This work was partially funded by FAI-UASLP grant number C19-FAI-05-89.89 and CONACYT grant number 316463 (Apoyos a la Ciencia de Frontera: Fortalecimiento y Mantenimiento de Infraestructuras de Investigaci&#243;n de Uso Com&#250;n y Capacitaci&#243;n T&#233;cnica 2021). The authors thank the technical assistance of Julian E. Mata-Morales in video edition.

1. Determination (in silico) of the ANS and SERCA N-domain interaction
<ol>
	<li>Generate a three-dimensional (3D) structure of the protein (SERCA N-domain) by molecular modeling using the preferred protein modeling software50.</li>
	<li>Identify the amino acid residues that form the nucleotide-binding site using the preferred molecular structure software51, and determine the presence of Arg and Lys residues35; these are required for ANS binding and to increase the fluorescence intensity (quantum yield).</li>
	<li>Perform molecular docking (using the preferred docking software)52,53,54 to determine the interactions of ATP, fluorescein isothiocyanate (FITC) (which forms a covalent bond with Lys515 labeling the nucleotide-binding site), and ANS with amino acids residues in the nucleotide-binding site (Figure 1).</li>
	<li>Calculate the molecular distance (&#197;) between Trp residue and bound ANS using the measurement tool in the preferred software.</li>
	<li>Perform molecular dynamics simulation of ANS-N-domain complex to determine the stability of the interaction52,54. Then, perform the in vitro experiments when the stability of the complex has been confirmed.</li>
</ol>
2. Expression and purification of the recombinant N-domain
<ol>
	<li>Synthesize the gene coding for N-domain40.</li>
	<li>Design and construct the plasmid that contains the synthetic gene that codes for the N-domain40.</li>
	<li>Express and purify by affinity chromatography (Ni-NTA), the engineered recombinant N-domain. Perform an SDS-PAGE of the purified protein to determine the purity (Figure 2)40.</li>
	<li>Determine the protein concentration by studying the absorbance at &#955; of 280 nm with the N-domain extinction coefficient (&#949;= 11,960 M-1&#183;cm-1)40.</li>
</ol>
3. Monitor the formation of the ANS-N-domain complex based on ANS and N-domain fluorescence intensity changes.
<ol>
	<li>Prepare an ANS stock solution in N,N-dimethylformamide.
	<ol>
		<li>Weigh a small amount (1-5 mg) of ANS, and dissolve it in 1 mL of the final volume of N,N-dimethylformamide, e. g., 3.2 mg (10.69 mM final concentration).</li>
		<li>Prepare a 100 &#181;M ANS aqueous stock solution using the ANS solution in N,N-dimethylformamide, e. g., add 9.4 &#181;L of the 10.69 mM ANS solution to 990.6 &#181;L of 50 mM phosphate buffer with pH 8.0 to obtain a final volume of 1 mL.</li>
		<li>Mix the solutions by vortexing 3 - 5 times for 15 s. 
		NOTE: In the following experiment, use only the ANS aqueous stock solution. Freshly prepare the ANS aqueous stock solution before initiating the experiments.</li>
