Name: using in vitro fluorescence resonance energy transfer to study the dynamics of protein complexes at a millisecond time scale
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We thank Shu-Ou Shan (California Institute of Technology) for insightful discussion on the development of the FRET assay. M.G., Y.Z., and X.L. were funded by startup funds from Purdue University to Y.Z. and X.L.This work was supported in part by a seed grant from Purdue University Center for Plant Biology.

1. Design the FRET assay.
<ol>
	<li>Download the structure file of the Cul1&#8226;Cand1 complex from the Protein Data Bank (file 1U6G).</li>
	<li>View the structure of the Cul1&#8226;Cand1 complex in PyMOL.</li>
	<li>Use the Measurement function under the Wizard menu of PyMOL to estimate the distance between the first amino acid of Cand1 and the last amino acid of Cul1 (Figure 1).</li>
	<li>Load the online spectra viewer (see Table of Materials</st...

<ol>
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FRET is a physical phenomenon that is of great interest for studying and understanding biological systems19. Here, we present a protocol for testing and using FRET to study the binding kinetics of two interacting proteins. When designing FRET, we considered three major factors: the spectral overlap between donor emission and acceptor excitation, the distance between the two fluorophores, and the dipole orientation of the fluorophores28. To choose the fluorophores for FRET, ...

Biological activities are ultimately carried out by proteins, most of which interact with others for proper biological functions. Using a computational approach, the total amount of protein-protein interactions in human is estimated to be ~650,0001, and disruption of these interactions often leads to diseases2. Due to their essential roles in controlling cellular and organismal processes, numerous methods have been developed to study protein-protein interactions, such as yeast-two-hybrid, bimolecular fluorescence complementation, split-luciferase complementation, and co-immunoprecipitation assay3. While these methods are good at discovering and confirming protein-protein interactions, they are usually non-quantitative and thus provide limited information about the affinity between the interacting protein partners. Quantitative pull-downs can be used to measure the binding affinity (e.g., the dissociation constant Kd), but it does not measure the kinetics of the binding, nor can it be applied when the Kd is very low due to an inadequate signal-to-noise ratio4. Surface plasmon resonance (SPR) spectroscopy quantifies the binding kinetics, but it requires a specific surface and immobilization of one reactant on the surface, which can potentially change the binding property of the reactant5. Moreover, it is difficult for SPR to measure fast association and dissociation rates5, and it is not appropriate to use SPR to characterize the event of exchanging protein subunits in a protein complex. Here, we describe a method that allows measuring rates of protein complex assembly and disassembly at a millisecond time scale. This method was essential for determining the role of Cullin-associated-Nedd8-dissociated protein 1 (Cand1) as the F-box protein exchange factor6,7.
Cand1 regulates the dynamics of Skp1&#8226;Cul1&#8226;F-box protein (SCF) E3 ligases, which belong to the large family of Cullin-RING ubiquitin ligases. SCFs consist of the cullin Cul1, which binds the RING domain protein Rbx1, and an interchangeable F-box protein, which recruits substrates and binds Cul1 through the adaptor protein Skp18. As an E3 ligase, SCF catalyzes the conjugation of ubiquitin to its substrate, and it is activated when the substrate is recruited by the F-box protein, and when Cul1 is modified by the ubiquitin-like protein Nedd89. Cand1 binds unmodified Cul1, and upon binding, it disrupts both the association of Skp1&#8226;F-box protein with Cul1 and the conjugation of Nedd8 to Cul110,11,12,13. As a result, Cand1 appeared to be an inhibitor of SCF activity in vitro, but Cand1 deficiency in organisms caused defects that suggests a positive role of Cand1 in regulating SCF activities in vivo14,15,16,17. This paradox was finally explained by a quantitative study that revealed the dynamic interactions among Cul1, Cand1, and Skp1&#8226;F-box protein. Using fluorescence resonance energy transfer (FRET) assays that detect the formation of the SCF and Cul1&#8226;Cand1 complexes, the association and dissociation rate constants (kon and koff, respectively) were measured individually. The measurements revealed that both Cand1 and Skp1&#8226;F-box protein form extremely tight complex with Cul1, but the koff of SCF is dramatically increased by Cand1 and the koff of Cul1&#8226;Cand1 is dramatically increased by Skp1&#8226;F-box protein6,7. These results provide the initial and critical support for defining the role of Cand1 as a protein exchange factor, which catalyzes the formation of new SCF complexes through recycling Cul1 from the old SCF complexes.
Here, we present the procedure of developing and using the FRET assay to study the dynamics of the Cul1&#8226;Cand1 complex7, and the same principle can be applied to study the dynamics of various biomolecules. FRET occurs when a donor is excited with the appropriate wavelength, and an acceptor with excitation spectrum overlapping the donor emission spectrum is present within a distance of 10-100 &#197;. The excited state is transferred to the acceptor, thereby decreasing the donor intensity and increasing the acceptor intensity18. The efficiency of FRET (E) depends on both the F&#246;rster radius (R0) and the distance between the donor and acceptor fluorophores (r), and is defined by: E = R06/(R06 + r6). The F&#246;rster radius (R0) depends on a few factors, including the dipole angular orientation, the spectral overlap of the donor-acceptor pair, and the solution used19. To apply the FRET assay on a stopped-flow fluorimeter, which monitors the change of the donor emission in real-time and enables measurements of fast kon and koff, it is necessary to establish efficient FRET that results in a significant reduction of donor emission. Therefore, designing efficient FRET by choosing the appropriate pair of fluorescent dyes and sites on the target proteins to attach the dyes is important and will be discussed in this protocol.

