Name: high-resolution neutron spectroscopy to study picosecond-nanosecond dynamics of proteins and hydration water
Uploaded: 2022-04-28T13:10:00
Description: Watch this Scientific Journal Video about High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water at JoVE.com

The authors are grateful to Michaela Zamponi at the J&#252;lich Centre for Neutron Science at the Heinz Maier-Leibnitz Zentrum, Garching, Germany, for part of the neutron scattering experiments conducted on the instrument SPHERES. This work has benefited from the activities of the Deuteration Laboratory (DLAB) consortium funded by the European Union under Contracts HPRI-2001-50065 and RII3-CT-2003-505925, and from UK Engineering and Physical Sciences Research Council (EPSRC)-funded activity within the Institut Laue Langevin EMBL DLAB under Grants GR/R99393/01 and EP/C015452/1. Support by the European Commission under the 7th Framework Programme through the Key Action: Strengthening the European Research Area, Research Infrastructures is acknowledged [Contract 226507 (NMI3)]. Kevin Pounot and Christian Beck thank the Federal Ministry of Education and Research (BMBF, grant number 05K19VTB) for funding of their postdoctoral fellowships.

1. Prepare the deuterated buffer for proteins in the liquid state
<ol>
	<li>Dissolve all components of the buffer in pure D2O.</li>
	<li>If the pH electrode was calibrated in H2O, adjust the pD according to the formula pD = pH + 0.4 using NaOD or DCl34. 
	NOTE: The use of D2O instead of H2O might affect protein solubility and the buffer conditions might need to be adapted, (e.g., by a slight change in salt concentration).</li>
</o...

Neutron spectroscopy is the only method that allows probing the ensemble-averaged ps-ns dynamics of protein samples regardless of the size of the protein or the complexity of the solution when deuteration is used6. Specifically, by probing self-diffusion of protein assemblies in solution, the hydrodynamic size of such assemblies can be unambiguously determined. Nonetheless, the method is commonly limited by the low neutron flux, which implies long acquisition times and the requirement of high amou...