	</ol>
	</li>
	<li>Prepare the NBS stock solution in N,N-dimethylformamide.
	<ol>
		<li>Weigh a small amount (1-5 mg) of NBS, and dissolve it in 1 mL of N,N-dimethylformamide, e. g., 5.3 mg in 1 mL (29.78 mM final concentration).</li>
		<li>Prepare a 1 mM NBS aqueous stock solution using the NBS solution in N,N-dimethylformamide, e. g., add 3.36 &#181;L of the 29.78 mM NBS solution to 96.64 &#181;L of 50 mM phosphate buffer with pH 8.0 to obtain a final volume 0.1 mL.</li>
		<li>Mix the solutions by vortexing 3 - 5 times for 15 s. 
		NOTE: Freshly prepare the NBS aqueous stock solution before starting the experiments.</li>
	</ol>
	</li>
	<li>Titrate the N-domain with ANS, and record the fluorescence spectra by excitation at &#955;=295 nm at 25 &#176;C.
	<ol>
		<li>Obtain the fluorescence spectrum baseline.
		<ol>
			<li>Place 1 mL of 50 mM phosphate buffer with pH 8.0 in a 1 mL fluorescence quartz cuvette.</li>
			<li>Position the cell in the thermo-stated cell chamber (25 &#176;C) of the spectrofluorometer and set the excitation &#955; to 295 nm.</li>
			<li>Record the fluorescence spectrum (305 - 550 nm). 
			NOTE: The fluorescence spectrum of the 50 mM phosphate buffer with pH 8.0, which serves as the blank sample, is subtracted from all obtained fluorescence spectra.</li>
		</ol>
		</li>
		<li>Obtain the intrinsic fluorescence spectrum of the N-domain.
		<ol>
			<li>Place 900 &#181;L of 50 mM phosphate buffer with pH 8.0 in a fluorescence quartz cuvette.</li>
			<li>Add 100 &#181;L of N-domain (10 &#956;M) suspension to obtain a 1 &#956;M N-domain final concentration in a 1 mL final volume.</li>
			<li>Gently homogenize using a micropipette 20 times to ensure the homogeneity of the solution. 
			NOTE: The protein should be freshly purified to obtain high-quality intrinsic fluorescence spectra, e. g., the purified recombinant N-domain may only be used for a week after purification.</li>
			<li>Position the cell in the thermo-stated cell chamber (25 &#176;C) of the spectrofluorometer and set the excitation &#955; to 295 nm.</li>
			<li>Record the N-domain intrinsic fluorescence spectrum (305-550 nm).</li>
		</ol>
		</li>
		<li>Add ANS, and obtain the fluorescence spectrum by excitation at &#955;=295 nm.
		<ol>
			<li>Add a 2 &#181;L aliquot of 100 &#181;M ANS aqueous stock solution to the suspended N-domain (1 &#956;M) to obtain a 0.2 &#181;M ANS final concentration.</li>
			<li>Gently homogenize using a micropipette 20 times to ensure the homogeneity of the solution.</li>
			<li>Position the cell in the thermo-stable cell chamber (25 &#176;C) of the spectrofluorometer and set the excitation &#955; to 295 nm.</li>
			<li>Record the fluorescence spectrum (305-550 nm).</li>
			<li>Repeat the ANS additions and fluorescence spectra recording above 1:1 molar relationship ANS:N-domain.</li>
			<li>Subtract the blank spectrum from each spectrum using suitable software.</li>
			<li>Plot all the spectra in a single graph.</li>