<table>
	<tbody>
		<tr>
			<td>Anion exchange chromatography column</td>
			<td>GE Healthcare</td>
			<td>17505301</td>
			<td>HiTrap Q FF anion exchange chromatography column</td>
		</tr>
		<tr>
			<td>Benchtop refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>2231000511</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent Cells</td>
			<td>ThermoFisher Scientific</td>
			<td>C600003</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C78-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cation exchange chromatography column</td>
			<td>GE Healthcare</td>
			<td>17505401</td>
			<td>HiTrap SP Sepharose FF</td>
		</tr>
		<tr>
			<td>Desalting Column</td>
			<td>GE Healthcare</td>
			<td>17085101</td>
			<td></td>
		</tr>
		<tr>
			<td>Floor model centrifuge (high speed)</td>
			<td>Beckman Coulter</td>
			<td>J2-MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Floor model centrifuge (low speed)</td>
			<td>Beckman Coulter</td>
			<td>J6-MI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence SpectraViewer</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html</td>
		</tr>
		<tr>
			<td>FluoroMax fluorimeter</td>
			<td>HORIBA</td>
			<td>FluoroMax-3</td>
			<td></td>
		</tr>
		<tr>
			<td>FPLC</td>
			<td>GE Healthcare</td>
			<td>29018224</td>
			<td></td>
		</tr>
		<tr>
			<td>GGGGAMC peptide</td>
			<td>New England Peptide</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione beads</td>
			<td>GE Healthcare</td>
			<td>17075605</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-D-thiogalactoside (IPTG)</td>
			<td>Fisher Scientific</td>
			<td>15-529-019</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA agarose</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin</td>
			<td>MilliporeSigma</td>
			<td>A2512</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX-4T-2 vector</td>
			<td>GE Healthcare</td>
			<td>28954550</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>MilliporeSigma</td>
			<td>4693132001</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced glutathione</td>
			<td>Fisher Scientific</td>
			<td>BP25211</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated shaker</td>
			<td>Eppendorf</td>
			<td>M1282-0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosetta Competent Cells</td>
			<td>MilliporeSigma</td>
			<td>70953-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Size exclusion chromatography column</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td>Superdex 200 10/300 GL column</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Stopped-flow fluorimeter</td>
			<td>Hi-Tech Scientific</td>
			<td>SF-61 DX2</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP&#183;HCl</td>
			<td>Fisher Scientific</td>
			<td>PI20490</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>MilliporeSigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrafiltration membrane</td>
			<td>MilliporeSigma</td>
			<td>UFC903008</td>
			<td>Amicon Ultra-15 Centrifugal Filter Units, Ultra-15, 30,000 NMWL</td>
		</tr>
	</tbody>
</table>

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.

