquired immunodeficiency syndrome (AIDS) and COVID-1947,48,49,50,51,52. While individual molecular visualization programs offer different interfaces and user experiences, most offer comparable features. It is important for the development of biomolecular visualization literacy that upper level biochemistry students become familiar with structure visualization and the tools to generate such images4,20,53. This allows students to move beyond the interpretation of two-dimensional images in textbooks and journal articles and to more easily develop their own hypotheses from structural data54, which will prepare developing scientists to address future public health issues and improve the understanding of biochemical processes.
In summary, this protocol details active site modeling using four leading free macromolecular modeling programs. Our community, BioMolViz, adopts a non-software-specific approach to biomolecular modeling. We specifically avoided a critique or comparison of program features, although a user sampling each program will likely find that they prefer certain aspects of macromolecular modeling in one program versus another. We invite readers to utilize the BioMolViz Framework, which details the biomolecular visualization-based learning goals and objectives targeted in this protocol, and explore resources for teaching and learning biomolecular visualization through the BioMolViz community website at&#160;http://biomolviz.org.

Understanding representations of the molecular world is critical to becoming an expert in the biomolecular sciences1, because the interpretation of such images is key to understanding biological function2. A learner&#39;s introduction to macromolecules usually comes in the form of two-dimensional textbook images of cell membranes, organelles, macromolecules, etc. but the biological reality is that these are three-dimensional structures, and an understanding of their properties requires ways to visualize and extract meaning from 3D models.
Accordingly, the development of biomolecular visual literacy in upper division molecular life science courses has gained attention, with a number of articles reporting on the importance and difficulties of teaching and assessing visualization skills1,3,4,5,6,7,8,9. The response to these articles has been an increase in the number of classroom interventions, typically within a semester in a single institution, wherein molecular visualization programs and models are used to target difficult concepts2,10,11,12,13,14,15. Additionally, researchers have sought to characterize how students use biomolecular visualization programs and/or models to approach a specific topic16,17,18,19. Our own group, BioMolViz, has described a Framework that subdivides overarching themes in visual literacy into learning goals and objectives to guide such interventions20,21, and we lead workshops that train faculty to use the Framework in the backward design of assessments to measure visual literacy skills22.
At the center of all of this work is a critical skill: the ability to manipulate structures of macromolecules using programs for biomolecular visualization. These tools were developed independently using a variety of platforms; therefore, they can be rather unique in their operation and use. This necessitates program-specific instructions, and the identification of a program that a user is comfortable with is important to facilitate continued implementation.
Beyond the very basics of manipulating structures in 3D (rotating, selecting, and altering the model), a major goal is to model the active site of a protein. This process allows a learner to develop their understanding in three overarching themes described by the BioMolViz Framework: molecular interactions, ligands / modifications, and structure-function relationships20,21.
Four popular choices of programs for biomolecular visualization include: Jmol/JSmol23, iCn3D24, PyMOL25, and UCSF Chimera26,27. We encourage those new to Chimera to use UCSF ChimeraX, the next generation of the Chimera molecular visualization program, which is the currently supported version of the program.