			<li>Determine whether the spectra form a FRET-like pattern. The ANS-N-domain fluorescence spectra form a FRET-like pattern (Figure 3A).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. N-domain intrinsic fluorescence titration by Trp chemical modification with NBS.
<ol>
	<li>Repeat steps 3.3.1 and 3.3.2.</li>
	<li>Add a 1 &#181;L aliquot of 1 mM NBS aqueous stock solution to the suspended N-domain (1 &#956;M) to obtain a final concentration of 1 &#181;M NBS.</li>
	<li>Gently homogenize by using a micropipette 20 times to ensure the homogeneity of the solution.</li>
	<li>Position the cell in the thermo-stable cell chamber (25 &#176;C) of the spectrofluorometer and set the excitation &#955; to 295 nm.</li>
	<li>Record the fluorescence spectrum (305-550 nm) (Figure 3B).</li>
	<li>Repeat the NBS addition and fluorescence spectra recording until minimal N-domain intrinsic fluorescence quenching is observed40. In the N-domain, this usually occurs at a molar ratio of 5-6 NBS/N-domain40. 
	NOTE: NBS rapidly quenches (&#60;5 s) the intrinsic fluorescence of the N-domain; a decrease in fluorescence intensity is observed. Proceed immediately to the next step, as NBS may also react with other amino acid residues47.</li>
	<li>Subtract the blank spectrum from each spectrum using suitable software.</li>
	<li>Plot all spectra in a single graph (Figure 3B).</li>
</ol>
5. Titrate the NBS-modified N-domain with ANS by recording fluorescence spectra at 25 &#176;C.
<ol>
	<li>Perform Step 3.3.3 using the NBS modified N-domain that was generated in Step 4.</li>
	<li>Subtract the blank spectrum from each spectrum using suitable software.</li>
	<li>Plot all spectra in a single graph (Figure 3C).</li>
	<li>The generated fluorescence spectra (Figure 3C) support or refute the occurrence of FRET, i.e., when FRET occurs, the ANS fluorescence does not increase and vice-versa.</li>
</ol>
6. Evidence of ANS binding to the chemically modified N-domain by excitation at &#955;=370 nm.
<ol>
	<li>Perform Step 3.3.3 using the NBS modified N-domain that was generated in Step 4 but changing the excitation &#955; to 370 nm.</li>
	<li>Subtract the blank spectrum from each spectrum using suitable software.</li>
	<li>Plot all spectra in a single graph (Figure 3D).</li>
	<li>Confirm ANS binding to the N-domain by observing the increase in ANS fluorescence intensity. ANS binding to the N-domain shows a fluorescence increase when excited at &#955;=370 nm (Figure 3D). As a control, the fluorescence spectrum of ANS (alone) in 50 mM phosphate buffer with pH 8.0 was obtained exciting at &#955; of 295 and 370 nm (Figure 4, not shown in video). 
	NOTE: The stoichiometric relationship of NBS:Trp that is required for chemical modification depends on the degree of burying of the Trp residue(s) in the protein under study46,47,55,56. Therefore, it is recommended to determine the NBS:protein/(Trp) molar ratio, beforehand.</li>
</ol>