To test the FRET between Cul1AMC and FlAsHCand1, we first determined the emission intensity of 70 nM Cul1AMC (the donor) and 70 nM FlAsHCand1 (the acceptor), respectively (Figure 3A-C, blue lines). In each analysis, only one emission peak was present, and the emission of FlAsHCand1 (the acceptor) was low. When 70 nM each of Cul1AMC and FlAsHCand1 were mixed to genera...

Watch this Scientific Journal Video about Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale at JoVE.com

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

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 ...

Our method helps study the kinetics of a protein complex formation. It describes how tight a protein complex is, and if the protein complex formation is interfered by other proteins. This technique enables studying protein-protein interaction in quantitative way using fluorescence as a reporter, our assay can monitor the association and disassociation of a protein complex in real-time.To begin, design the FRET assay starting with the data for the COL1 CAND1 complex from the protein database. Load the structure in PyMOL, and then go to the wizard menu and use the measurement function to estimate the distance between the first amino acid of CAND1 and the last amino acid of COL1. Then, load the online spectra viewer and view the excitation and emission spectra of 7-amino-4-methylcoumarin and FlASH simultaneously.AMC is the FRET donor and FlASH is the FRET acceptor. Prepare cells with the FRET donor protein by following along in the accompanying text protocol. Then, purify the COL1 sortase RBX1 complex by first adding 50 milliliters of lysis buffer to a pellet of E.Coli cells expressing the COL1 sortase RBX1 complex.Ice the cells on ice with sonication at 50%amplitude. Alternate the power for three minutes so that it is one second on, and then one second off to prevent overheating the sample. Transfer the resulting cell lysate into a 50 milliliter centrifugation tube and remove the cell debris by centrifugation at 25, 000 times g for 45 minutes.Then, add the supernatant to five milliliters of glutathione beads and incubate them at four degrees Celsius to clear the cell lysate. Following incubation, centrifuge the bead lysate mixture at 1, 500 times g for two minutes at four degrees Celsius. Then, remove the supernatant.Next, wash the beads with 5 milliliters of lysis buffer having no protease inhibitor. After centrifuging the solution, remove the supernatant. Now, add three milliliters of lysis buffer to the washed beads and transfer the bead slurry into an empty column.To the new column, also add five milliliters of elution buffer and then incubate the column for 10 minutes and collect the eluate. Next, add 200 microliters of thrombin at five milligrams per milliliter to the eluate from the glutathione beads. Following an overnight incubation at four degrees Celsius dilute the protein sample three fold with buffer A.Load the protein sample to a buffer A equilibrated cationic exchange chromatography column at zero point five milliliters per minute.Then, add a gradient of zero to 50%of buffer B in buffer A to elute the protein with a flow rate of 1 milliliter per minute. Next, pool the eluate fractions containing the FRET donor protein and concentrate it down to two point five milliliters using an ultrafiltration membrane with a 30 kilodalton cutoff. Now, add 7-amino-4-methylcoumarin to the c-terminus of COL1 through sortase mediated transpeptidation by first changing the buffer in the FRET donor protein sample into the sortase buffer using a desalting column.To accomplish this, first equilibrate a desalting column with 25 milliliters of sortase buffer. Then, load two point five milliliters of the FRET donor protein solution into the column and discard the flow through. Next, elute the sample with three point five milliliters of sortase buffer and collect the flow through.In 900 microliters of the eluate, add 100 microliters of 600 micromolar purified sortase A solution and 10 microliters of a 25 millimolar GGGG-AMC peptide. Incubate the reaction mixture at 30 degrees Celsius in the dark overnight to generate COL1-AMC-RBX1. Now, load all of the sample onto the size exclusion chromatography column and elute it with one point five times the column volume of buffer.In the end, pool the eluate fractions containing COL1-AMC-RBX1. Follow along in the text protocol to prepare the FRET acceptor protein. Many of the steps are similar to the FRET donor protein preparation.Once finished, prepare the FlASH CAND1 solution. First, add one microliter of the FlASH solution to 50 microliters of the freshly prepared tetra-cys-CAND1 solution. Mix the combined solutions well and incubate the mixture at room temperature in the dark for one to two hours to form the FlASH CAND1 solution.In 