This study was supported by the State Key Development Program for Basic Research of China (2017YFA0700500) and the National Natural Science Foundation of China (21574020). The Fundamental Research Funds for the Central Universities, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the National Demonstration Center for Experimental Biomedical Engineering Education (Southeast University) were also greatly appreciated.

1. SFG experimental
<ol>
	<li>Use a commercial picosecond SFG system (Table of Materials), which provides a fundamental 1064 nm beam with a pulse width of ~20 ps and a frequency of 50 Hz, based on an Nd:YAG laser.</li>
	<li>Convert the fundamental 1064 nm beam into a 532 nm beam and a 355 nm beam by using second and third harmonic modules. Directly guide the 532 nm beam as an input light beam and generate the other input mid-infrared (IR) beam covering the frequency range from 1000 to 4000 cm-1 through the optical parametric generation (OPG)/optical parametric amplification (OPA)/difference frequency generation (DFG) process.</li>
	<li>Set the incident angles of two input beams to be 53&#176; (IR) and 64&#176; (visible), respectively, versus the surface normal.</li>
	<li>To detect the polymer interfacial structures (either film/substrate interface or film/the other medium interface), use ssp (s-polarized sum-frequency beam, s-polarized visible beam and p-polarized infrared beam) and ppp polarization combinations.</li>
	<li>To detect the interfacial protein secondary structures and water structures surrounding DNA, besides ssp and ppp, use chiral spp and psp polarization combinations were used.</li>
	<li>To ensure the samples were not damaged, control the infrared and visible pulse energies to be ~70 and ~30 &#956;J, respectively. A schematic of the SFG process with the energy level diagram was shown in Figure 1. Figure 2 shows the SFG system in our clean room.</li>
</ol>
2. Fresnel coefficients
<ol>
	<li>Use right-angle prisms as substrates for all the experiments discussed here. There exist two interfaces for a polymer film on the solid substrate, i.e., polymer surface in air and polymer/substrate interface. Both can generate SFG signals since inversion symmetry is broken at both interfaces. Therefore, a collected SFG spectrum is an interfered one. However, the local field coefficients or the Fresnel coefficients at the two interfaces can be adjustable by varying either the incident angles or the film thickness one at a time or simultaneously31,32. This provides the opportunity for us to probe the SFG vibrational signal from only one interface. Here, the PHEMA film on the CaF2 prism was taken as an example9.</li>
	<li>As shown in Figure 3, employ the right-angle prism geometry to detect the SFG signals generated from the bottom PHEMA film. The SFG output intensity in the reflected mode is expressed as 
	<img alt="Equation 1" src="/files/ftp_upload/59380/59380eq1.jpg" style="opacity: 0.9;" />&#160;(1) 
	where &#160;<img alt="Equation 2" src="/files/ftp_upload/59380/59380eq2.jpg" style="opacity: 0.9;" />&#160;denotes the effective second-order nonlinear susceptibility tensor. 
	<img alt="Equation 2" src="/files/ftp_upload/59380/59380eq2.jpg" style="opacity: 0.9;" />&#160;consists of three parts, namely, the prism/polymer interface, the polymer/bottom medium interface (bottom medium includes gas, liquid or solid.) and the nonresonant background, as shown in the following equation. 
	<img alt="Equation 3" src="/files/ftp_upload/59380/59380eq3.jpg" style="opacity: 0.9;" />&#160;(2) 
	&#8203;Here the bottom medium could be air, water or something else. F represents the corresponding Fresnel coefficient responsible for the local field correction.</li>
	<li>Apply a thin-film model to calculate the Fresnel coefficients in this case. Here only brief calculation procedures are presented.
	<ol>
		<li>For the prism/polymer interface, use 
		<img alt="Equation 4" src="/files/ftp_upload/59380/59380eq4.jpg" style="opacity: 0.9;" />&#160; (3) 
		<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq5.jpg" />&#160; (4) 
		<img alt="Equation 6" src="/files/ftp_upload/59380/59380eq6.jpg" />&#160; (5) 
		The meaning of each parameter shown is presented below.