The authors declare that they have no competing financial interests.

Fluorescence spectra of the ANS-N-domain complex display a FRET-like pattern when excited at a &#955; of 295 nm, while the molecular distance (20 &#197;) between the Trp residue and ANS seems to support the occurrence of FRET (Figure 1). Trp chemical modification by NBS results in a less fluorescent N-domain (Figure 3B, Spectrum f); hence, energy transfer is not possible. The ANS fluorescence spectra are similar to that of the nonmodified N-domain when excited a...

F&#246;ster resonance energy transfer (FRET) has become a standard technique for determining the distance between molecular structures after binding or interaction in protein structure and function studies1,2,3,4. In P-type ATPases, FRET has been used to investigate the structure and function of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)2,5,6,7,8, e. g., structural fluctuations during the catalytic cycle have been analyzed in the whole protein by FRET7.
FRET donors are diverse, and range from small fluorescent (extrinsic) molecules to fluorescent proteins9,10. Tryptophan (Trp) residues (due to their fluorescence) are useful for identifying structural changes in protein amino acid sequences11,12. The fluorescence intensity of Trp depends substantially on the polarity of its surrounding environment13,14. Ligand binding usually generates structural rearrangements in proteins/enzymes15,16. If Trp is present at or located close to the protein binding site, structural fluctuations frequently affect the degree of Trp exposure to aqueous media13,14; thus, the change in polarity results in quenching of the Trp fluorescence intensity13,14. Hence, the fluorescent property of Trp is useful for performing ligand binding studies for enzymes. Other physical phenomena may also lead to Trp fluorescence quenching17,18,19,20, e. g., FRET and changes in medium polarity. Energy transfer from the excited state of Trp to a fluorophore also has potential applications, e. g., affinity determination of small ligands in proteins21. Indeed, Trp has been primarily used as a fluorescence donor in FRET studies in proteins22,23,24, e. g., in terbium (Tb3+) FRET studies, a Trp residue is used frequently as an antenna for energy transfer to Tb3+ 25,26,27. Trp displays various advantages over other FRET donors due to its inherent constitutive character in the protein structure, which eliminates the need for preparative processes that may affect the function/structure of the studied protein24. Thus, the identification of radiative decays (energy transfer and changes in the medium polarity that are induced by protein structural rearrangements) is important for drawing accurate conclusions regarding ligand binding in protein structural studies13,14,19,28.
In protein structural studies, an extrinsic fluorophore, namely, 8-anilino-1-naphthalene sulfonate (ANS), has been primarily used in experiments related to protein folding/unfolding28,29. ANS binds to proteins/enzymes in the native state, usually in the binding sites of substrates31,32,33; an increase in ANS fluorescence quantum yield (&#934;F) (namely, an increase in fluorescence intensity) is induced by exciting the protein at &#955;=370 nm when suitable interactions of ANS with Arg and His residues in hydrophobic pockets occur34,35,36,37. In various studies, the occurrence of FRET (when exciting at &#955; within 280-295 nm) between Trp residues (donors) and ANS (acceptor) has been reported, which is based on the following: 1) overlap of the fluorescence emission spectrum of Trp and excitation spectrum of ANS, 2) identification of a suitable distance between one or more Trp residue(s) and ANS for energy transfer, 3) high ANS quantum yield when bound in protein pockets, and 4) characteristic FRET pattern in the fluorescence spectra of the protein in the presence of ANS3,17,27,37,38.
Recently, ligand binding to the nucleotide-binding domain (N-domain) in SERCA and other P-type ATPases have been investigated using engineered recombinant N-domains40,41,42,43,44,45,46. Molecular engineering of the SERCA N-domain has been used to move the sole Trp residue (Trp552Leu) to a more dynamic structure (Tyr587Trp) that is close to the nucleotide-binding site, where fluorescence variations (quenching) may be used to monitor structural changes upon ligand binding34. Experimental results have demonstrated that ANS binds (as ATP) to the nucleotide-binding site in the purified recombinant SERCA N-domain34. Interestingly, the ANS fluorescence increases upon binding to the N-domain upon excitation at a &#955; of 295 nm, while the intrinsic fluorescence of the N-domain decreases34, thereby producing a FRET pattern that suggests the formation of a Trp-ANS FRET pair.
The use of NBS has been proposed to determine the content of Trp residues in proteins47 by absorbance assay of modified proteins. NBS modifies the highly absorbing indole group of Trp to the less absorbent oxindole47,48. This results in the loss (quenching) of the Trp fluorescent property40. Hence, NBS-mediated chemical modification of Trp residues may be used as an assay to define the role of Trp (as a donor) when FRET is hypothesized.
This protocol describes the chemical modification of the sole Trp residue by NBS in the engineered recombinant N-domain of SERCA as a protein model. Experimental results demonstrate that the ANS fluorescence intensity still increases in the chemically NBS-modified N-domain34, which lacks intrinsic fluorescence. Therefore, the assay is useful for demonstrating the absence of FRET between the Trp residue and ANS when bound to the N-domain34,40,49. Hence, this assay (NBS chemical modification of Trp) is useful in proving the presence of the Trp-ANS FRET pair in proteins.