The neutron is a charge-less and massive particle that has been successfully used over the years to probe samples in various fields from fundamental physics to biology1. For biological applications, small-angle neutron scattering, inelastic neutron scattering, and neutron crystallography and reflectometry are extensively used2,3,4. Inelastic neutron scattering provides an ensemble-averaged measurement of the dynamics without requiring specific labeling per se, and a signal quality that does not depend on the size or the protein5.&#160;The measurement can be done using a highly complex environment for the protein under study that mimics the intracellular medium, such as a deuterated bacterial lysate or even in vivo3,6,7. Different experimental setups can be used to study the dynamics, namely i) time-of-flight-giving access to sub-ps-ps dynamics, ii) backscattering-giving access to ps-ns dynamics, and iii) spin-echo-giving access to dynamics from ns to hundreds of ns. Neutron backscattering makes use of the Bragg&#39;s law 2d sin&#952; = n&#955;, where d is the distance between planes in a crystal,&#160;&#952; the scattering angle, n the scattering order, and&#160;&#955; the wavelength. The use of crystals for backscattering toward the detectors allows for achieving a high resolution in energy, typically ~0.8 &#181;eV. To measure the energy exchange, either a Doppler drive carrying a crystal in backscattering is used to define and tune the incoming neutron wavelength8,9,10 (Figure 1), or a time-of-flight setup can be used at the cost of a decrease in energy resolution11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63664/63664fig01.jpg" /> 
Figure 1: Sketch of a neutron backscattering spectrometer with a Doppler drive. The incoming beam hits the phase space transformation (PST) chopper42, which increases the flux at the sample position. It is then backscattered towards the sample by the Doppler drive, which selects an energy E1 (cyan arrow). The neutrons are then scattered by the sample (with different energies represented by the color of the arrows) and the analyzers, made of Si 111 crystals, will only backscatter neutrons with a specific energy E0 (red colored arrows here). Hence, the momentum transfer q is obtained from the detected position of the neutron on the detector array, and the energy transfer is obtained from the difference E1- E0. The time-of-flight expected for the neutron pulse produced by the PST is used to discard the signal from the neutrons scattered directly toward the detector tubes. Abbreviation: PST = phase space transformation. <a href="https://www.jove.com/files/ftp_upload/63664/63664fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For backscattering spectroscopy, the main contribution to the signal from hydrogen proton-rich samples, such as proteins, comes from incoherent scattering, for which the scattering intensity Sinc(q, &#969;) is shown by Eq (1)12
<img alt="Equation 1" src="/files/ftp_upload/63664/63664eq01.jpg" style="margin: auto;" /> (1)
Where &#963;inc is the incoherent cross-section of the element considered, k&#39; is the norm of the scattered wavevector, k the norm of the incoming wavevector, q (= k - k&#39;) the momentum transfer, rj(t) the position vector of atom j at time t, and&#160;&#969; the frequency corresponding to the energy transfer between the incoming neutron and the system. The angular brackets denote the ensemble average. Hence, incoherent scattering probes the ensemble-averaged single-particle self-correlation of atom positions with time and gives the self-dynamics averaged over all atoms in the system and different time origins (ensemble average). The scattering function is the Fourier transform in time of the intermediate scattering function I(q, t), which can be viewed as the Fourier transform in space of the van Hove correlation function shown by Eq (2):
<img alt="Equation 2" src="/files/ftp_upload/63664/63664eq02.jpg" style="margin: auto;" /> (2)
Where &#961;(r,t) is the probability density of finding an atom at position r and time t13.
For a Fickian diffusion process, the self-diffusion function results (see Eq (3)) after a double Fourier transform in a scattering function consisting in a Lorentzian of line width given by &#947; = Dq2.
<img alt="Equation 10" src="/files/ftp_upload/63664/63664eq10.jpg" style="margin: auto;" /> (3)