In this protocol, we demonstrate how to use each of these four programs to model the active site of human glucokinase with a bound substrate analog complex (PDB ID: 3FGU), and to display measurements to illustrate specific binding interactions28. The model represents a catalytic complex of the enzyme. To capture the active site in the pre-catalysis state, a non-hydrolyzable analog of ATP was bound to the glucokinase active site. This phosphoaminophosphonic acid-adenylate ester (ANP) contains a phosphorous-nitrogen bond instead of the usual phosphorous-oxygen linkage at this position. The active site also contains glucose (denoted BCG in the model) and magnesium (denoted MG). Additionally, there is a potassium ion (K) in the structure, resulting from potassium chloride used in the crystallization solvent. This ion is not critical for biological function and is located outside of the active site.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63170/63170fig01.jpg" /> 
Figure 1: ATP/ANP structures. Adenosine triphosphate (ATP) structure compared to the phosphoaminophosphonic acid-adenylate ester (ANP). <a href="https://www.jove.com/files/ftp_upload/63170/63170fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The protocol demonstrates the selection of the bound ligands of the substrate analog complex and the identification of active-site residues within 5 &#197; of the bound complex, which captures amino acids and water molecules capable of making relevant molecular interactions, including hydrophobic and van der Waals interactions.
The display is initially manipulated to show the majority of the protein in a cartoon representation, with the active site amino acid residues in stick representation to show the relevant atoms of the protein and highlight the molecular interactions. After step 3 of the protocol for each program, these representations have been applied and the view of the protein is similar across programs (Figure 2). At the end of the protocol, the protein cartoon is hidden to simplify the view, and focus on the active site.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63170/63170fig02.jpg" /> 
Figure 2: Structure comparison across programs. Comparison of the structure of 3FGU in each program following the Adjust the Representation step (step 2 or 3 of each protocol). <a href="https://www.jove.com/files/ftp_upload/63170/63170fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
CPK coloring is applied to the active site amino acids and bound ligands29,30. This coloring scheme distinguishes atoms of different chemical elements in molecular models shown in line, stick, ball and stick, and space-filling representations. Hydrogen is white, nitrogen is blue, oxygen is red, sulfur is yellow, and phosphorus is orange in the CPK coloring scheme. Traditionally, black is used for carbon, although in modern use, carbon coloring may vary.
Hydrogen atoms are not visible in crystal structures, although each of these programs is capable of predicting their location. Adding the hydrogen atoms to a large macromolecular structure can obscure the view, thus they are not displayed in this protocol. Accordingly, hydrogen bonds will be shown by measuring from the center of two heteroatoms (e.g., oxygen to oxygen, oxygen to nitrogen) in these structures.
Program Overviews 
Downloadable Graphical User Interfaces (GUIs): PyMOL (Version 2.4.1), ChimeraX (Version 1.2.5), and Jmol (Version 1.8.0_301) are GUI-based molecular modeling tools. These three interfaces feature command lines to input typed code; many of the same capabilities are available through menus and buttons in the GUI. A common feature in the command line of these programs is that the user may load and re-execute previous commands using the up and down arrow keys on the keyboard.
Web-based GUIs: iCn3D (I-see-in-3D) is a WebGL-based viewer for interactive viewing of three-dimensional macromolecular structures and chemicals on the Web, without the need to install a separate application. It does not use a command line, though the full web version features an editable command log. JSmol is a JavaScript or HTML5 version of Jmol for use on a website or in a web browser window, and is very similar in operation to Jmol. JSmol can be used to create online tutorials, including animations.
Proteopedia31,32, FirstGlance in Jmol33, and the JSmol web interface (JUDE) at the Milwaukee School of Engineering Center for BioMolecular Modeling are examples of such Jmol-based online design environments34. The Proteopedia wiki is a teaching tool that allows the user to model a macromolecule structure and create pages featuring these models within the website35. The Proteopedia scene authoring tool, built using JSmol, integrates a GUI with additional features not available in the Jmol GUI.