<table><tbody><tr><td>Acrylamide</td><td>Bio-Rad</td><td>1610107</td><td>SDS-PAGE</td></tr><tr><td>Ammonium persulfate</td><td>Bio-Rad</td><td>1610700</td><td>SDS-PAGE</td></tr><tr><td>8-Anilino-1-naphthalenesulfonic acid</td><td>Sigma-Aldrich</td><td>A1028</td><td>Fluorophore</td></tr><tr><td>Bis-acrylamide</td><td>Bio-Rad</td><td>1610125</td><td>SDS-PAGE</td></tr><tr><td>N-Bromosuccinimide</td><td>Sigma-Aldrich</td><td>B81255</td><td>Chemical modification</td></tr><tr><td>N,N-dimethylformamide</td><td>J.T. Baker</td><td>9213-12</td><td>Stock solution preparation</td></tr><tr><td>Fluorescein isothiocyanate</td><td>Sigma-Aldrich</td><td>F7250</td><td>Chemical fluorescence label</td></tr><tr><td>Fluorescence cuvette</td><td>Hellma</td><td>Z801291</td><td>Fluorescence assay</td></tr><tr><td>Fluorescence Spectrofluorometer</td><td>Shimadzu</td><td>RF 5301PC</td><td>Fluorescence assay</td></tr><tr><td>HisTrap&amp;trade; FF</td><td>GE Healtcare</td><td>11-0004-59</td><td>Protein purification</td></tr><tr><td>IPTG, Dioxane free</td><td>American Bionalytical</td><td>AB00841-00010</td><td>Protein expression</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I5513-25G</td><td>Protein purification</td></tr><tr><td>LB media</td><td>Fisher Scientific</td><td>10000713</td><td>Cell culture</td></tr><tr><td>Pipetman L P10L</td><td>Gilson</td><td>FA10002M</td><td>Fluorescence assay</td></tr><tr><td>Pipetman L P100L</td><td>Gilson</td><td>FA10004M</td><td>Fluorescence assay</td></tr><tr><td>Pipetman L P200L</td><td>Gilson</td><td>FA10005M</td><td>Fluorescence assay</td></tr><tr><td>Pipetman L P1000L</td><td>Gilson</td><td>FA10006M</td><td>Fluorescence assay</td></tr><tr><td>Pipetman L P5000L</td><td>Gilson</td><td>FA10007</td><td>Fluorescence assay</td></tr><tr><td>Precision plus std</td><td>Bio-Rad</td><td>1610374</td><td>SDS-PAGE</td></tr><tr><td>Sodium dodecyl sulphate</td><td>Bio-Rad</td><td>1610302</td><td>SDS-PAGE</td></tr><tr><td>Sodium phosphate dibasic</td><td>J.T. Baker</td><td>3828-19</td><td>Buffer preparation</td></tr><tr><td>Sodium phosphate monobasic</td><td>J.T. Baker</td><td>3818-01</td><td>Buffer preparation</td></tr><tr><td>Syringe filter 0.2 um</td><td>Millipore</td><td>GVWP04700</td><td>Solution filtration</td></tr><tr><td>Temed</td><td>Bio-Rad</td><td>1610801</td><td>SDS-PAGE</td></tr><tr><td>Tris</td><td>Bio-Rad</td><td>1610719</td><td>SDS-PAGE</td></tr></tbody></table>

chemical modification of the tryptophan residue in a recombinant ca2+-atpase n-domain for studying tryptophan-ans fret

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that has been crystallized in various conformations. Detailed functional information may nonetheless be obtained from isolated recombinant domains. The engineered (Trp552Leu and Tyr587Trp) recombinant nucleotide-binding domain (N-domain) displays fluorescence quenching upon ligand binding. An extrinsic fluorophore, namely, 8-anilino-1-naphthalene sulfonate (ANS), binds to the nucleotide-binding site via electrostatic and hydrophobic interactions with Arg, His, Ala, Leu, and Phe residues. ANS binding is evidenced by the increase in fluorescence intensity when excited at a wavelength (&#955;) of 370 nm. However, when excited at &#955; of 295 nm, the increase in fluorescence intensity seems to be coupled to the quenching of the N-domain intrinsic fluorescence. Fluorescence spectra display a F&#246;ster resonance energy transfer (FRET)-like pattern, thereby suggesting the presence of a Trp-ANS FRET pair, which appears to be supported by the short distance (~20 &#197;) between Tyr587Trp and ANS. This study describes an analysis of the Trp-ANS FRET pair by Trp chemical modification (and fluorescence quenching) that is mediated by N-bromosuccinimide (NBS). In the chemically modified N-domain, ANS fluorescence increased when excited at a &#955; of 295 nm, similar to when excited at a &#955; of 370 nm. Hence, the NBS-mediated chemical modification of the Trp residue can be used to probe the absence of FRET between Trp and ANS. In the absence of Trp fluorescence, one should not observe an increase in ANS fluorescence. The chemical modification of Trp residues in proteins by NBS may be useful for examining FRET between Trp residues that are close to the bound ANS. This assay will likely also be useful when using other fluorophores.

Molecular docking shows the binding of ANS to the nucleotide-binding site of the N-domain via electrostatic as well as hydrophobic interactions (Figure 1). Molecular distance (20 &#197;) between the Trp residue and ANS (bound to the nucleotide-binding site) supports the occurrence of FRET (Figure 1). The designed (engineered) recombinant N-domain was obtained at high purity by affinity chromatography (Figure 2) and was suitable for ...