More sophisticated models were developed and found useful such as the jump diffusion model by Singwi and Sj&#246;lander for ps-ns internal protein dynamics14 or the rotation model by Sears for hydration water15,16,17.
On the neutron backscattering (NBS) instrument IN16B8,9 at the ILL, Grenoble, France (Supplemental Figure S1), a setup commonly used with proteins consists of Si 111 crystals for the analyzers with a Doppler drive for tuning the incoming wavelength (Supplemental Figure S2A), thereby giving access to the momentum transfer range ~0.2 &#197;-1 &#60; q &#60; ~2 &#197;-1 and energy transfer range of -30 &#181;eV &#60; <img alt="Equation 3" src="/files/ftp_upload/63664/63664eq03.jpg" style="margin: auto;" /> &#60; 30 &#181;eV-corresponding to timescales ranging from a few ps to a few ns and distances of a few &#197;. In addition, IN16B offers the possibility to perform elastic and inelastic fixed-window scans (E/IFWS)10, which include data acquisition at a fixed energy transfer. As the flux is limited when working with neutrons, E/IFWS allows maximization of the flux for one energy transfer, thus reducing the acquisition time needed to obtain a satisfying signal-to-noise ratio. A more recent option is the backscattering and time-of-flight spectrometer (BATS) mode11, which allows measurement of a wide range of energy transfers, (e.g., -150 &#181;eV &#60; <img alt="Equation 3" src="/files/ftp_upload/63664/63664eq03.jpg" style="margin: auto;" /> &#60; 150 &#181;eV), with a higher flux than with the Doppler drive, yet at the cost of a lower energy resolution (Supplemental Figure S2B).
An important property of neutron scattering is that the incoherent cross section&#160;&#963;inc has a 40 times higher value for hydrogen than for deuterium and is negligible for other elements commonly found in biological samples. Therefore, the dynamics of proteins in a liquid environment can be studied by using a deuterated buffer, and the powder state allows for the study of either protein internal dynamics with hydrogenated protein powder hydrated with D2O, or the study of hydration water for perdeuterated protein powder hydrated with H2O. In the liquid state, neutron backscattering typically allows simultaneously accessing of the center-of-mass self-diffusion of proteins (Fickian-type diffusion) and their internal dynamics. The latter are backbone and side-chain motions usually described by the so-called jump diffusion model or others3,18. In hydrogenated protein powders, the protein diffusion is absent and only internal dynamics needs to be modeled. For hydration water, the contributions of translational and rotational motions of water molecules present a different dependence on the momentum transfer q, which allows for their distinction in the data analysis process17.
This paper illustrates the neutron backscattering method with the study of proteins that were found to be able to unfold, aggregate into a canonical form consisting of stacks of &#946;-strands-the so-called cross-&#946; pattern19,20-and form elongated fibers. This is the so-called amyloid aggregation, which is extensively studied due to its central role in neurodegenerative disorders such as Alzheimer&#39;s or Parkinson&#39;s diseases21,22. The study of the amyloid proteins is also motivated by the functional role they can play23,24 or their high potential for the development of novel biomaterials25. The physicochemical determinants of the amyloid aggregation remain unclear, and no general theory of amyloid aggregation is available, despite tremendous progress during the past years21,26.
Amyloid aggregation implies changes in protein structure and stability with time, the study of which naturally implies dynamics, linked to protein conformation stability, protein function, and protein energy landscape27. Dynamics is directly linked to the stability of a specific state through the entropic contribution for the fastest motions28, and protein function can be sustained by motions on various timescales from sub-ps for light-sensitive proteins29 to ms for domain motions, which can be facilitated by picosecond-nanosecond dynamics30.
Two examples of using neutron backscattering spectroscopy to study amyloid proteins will be presented, one in the liquid state to study protein dynamics and one in the hydrated powder state to study hydration water dynamics. The first example concerns the aggregation of lysozyme into &#181;m sized spheres (called particulates) followed in real time5, and the second a comparison of water dynamics in native and aggregated states of the human protein tau31.