300 microliters of FRET buffer, add COL1-AMC-RBX1 to a final concentration of 70 nanomolar and transfer the solution into a cuvette. Place the cuvette in the sample holder of a fluoriometer. Excite the sample with 350 nanometer excitation light and scan the emission signals from 400 to 600 nanometers at one nanometer increments.Then, in 300 microliters of FRET buffer, add both COL1-AMC-RBX1 and FlASH-CAND1 to a final concentration of 70 nanomolar. Excite the sample with 350 nanometer excitation light and scan the emission signals from 400-600 nanometers at one nanometer increments. Set the excitation light at 350 nanometers and use a band pass filter that allows 450 nanometers emission light to pass and blocks 500 to 650 nanometer emission light.With the sample valves at the fill position connect a three milliliter syringe filled with water. Then, wash the two sample syringes with water by moving the sample syringe drive up and down several times, discarding the water when finished. Next, connect a three milliliter syringe filled with FRET buffer.Wash the two sample syringes with the FRET buffer by moving the sample syringe drive up and down several times, discarding the water when finished. Now, connect a three milliliter syringe and load the first sample syringe, syringe A, with 100 nanomolar COL1-AMC-RBX1 in the FRET buffer. Then turn the sample valve to the drive position Also, connect a three milliliter syringe to syringe B and load syringe B with 100 nanomolar of FlASH CAND1 in the FRET buffer and turn its valve to the drive position.Open the control panel under acquire in the software and record the emission of COL1-AMC over the course of 60 seconds. Then, take a single shot. Fit the decrease and the fluorescent signals measured over time from each shot to a single exponential curve.Wash the system as previously shown and then add a solution of 100 nanomolar COL1-AMC-RBX1 and 100 nanomolar FlASH CAND1 in the FRET buffer into syringe A.Connect it to the system while it is in the fill position Load syringe A and then turn the sample valve to the drive position. Next, load a solution of Skp1-Skp2 into syringe B.Connect it to the system while it is in the fill position. Load syringe B and then turn the sample to the drive position.In the software, go to acquire and open the control panel. Set up the program to record the emission of COL1-AMC over 30 seconds. Then take a single shot.The fluorescence signals increase over time after mixing solutions from syringe A and syringe B.When 70 nanomolar each of COL1-AMC and FlASH CAND1 were mixed to generate FRET, two emission peaks were present in the emission spectra. And the peak of COL1-AMC became lower, and the peak of FlASH CAND1 became higher. When the full length CAND1 was used for FRET the donor peak showed a 10%reduction in intensity.However, when CAND1 with its first helix truncated was used the reduction of donor peak intensity was increased to 30%suggesting higher FRET efficiency To confirm that the signal changes were resulted from FRET between COL1-AMC and FlASH CAND1, the donor FRET was mixed with excess unlabelled CAND1 known as the Chase in the acceptor. As a result the donor peak was fully restored and the acceptor peak was decreased. Shown here is a plot of the average observed K value with the CAND1 concentration following linear regression analysis.To measure the on constant of the complex, 50 nanomolar COL1-AMC was used in a series of observed dissociation rate constants were measured by mixing 50 nanomolar COL1-AMC with increasing concentrations of FlASH CAND1. Similar to the measurement of the on constant, the observed dissociation constant of COL1 and CAND1 was found by monitoring the restoration of the donor signal over time on the stopped flow fluorometer. Here the donor signal increased quickly and it revealed an observed K value of zero point four per second.In contrast, when buffer was added to the pre assembled complex, no signal increase was observed suggesting the fast dissociation of COL1 CAND1 was triggered by Skp1 Skp2. Make sure to minimize the amount of light the donor and acceptor proteins are exposed to. Try to keep fluorophores in the dark as much as possible.

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Research

JoVE Journal

Biochemistry

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 ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &amp;#967;CRAC

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &amp;#945;-Synuclein Monomer at a Time

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. 
Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

F&amp;#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo F&#246;rster resonance energy transfer measurements, followed by data evaluation.

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...