		<ol>
			<li>&#969;i denotes the beam frequency.</li>
			<li>tp and ts denote the overall transmission coefficients and can be expressed as 
			<img alt="Equation 7" src="/files/ftp_upload/59380/59380eq7.jpg" style="opacity: 0.9;" />&#160; (6) 
			<img alt="Equation 8" src="/files/ftp_upload/59380/59380eq8.jpg" />&#160; (7)</li>
			<li>tp12 and ts12 denote the linear transmission coefficients of the light beam at the prism/polymer interface.</li>
			<li>rp23 and rs23 denote the linear reflection coefficients of the light beam at the polymer/medium interface.</li>
			<li>&#945; represents the phase difference between a reflective beam and its secondary reflective beam after it propagates across the polymer thin film and then reflects back, which can be expressed as 
			<img alt="Equation 9" src="/files/ftp_upload/59380/59380eq9.jpg" style="opacity: 0.9;" />&#160; (8)</li>
			<li>&#955;&#160;represents the wavelength of the light beam and d is the polymer film thickness.</li>
			<li>&#934;1 and &#934;2 represent the incident angles at the prism/polymer interface and the polymer/medium interface respectively.</li>
			<li>n1 and n2 represent the refractive indices of the prism and polymer film respectively.</li>
			<li>n12 represents the refractive indices of the polymer interfacial layers for the prism/polymer.</li>
		</ol>
		</li>
		<li>For the polymer/medium interface, use 
		<img alt="Equation 10" src="/files/ftp_upload/59380/59380eq10.jpg" />&#160; (9) 
		<img alt="Equation 11" src="/files/ftp_upload/59380/59380eq11.jpg" />&#160; (10) 
		<img alt="Equation 12" src="/files/ftp_upload/59380/59380eq12.jpg" />&#160; (11)
		<ol>
			<li>&#916; represents the phase difference of the light electrical fields at two interfaces.</li>
			<li>Because the pulse width for our input beams is ~20 ps, the error from the time delay associated with the dispersion effect can be neglected.</li>
			<li>The expression of such phase difference for the output SFG, the input visible and the input infrared beams can be separately written as 
			<img alt="Equation 13" src="/files/ftp_upload/59380/59380eq13.jpg" />&#160; (12) 
			<img alt="Equation 14" src="/files/ftp_upload/59380/59380eq14.jpg" />&#160; (13) 
			<img alt="Equation 15" src="/files/ftp_upload/59380/59380eq15.jpg" />&#160; (14) 
			&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>From the above discussion, for the prism-polymer film-medium (1-2-3) system, express the total Fresnel coefficients for the prism/polymer and polymer/medium interfaces as the following equations, for ssp and ppp polarization combinations. Of course, both interfaces are considered azimuthally isotropic.
	<ol>
		<li>For the prism/polymer interface, the expressions of the total Fresnel coefficients for both ssp and ppp polarization combinations are presented as follows.
		<ol>
			<li>&#160;For ssp, the equation is 
			<img alt="Equation 16" src="/files/ftp_upload/59380/59380eq16.jpg" />(15)</li>
			<li>And for ppp, the equation is&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq17v3.jpg" style="opacity: 0.9;" /> (16) 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq18v3.jpg" style="opacity: 0.9;" />&#160; (17)&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq19v3.jpg" style="opacity: 0.9;" /> (18) 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq20v3.jpg" style="opacity: 0.9;" />&#160; (19) 
			&#160;</li>
			<li>t10 and t01 denote the linear transmission coefficients at the air/prism and prism/air interfaces respectively.</li>
		</ol>
		</li>
		<li>For the polymer/medium interface, the expressions of the total Fresnel coefficients for both ssp and ppp polarization combinations are described as follows.
		<ol>
			<li>For ssp, the equation is 
			<img alt="Equation 21" src="/files/ftp_upload/59380/59380eq21.jpg" style="opacity: 0.9;" />&#160; (20)</li>
			<li>For ppp, the equations are&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq22v3.jpg" style="opacity: 0.9;" />&#160;(21)&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq23v3.jpg" style="opacity: 0.9;" />&#160;(22)&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq24v3.jpg" style="opacity: 0.9;" /> (23)&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq25v3.jpg" style="opacity: 0.9;" /> (24) 
			&#160;&#160;&#160; 
			&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After calculating the Fresnel coefficients using the sandwiched model, plot them as a function of the film thickness, as shown in Figure 4. 