Lysozyme is an enzyme involved in immune defense and is composed of 129 amino acid residues. Lysozyme can form particulates in deuterated buffer at pD of 10.5 and at a temperature of 90 &#176;C. With neutron scattering, we showed that the time evolution of the center-of-mass diffusion coefficient of lysozyme follows the single exponential kinetics of thioflavin T fluorescence (a fluorescent probe used to monitor the formation of amyloid cross-&#946; patterns32), indicating that the formation particulate superstructures and cross-&#946; patterns occur in a single step with the same rate. Moreover, the internal dynamics remained constant throughout the aggregation process, which can be explained either by a fast conformational change that cannot be observed on NBS instruments, or by the absence of significant change in protein internal energy upon aggregation.
The human protein tau is an intrinsically disordered protein (IDP) consisting of 441 amino acids for the so-called 2N4R isoform, which is notably involved in Alzheimer&#39;s disease33. Using neutron backscattering on powders of perdeuterated protein tau, we showed that hydration water dynamics is increased in the fiber state, with a higher population of water molecules undergoing translational motions. The result suggests that an increase in hydration water entropy might drive the amyloid fibrillation of tau.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum sample holder</td>
			<td></td>
			<td></td>
			<td>Not commercially available. Either the local contact on the instrument can provide them or they can be manufactured based on a technical drawing that can be provided by the local contact.</td>
		</tr>
		<tr>
			<td>Deuterium chloride, 35 wt. % in D2O, &#8805;99 atom % D</td>
			<td>Sigma-Aldrich</td>
			<td> 
			543047</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide (D, 99.9%)</td>
			<td>Eurisotop</td>
			<td>DLM-4DR-PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning high-vacuum silicone grease</td>
			<td>Sigma-Aldrich</td>
			<td>Z273554-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%, EMSURE Reag. Ph Eur</td>
			<td>Sigma-Aldrich</td>
			<td>1.5901</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass dessicator</td>
			<td>VWR</td>
			<td>&#160; 75871-660</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass dessicator plate, 140 mm</td>
			<td>VWR</td>
			<td>89038-068</td>
			<td></td>
		</tr>
		<tr>
			<td>Indium wire, 1.0 mm (0.04 in) dia, Puratronic, 99.999%</td>
			<td>Alfa Aesar</td>
			<td>00470.G1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme from chicken egg white dialyzed, lyophilized, powder, ~100,000 U/mg</td>
			<td>Sigma-Aldrich</td>
			<td>62970</td>
			<td></td>
		</tr>
		<tr>
			<td>nPDyn</td>
			<td></td>
			<td>v3.x</td>
			<td>see github.com/kpounot/nPDyn, model functions fot fitting also included in the software</td>
		</tr>
		<tr>
			<td>OHAUS AX324 Adventurer balance, internal calibration</td>
			<td>Dutscher</td>
			<td>92641</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorus pentoxide, ReagentPlus, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>214701</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette ErgoOne 0.5-10 &#956;L</td>
			<td>Starlab</td>
			<td>S7100-0510</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette ErgoOne 100-1,000 &#956;L</td>
			<td>Starlab</td>
			<td>S7100-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette ErgoOne 20-200 &#956;L</td>
			<td>Starlab</td>
			<td>S7100-2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip TipOne 1,000 &#956;L</td>
			<td>Starlab</td>
			<td>S1111-6001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip TipOne 10 &#956;L</td>
			<td>Starlab</td>
			<td>S1111-3200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip TipOne 200 &#956;L</td>
			<td>Starlab</td>
			<td>S1111-0206</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deuteroxide solution, 40 wt. % in D2O, 99.5 atom % D</td>
			<td>Sigma-Aldrich</td>
			<td>372072</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-resolution neutron spectroscopy to study picosecond-nanosecond dynamics of proteins and hydration water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