Jmol and iCn3D are based on the Java programming language; JSmol uses either Java or HTML5, and PyMOL and ChimeraX are based on the Python programming language. Each of these programs loads protein data bank files, which can be downloaded from the RCSB Protein Data Bank under a 4-digit alphanumeric PDB ID36,37. The most common file types are Protein Data Bank (PDB) files containing the .pdb extension and Crystallographic Information File (CIF or mmCIF) containing the .cif extension. CIF has superseded PDB as the default file type for the Protein Data Bank, but both file formats function in these programs. There can be slight differences in the way the sequence/structure is displayed when using CIF as opposed to PDB files; however, the files function similarly and the differences will not be addressed in detail here. The Molecular Modeling Database (MMDB), a product of the National Center for Biotechnology Information (NCBI), is a subset of PDB structures to which categorical information has been associated (e.g., biological features, conserved protein domains)38. iCn3D, a product of the NCBI, is capable of loading PDB files containing the MMDB data.
To view a model, the user can download the desired file from the dedicated Protein Data Bank page for the structure (e.g.,&#160;https://www.rcsb.org/structure/3FGU), and then use the dropdown&#160;File menu of the program to open the structure. All of the programs are also capable of loading a structure file directly through the interface, and that method is detailed within the protocols.
The ChimeraX, Jmol, and PyMOL GUIs each contain one or more windows of the console that may be resized by dragging the corner. iCn3D and JSmol are entirely contained in a web browser. When using iCn3D, the user may need to scroll within the pop-up windows to reveal all menu items, depending on screen size and resolution.
The protocols detailed here provide a simple method to display the active site of the enzyme using each program. It should be noted that there are multiple ways to execute the steps in each program. For example, in ChimeraX, the same task may be executed using dropdown menus, the toolbar at the top, or the command line. Users interested in learning a specific program in detail are encouraged to explore the online tutorials, manuals, and Wikis available for these programs39,40,41,42,43,44,45,46.
Existing manuals and tutorials for these programs present the items in this protocol as discrete tasks. To display an active site, the user must synthesize the required operations from the various manuals and tutorials. This manuscript augments existing tutorials available by presenting a linear protocol for modeling a labeled active site with molecular interactions, providing the user with a logic for active site modeling that can be applied to other models and programs.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63170/63170fig03.jpg" /> 
Figure 3: ChimeraX GUI. ChimeraX GUI interface with the dropdown menus, toolbar, structure viewer, and command line labeled. <a href="https://www.jove.com/files/ftp_upload/63170/63170fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63170/63170fig04.jpg" /> 
Figure 4: iCn3D GUI. iCn3D GUI interface with the dropdown menus, toolbar, structure viewer, command log, select sets pop-up, and sequence and annotations pop-up menus labeled. <a href="https://www.jove.com/files/ftp_upload/63170/63170fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63170/63170fig05.jpg" /> 
Figure 5: Jmol GUI. Jmol GUI interface with the dropdown menus, toolbar, structure viewer, pop-up menu, and console/command line labeled. <a href="https://www.jove.com/files/ftp_upload/63170/63170fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63170/63170fig06.jpg" /> 
Figure 6: PyMOL GUI. PyMOL GUI interface with the dropdown menus, structure viewer, names/object panel, mouse controls menu, and command line labeled. <a href="https://www.jove.com/files/ftp_upload/63170/63170fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ChimeraX (Version 1.2.5) https://www.rbvi.ucsf.edu/chimerax/</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td>Any</td>
		</tr>
		<tr>
			<td>iCn3D (web-based only: https://www.ncbi.nlm.nih.gov/Structure/icn3d/full.html)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Java (for Jmol) https://java.com/en/download/</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jmol (Version 1.8.0_301) http://jmol.sourceforge.net/</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse (optional)</td>
			<td></td>
			<td></td>
			<td>Any</td>
		</tr>
		<tr>
			<td>PyMOL (Version 2.4.1 - educational): https://pymol.org/2 educational use only version: https://pymol.org/edu/?q=educational</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling an enzyme active site using molecular visualization freeware