	NOTE: In this case, there exists a thickness range for collecting the SFG signal from the CaF2 prism/PHEMA interface with neglectable contribution from the other interface, which is around 150 nm. Similarly, a suitable thickness can be chosen for detection of the PHEMA/bottom medium interface with neglectable contribution from the CaF2 prism/PHEMA interface.</li>
</ol>
3. Chiral SFG polarization combination
<ol>
	<li>For the normal achiral interface, commonly, use C&#8734;v symmetry in terms of ensemble average33,34. With operation&#160;of inversion symmetry, the nonzero second-order nonlinear susceptibility tensor components can be deduced, which are cxxz, cxzx, czxx, cyyz, cyzy, czyy and czzz (the existing terms can be further reduced if an isotropic interface is assumed, which means x and y are the same). However, for the chiral interface, situation will be different. The chiral interface possesses the C&#8734;&#160;symmetry, only the rotation symmetry operation is allowed. In this case, besides the normal achiral terms, more second-order nonlinear susceptibilities will be nonzero, which can be termed as the chiral terms, namely, czyx, czxy and cyzx under the consideration of non-electronic resonance. Therefore, by using psp, pps and spp polarization combinations, chiral SFG spectra can be collected33,34.</li>
</ol>
4. Sample preparation
<ol>
	<li>Preparation of PHEMA film
	<ol>
		<li>Dissolve PHEMA powder (see Table of Materials) in anhydrous ethanol to prepare the solution with 2 wt% and 4 wt% respectively.</li>
		<li>Before deposition of the PHEMA films, soak the CaF2 right-angle prisms in the toluene solvent firstly and then wash them with ethanol and ultrapure water (18.2 M&#937;&#183;cm).</li>
		<li>Afterwards, expose the substrates (CaF2 right-angle prisms) to oxygen plasma to remove possible organic contaminants by plasma cleaner (see Table of Materials).
		<ol>
			<li>First turn on the plasma cleaner and put the substrates in it.</li>
			<li>Then turn on the vacuum pump to vacuumize the cleaner. Input the oxygen in it.</li>
			<li>Finally set 4 minutes for cleaning. After that, preserve the clean substrates for the sequential PHEMA film preparation.</li>
			<li>Then prepare the PHEMA films on the CaF2 prisms by a spin-coater (see Table of Materials). Adjust the film thicknesses by the solution concentration and spin speed.
			<ol>
				<li>Immobilize the CaF2 prism on the sucking disc of spin-coater.</li>
				<li>Drop one drop of the PHEMA solution prepared before onto the clean substrates at 1,500 rpm for 1 min (film thickness 2 wt% for 100 nm and 4 wt% for 200 nm).</li>
			</ol>
			</li>
			<li>Anneal all the prepared PHEMA films in a vacuum oven at 80 &#176;C overnight.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of silk fibroin (SF) 
	&#8203;NOTE: The protocol suggested by Kaplan et al.35 was adopted.
	<ol>
		<li>Place 7.5 g of silk cocoons of B. mori in the boiling sodium carbonate (Na2CO3, 0.02 M) aqueous solution (3 L) for 30 min. Remove the fibrous SF to a clean container.</li>
		<li>Wash the obtained fibrous SF with deionized water for three times under stirring in order to remove the sericin molecules and leave only the SF molecules in the fibrous sample.</li>
		<li>Dry the fibrous SF sample in a vacuum oven at 60 &#176;C overnight.</li>
		<li>Afterwards, dissolve the degummed fibrous SF sample in a lithium bromide (LiBr, 9.3 M) aqueous solution (1 g of&#160; SF was solved in ~4 mL of LiBr solution.) and incubate it at 60 &#176;C for 2 h under stirring.</li>
		<li>Dialyze the SF solution against deionized water (3,500 Da dialysis bags) for 3 days to remove the dissolved LiBr. Change new deionized water three times every day. Finally store the processed SF solution at 4 &#176;C for later SFG experiments.</li>
	</ol>
	</li>
	<li>Preparation of short-chain oligonucleotide duplex
	<ol>
		<li>Order the single-stranded oligonucleotide sample with its 3&#8217;-end modified by cholesterol-triethylene glycol (Chol-TEG) (5&#8217;-GCTTCCGAAGGTCGA-3&#8217;) from a commercial corporation (see Table of Materials) as well as the complementary one. For each single strand, dissolve 10 nmol of the sample powder in 0.5 ml ultrapure water. Then mix them together to form the duplex oligonucleotide solution (10 nmol/mL).</li>
		<li>Mix 2 mg of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 2 mg of deuterated DPPC (d-DPPC) and dissolve them in 1 mL of chloroform to prepare the lipid solution.</li>
		<li>Preparation of DPPC &#38; d-DPPC monolayer by a Langmuir&#8722;Blodgett (LB) trough
		<ol>
			<li>Attach the right-angle CaF2 prism to a homemade sample holder with one prism face perpendicularly dipped into the aqueous environment of the LB trough.</li>
			<li>Afterwards, inject the mixed lipid solution prepared before onto the water surface until the surface pressure reached a certain value below 34 mN&#183;m&#8722;1.</li>
			<li>After the surface pressure levels off, use two Teflon barriers to compress the lipid monolayer at a ratio of 5 mm/min until a surface pressure of 34 mN&#183;m&#8722;1 was reached.</li>
			<li>Lift the prism with a lipid monolayer out of the water at a rate of 1 mm/min vertically.</li>
		</ol>
		</li>
		<li>Preparation of the other lipid monolayer
		<ol>
			<li>To facilitate the assembly of the duplex oligonucleotide and the lipid molecules via the hydrophobic interaction (cholesterol and a lipid alkyl chain), mix the duplex oligonucleotide solution with the lipid solution in a molar ratio of 1:100 (oligonucleotide to lipid).</li>
			<li>Inject the mixed lipid and duplex oligonucleotide solution onto the water surface in a homemade Teflon container until a surface pressure of 34 mN&#183;m&#8722;1 was reached.</li>
		</ol>
		</li>
		<li>Finally, put the lipid monolayer at the bottom of the prism in contact with the lipid monolayer with inserted duplex oligonucleotides on the water surface to form the final sample for the SFG measurement.</li>
	</ol>
	</li>
	<li>Lorentz equation
	<ol>
		<li>Use the Lorentz equation to fit the SFG spectra to extract the vibrational information for a specific vibrational mode. 