The aggregation of lysozyme into particulates was performed at 90 &#176;C with a protein concentration of 50 mg/mL in a deuterated buffer (0.1 M NaCl at pD 10.5). The formation of particulates is triggered by the temperature increase to 90 &#176;C and occurs within 6 h (Supplemental Figure S8). The data acquisition was performed on IN16B, as described in the protocol above (data are permanently curated by the ILL and accessible at&#160;http://dx.doi.org/10.5291/ILL-DATA.8-04-811).
<p class="jove_cont...

Watch this Scientific Journal Video about High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water at JoVE.com

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Neutron backscatterings measures global diffusion and internal dynamics of proteins and hydration water without radiation damage. Neutron spectroscopy simultaneously accesses spatial and time correlations on a molecular level. In soft matter and biological materials, it's particularly interesting to obtain indirect information on local order on these long range disorder systems.So neutron backscattering can be used to study dynamics in biology, chemistry, or material sense. Another strong field is energy, with studies, for instance, on batteries and fuel cells. Neutron backscattering has been used successfully to study the molecular dynamics of proteins involved in neurodegenerative diseases such as Alzheimer's and Parkinson's.The results obtained help us understand better the molecular hallmarks of these pathologies and might pave the way to develop better diagnostics. To prepare the sample holder, start by thoroughly cleaning a flat aluminum sample holder and its indium wire seal and screws with water, followed by ethanol, and then let it dry. Weigh the different parts of the sample holder, including the bottom, lid, and indium wire, separately on a precision balance.Place the one millimeter indium wire seal into the groove of the bottom part of the sample holder, leaving a small overlap where the two ends join. Add around 100 milligrams of lyophilized protein such that it fills the inner surface of the bottom part of the sample holder. Next, place the sample holder in a desiccator with a Petri dish containing phosphorus pentoxide powder for 24 hours to completely dry the protein powder.Weigh the dried bottom part of the sample holder containing the indium seal and the dried powder to obtain the mass of the dried sample. Remove the phosphorus pentoxide from the desiccator and put a Petri dish with deuterium oxide as well as the sample inside for hydrating the powder. Repeat the drying and hydrating three times for complete hydrogen to deuterium exchange.Measure the powder mass regularly to check the hydration level and when the hydration is slightly above the desired level, wait for the mass to decrease slowly. Once the desired hydration is obtained, quickly put the lid on the bottom part and close the sample holder first with four screws to stop the vapor exchange. Place and tighten all the remaining screws until no gap is visible between the bottom part and the lid.Weigh the sealed sample holder to check for any potential hydration loss via leaks after the neutron experiment. To prepare the liquid state sample, dissolve the protein in the deuterated buffer. Using water, determine the appropriate volume of liquid to be put in the sample holder.Before handling any material, ensure the ionizing radiation dose is less than 100 microsieverts per hour. Then, thoroughly dry the sample stick and remove any previous sample. Place the sample on the sample stick, check for proper centering relative to the beam center, and insert the sample stick into the cryo furnace.To acquire data in Nomad, go to the execution tab and drag and drop a furnace cryostat controller into the launchpad. Set the temperature to 200 Kelvin. Use the fast mode and a timeout of 30 minutes so that the temperature has time to stabilize.Click on the refresher icon to run the temperature ramp in the background for data acquisition during the temperature decrease. Drag and drop the IN16 Doppler settings controller into the launchpad. Set the speed profile to accurate velocity, the set by to max delta E, the energy offset to 0.00 micro electronvolt, and the number of channels to 128 to obtain an elastic fixed window scans configuration.Drag and drop a count controller into the launchpad, fill the subtitle field with a name that allows for easy identification of the data, and set scans of 30 seconds with 60 repetitions. Next, drag and drop the IN16 Doppler settings controller into the launchpad. Set the speed profile to sign, the set by to speed with a value of 4.5 meters per second, and the number of channels to 2048 to obtain a quasi elastic neutron scattering configuration.Drag and drop a count controller and fill the subtitle field with a name that allows for easy identification of the data. Set scans of 30 minutes with four repetitions. For the temperature ramp, drag and drop a furnace cryostat controller, set the temperature to 310 Kelvin, and set the ramp to set point with a delta of 0.05 Kelvin for six seconds.Use a timeout of 200 minutes. Use a for loop with 65 repetitions. Inside, insert an IN16 doppler settings controller, followed by the count controller and set a single scan of 30 seconds.Subsequently, insert IN16 Doppler settings as described previously, but using an energy offset of 1.5 micro electronvolt and 1024 channels. Then, insert the count controller and set a single scan of three minutes. To acquire the last quasi elastic neutron scattering of 312 Kelvin, drag and drop the IN16 Doppler settings and count controllers configured as demonstrated previously.Then, press the start button to run the script. The lysosome elastic and inelastic fixed window scans showed an increase in signal at low momentum transfer and low energy transfer, while the signal at high energy transfer and low momentum transfer decreased. The initial center of mass diffusion coefficient was 15 angstrom squared per nanosecond, which then exponentially decreased over time.At temperatures higher than 220 Kelvin, the hydration water around the tau fibers was significantly more mobile than around the tau monomers. The fraction of water molecules undergoing translational motion and both the translational and rotation diffusion coefficients of the water molecules were increased around fibers. The protein concentration should be 2000 milligram per ml or at least 20 milligram per ml for proteinated proteins due to neutron back scattering limitations from neutron flux or sample holder buffer background.Neutron backscattering has been recently applied, for example, to understand how water, which is the natural environmental proteins, can be fully replaced by a synthetic polymer for bio technological application like, for example, drug delivery.

high-resolution-neutron-spectroscopy-to-study-picosecond-nanosecond

Research

JoVE Journal

Biochemistry

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.