Biomolecular visualization skills are paramount to understanding key concepts in the biological sciences, such as structure-function relationships and molecular interactions. Various programs allow a learner to manipulate 3D structures, and biomolecular modeling promotes active learning, builds computational skills, and bridges the gap between two dimensional textbook images and the three dimensions of life. A critical skill in this area is to model a protein active site, displaying parts of the macromolecule that can interact with a small molecule, or ligand, in a way that shows binding interactions. In this protocol, we describe this process using four freely available macromolecular modeling programs: iCn3D, Jmol/JSmol, PyMOL, and UCSF ChimeraX. This guide is intended for students seeking to learn the basics of a specific program, as well as instructors incorporating biomolecular modeling into their curriculum. The protocol enables the user to model an active site using a specific visualization program, or to sample several of the free programs available. The model chosen for this protocol is human glucokinase, an isoform of the enzyme hexokinase, which catalyzes the first step of glycolysis. The enzyme is bound to one of its substrates, as well as a non-reactive substrate analog, which allows the user to analyze interactions in the catalytic complex.

A successfully executed protocol for each of the programs will result in a molecular model zoomed in on the active site, with active site residues and ligands shown as sticks, the protein cartoon hidden, and ligands displayed with a contrasting color scheme. Interacting amino acid residues should be labeled with their identifiers, and hydrogen bonding and ionic interactions shown with lines. The presence of these features can be determined by visual inspection of the model.
To facilitate this inspection and enable the user to determine whether they have performed the steps of the protocol correctly, we&#39;ve provided animated figures that present an image of the structure following each step. For ChimeraX, iCn3D, Jmol, and PyMOL, this is illustrated in Figures 7-10, respectively.
Figure 7: ChimeraX protocol output. Animated figure illustrating steps 1.1-1.8 of the ChimeraX protocol. <a href="https://www.jove.com/files/ftp_upload/63170/Figure 7.mp4" target="_blank">Please click here to download this figure.</a>
Figure 8: iCn3D protocol output. Animated figure illustrating steps 2.1-2.8 of the iCn3D protocol. <a href="https://www.jove.com/files/ftp_upload/63170/Figure 8.mp4" target="_blank">Please click here to download this figure.</a>
Figure 9: Jmol protocol output. Animated figure illustrating steps 3.1-3.8 of the Jmol protocol. <a href="https://www.jove.com/files/ftp_upload/63170/Figure 9.mp4" target="_blank">Please click here to download this figure.</a>
Figure 10: PyMOL protocol output. Animated figure illustrating steps 4.1-4.8 of the PyMOL protocol. <a href="https://www.jove.com/files/ftp_upload/63170/Figure 10.mp4" target="_blank">Please click here to download this figure.</a>
The most common error that can influence the outcome of these protocols is erroneous selection, resulting in part of the structure being displayed in an undesired rendering. This is typically a result of mis-clicking, either on the structure itself, or in one of the display menu buttons. An example of a suboptimal result would be a model containing residues outside of the active site displayed as sticks. The user can begin to analyze if this error has occurred by visually inspecting the residues displayed as sticks and ensuring that they are in the proximity of the active site ligands. An advanced method to evaluate whether or not the displayed residues are within 5&#197; of the active site ligands is to use the measurement tools built into each program to measure the distance between a nearby ligand and the active site residue. The measurement tools are beyond the scope of this manuscript; however, we encourage interested users to explore the many online tutorials detailing this type of analysis.
We present a specific example of a sub-optimal execution of this protocol, resulting from a mis-click on the names/objects panel in the PyMOL. This error displays the entire protein as sticks, rather than showing only the active site using this representation, as illustrated in Figure 11.
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/63170/63170fig11.jpg" /> 
Figure 11: Negative result. Example of a negative result. Mis-selecting the full cartoon in PyMOL and displaying sticks. <a href="https://www.jove.com/files/ftp_upload/63170/63170fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To troubleshoot, the user will need to hide the sticks for the entire model (labeled 3FGU in the names/object panel), and then show the stick representation for only the selection named &#34;active,&#34; using the hide and show buttons/commands in PyMOL. Recovering the model from this type of error is relatively straightforward once the user is able to create appropriate selections for different parts of the model and display and hide them effectively. It is tempting to restart the protocol and work through the steps another time; however, we encourage the user to not be afraid to go &#34;off script&#34; and experiment with the model. In our experience, working through display errors facilitates progress in understanding the modeling program.
A side-by-side display of the final output from a successfully-executed protocol for each program is shown in Figure 12. The views are oriented similarly to allow the user to compare the appearance of the models created in different programs.
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/63170/63170fig12.jpg" /> 
Figure 12: Final structure comparison across programs. Comparison of the structure of each active site rendering at the end of the protocol. A: ChimeraX, B: iCn3D, C: Jmol, D: PyMOL. The PyMOL active site label includes all active site residues and the ligands. The other outputs have only hydrogen bonded side chains labeled. <a href="https://www.jove.com/files/ftp_upload/63170/63170fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Modeling an Enzyme Active Site using Molecular Visualization Freeware at JoVE.com

Modeling an Enzyme Active Site using Molecular Visualization Freeware

A key skill in biomolecular modeling is displaying and annotating active sites in proteins. This technique is demonstrated using four popular free programs for macromolecular visualization: iCn3D, Jmol, PyMOL, and UCSF ChimeraX.

Biomolecular visualization skills are paramount to understanding key concepts in the biological sciences, such as structure-function relationships and ...

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JoVE Journal

Biochemistry

9.4K Views.  The University of Texas at Austin. A key skill in biomolecular modeling is displaying and annotating active sites in proteins. This technique is demonstrated using four popular free programs for macromolecular visualization: iCn3D, Jmol, PyMOL, and UCSF ChimeraX.

Video: Modeling an Enzyme Active Site using Molecular Visualization Freeware

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...