		<img alt="Equation 26" src="/files/ftp_upload/59380/59380eq26.jpg" style="opacity: 0.9;" />&#160; (25) 
		where&#160;<img alt="Equation 27" src="/files/ftp_upload/59380/59380eq27.jpg" />&#160;represents the intensity of the qth vibrational mode, <img alt="Equation 28" src="/files/ftp_upload/59380/59380eq28.jpg" />&#160;represents the resonant frequency,&#160;<img alt="Equation 29" src="/files/ftp_upload/59380/59380eq29.jpg" />&#160;denotes the half width at half maximum (HWHM) and <img alt="Equation 30" src="/files/ftp_upload/59380/59380eq30.jpg" />&#160;represents the scanning frequency of the incident IR beam.</li>
	</ol>
	</li>
</ol>

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N., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Ordering+of+Phenyl+Groups+at+the+Buried+Polystyrene/Metal+Interface.">Molecular Ordering of Phenyl Groups at the Buried Polystyrene/Metal Interface.</a> Langmuir. 30, 9418-9422 (2014).</li><li>Li, B., Lu, X., Ma, Y., Han, X., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+to+Probe+Glass+Transition+Temperatures+of+Polymer+Thin+Films.">Method to Probe Glass Transition Temperatures of Polymer Thin Films.</a> ACS Macro Letters. 4, 548-551 (2015).</li><li>Li, X., Deng, G., Ma, L., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interchain+Overlap+Affects+Formation+of+Silk+Fibroin+Secondary+Structure+on+Hydrophobic+Polystyrene+Surface+Detected+via+Achiral/Chiral+Sum+Frequency+Generation.">Interchain Overlap Affects Formation of Silk Fibroin Secondary Structure on Hydrophobic Polystyrene Surface Detected via Achiral/Chiral Sum Frequency Generation.</a> Langmuir. 34, 9453-9459 (2018).</li><li>Kai, S., Li, X., Li, B., Han, X., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+hydrolysis+of+supported+planar+lipids+was+triggered+by+honey+bee+venom+phospholipase+A2+with+the+right+orientation+at+the+interface.">Calcium-dependent hydrolysis of supported planar lipids was triggered by honey bee venom phospholipase A2 with the right orientation at the interface.</a> Physical Chemistry Chemical Physics. 20, 63-67 (2018).</li><li>Wang, J., Buck, S., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum+Frequency+Generation+Vibrational+Spectroscopy+Studies+on+Protein+Adsorption.">Sum Frequency Generation Vibrational Spectroscopy Studies on Protein Adsorption.</a> Journal of Physical Chemistry B. 106, 11666-11672 (2002).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Amide+I+Signals+of+Interfacial+Proteins+in+Situ+Using+SFG.">Detection of Amide I Signals of Interfacial Proteins in Situ Using SFG.</a> Journal of American Chemical Society. 125, 9914-9915 (2003).</li><li>Nguyen, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+Spontaneous+Membrane+Insertion+of+a+Tall-Anchored+Membrane+Protein+by+Sum+Frequency+Generation+Spectroscopy.">Probing the Spontaneous Membrane Insertion of a Tall-Anchored Membrane Protein by Sum Frequency Generation Spectroscopy.</a> Journal of American Chemistry Society. 132, 15112-15115 (2010).</li><li>Li, X., Ma, L., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Ions+Affect+Water+Molecular+Structures+Surrounding+an+Oligonucleotide+Duplex+as+Revealed+by+Sum+Frequency+Generation+Vibrational+Spectroscopy.">Calcium Ions Affect Water Molecular Structures Surrounding an Oligonucleotide Duplex as Revealed by Sum Frequency Generation Vibrational Spectroscopy.</a> Langmuir. , (2018).</li><li>Sartenaer, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum-frequency+generation+spectroscopy+of+DNA+monolayers.">Sum-frequency generation spectroscopy of DNA monolayers.</a> Biosensors &amp; Bioelectronics. 