Watch this Scientific Journal Video about Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET at JoVE.com

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

ANS binds to the Ca2+-ATPase recombinant N-domain. Fluorescence spectra display a FRET-like pattern upon excitation at a wavelength of 295 nm. NBS-mediated chemical modification of Trp quenches the fluorescence of the N-domain, which leads to the absence of energy transfer (FRET) between the Trp residue and ANS.

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that has been crystallized in various conformations. Detailed functional ...

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3.4K Views.  Universidad Autónoma de San Luis Potosí. Deze testen kunnen helpen om de rol van tryptofaanresiduen in eiwitten te verduidelijken bij het uitvoeren van ANS-bindende studies. De ANS-fluorescentie neemt toe bij binding aan specifieke structurele locaties in eiwitten. Soms lokaliseert ANS-bindingsplaats dicht bij een tryptofaanresidu met een afstand die het bestaan van FRET tussen het tryptofaan en ANS suggereert.NBS bemiddelt chemische modificatie van tryptofaanresiduen in eiwitten, waardoor de fluorescentie wordt geblust. Daar...

Video: Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Chemistry

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

F&#246;rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between ...

Monitoring GTPase Atlastin in Different Nucleotide Loading States

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver of homotypic membrane fusion within the endoplasmic reticulum (ER). Disruptions in this process have been associated with hereditary spastic paraplegia (HSP), a neurodegenerative disorder. Structural and biochemical studies suggest that the conformational changes in ATL are linked to GTP hydrolysis, but real-time visualization of these conformational dynamics during the GTP hydrolysis cycle remains challenging. To better understand the mechanical mechanisms behind ATL function, single-molecule F&#246;rster resonance energy transfer (smFRET) was utilized. Three specific strategies were employed to immobilize the N-terminal cytosolic region of human ATL1 (ATL1cyto) in a streptavidin-coated microfluidic chamber, facilitating the application of intramolecular and intermolecular smFRET imaging. This allowed precise monitoring of protein conformations in various nucleotide-loading states, providing direct insights into individual molecular behaviors. This method can be applied to study other mechanochemical proteins as well.

The functions of dynamin superfamily proteins depend on conformational changes coupled with GTP hydrolysis. A system is described using the single-molecule FRET (smFRET) technique to monitor the conformational dynamics of dynamin-like GTPase atlastin in different nucleotide-loading states.

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Biology

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

To measure [Ca2+] quantitatively, fura-2 analogs, which are ratiometric fluoroprobes, are frequently used. However, dye usage is intrinsically limited in live cells because of autofluorescence interference, mainly from nicotinamide adenine dinucleotide (NADH). More specifically, this is a major obstacle when measuring the mitochondrial [Ca2+] quantitatively using fura-2 analogs because the majority of NADH is in the mitochondria. If the fluorescent dye concentration is the same, a certain excitation intensity should produce the same emission intensity. Therefore, the emission intensity ratio of two different excitation wavelengths should be constant. Based on this principle, a novel online correction method of NADH signal interference to measure [Ca2+] was developed, and the real signal intensity of NADH and fura-2 can be obtained. Further, a novel equation to calculate [Ca2+] was developed with isosbestic excitation or excitation at 400 nm. With this method, changes in mitochondrial [Ca2+] could be successfully measured. In addition, with a different set of the excitation and emission wavelengths, multiple parameters, including NADH, [Ca2+], and pH or mitochondrial membrane potential (&#936;m), could be simultaneously measured. Mitochondrial [Ca2+] and &#936;m or pH were measured using fura-2-FF and tetramethylrhodamine ethyl ester (TMRE) or carboxy-seminaphtorhodafluor-1 (carboxy-SNARF-1).

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

Due to the spectral overlapping of the excitation and emission wavelengths of NADH and fura-2 analogs, the signal interference from both chemicals in live cells is unavoidable during quantitative measurement of [Ca2+]. Thus, a novel online correction method of NADH signal interference to measure [Ca2+] was developed.

To measure [Ca2+] quantitatively, fura-2 analogs, which are ratiometric fluoroprobes, are frequently used. However, dye usage is intrinsically limited in ...

Chemical Modification of the Tryptophan Residue in a Recombinant Ca²⁺-ATPase N-domain for Studying Tryptophan-ANS FRET

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