22, 2179-2183 (2007).</li><li>Asanuma, H., Noguchi, H., Uosaki, K., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+Cation-induced+Deformation+of+DNA+Self-Assembled+Monolayers+on+Silicon:+Vibrational+Sum+Frequency+Generation+Spectroscopy.">Metal Cation-induced Deformation of DNA Self-Assembled Monolayers on Silicon: Vibrational Sum Frequency Generation Spectroscopy.</a> Journal of American Chemistry Society. 130, 8016-8022 (2008).</li><li>Howell, C., Schmidt, R., Kurz, V., Koelsch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum-frequency-generation+spectroscopy+of+DNA+films+in+air+and+aqueous+environments.">Sum-frequency-generation spectroscopy of DNA films in air and aqueous environments.</a> Biointerphases. 3 (3), FC47 (2008).</li><li>Walter, S. R., Geiger, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+on+Stage:+Showcasing+Oligonucleotides+at+Surfaces+and+Interfaces+with+Second+Harmonic+and+Vibrational+Sum+Frequency+Generation.">DNA on Stage: Showcasing Oligonucleotides at Surfaces and Interfaces with Second Harmonic and Vibrational Sum Frequency Generation.</a> Journal of Physical Chemistry Letters. 1, 9-15 (2010).</li><li>Li, Z., Weeraman, C., Azam, M. S., Osman, E., Gibbs-Davis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thermal+reorganization+of+DNA+immobilized+at+the+silica/buffer+interface:+a+vibrational+sum+frequency+generation+investigation.">The thermal reorganization of DNA immobilized at the silica/buffer interface: a vibrational sum frequency generation investigation.</a> Physical Chemistry Chemical Physics. 17, 12452-12457 (2015).</li><li>Lambert, A. G., Neivandt, D. J., Briggs, A. M., Usadi, E. W., Davies, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+Effects+in+Sum+Frequency+Spectra+from+Monolayers+on+Composite+Dielectric/Metal+Substrates.">Interference Effects in Sum Frequency Spectra from Monolayers on Composite Dielectric/Metal Substrates.</a> Journal of Physical Chemistry B. 106, 5461-5469 (2002).</li><li>Tong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+effects+in+the+sum+frequency+generation+spectra+of+thin+organic+films.+I.+Theoretical+modeling+and+simulation.">Interference effects in the sum frequency generation spectra of thin organic films. I. Theoretical modeling and simulation.</a> Journal of Chemical Physics. 133, 034704 (2010).</li><li>McGall, S. J., Davies, P. B., Neivandt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+Effects+in+Sum+Frequency+Vibrational+Spectra+of+Thin+Polymer+Films:+An+Experimental+and+Modeling+Investigation.">Interference Effects in Sum Frequency Vibrational Spectra of Thin Polymer Films: An Experimental and Modeling Investigation.</a> Journal of Physical Chemistry B. 108, 16030-16039 (2004).</li><li>Li, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+Fresnel+Coefficients+and+Molecular+Structures+of+Model+Cell+Membranes:+From+a+Lipid+Monolayer+to+a+Lipid+Bilayer.">Interfacial Fresnel Coefficients and Molecular Structures of Model Cell Membranes: From a Lipid Monolayer to a Lipid Bilayer.</a> Journal of Physical Chemistry C. 118, 28631-28639 (2014).</li><li>Zhou, J., Anim-Danso, E., Zhang, Y., Zhou, Y., Dhinojwala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+Water+at+Polyurethane-Sapphire+Interface.">Interfacial Water at Polyurethane-Sapphire Interface.</a> Langmuir. 31 (45), 12401-12407 (2015).</li><li>Gautam, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Structure+of+Polystyrene+at+Air/Polymer+and+Solid/Polymer+Interfaces.">Molecular Structure of Polystyrene at Air/Polymer and Solid/Polymer Interfaces.</a> Physical Review Letters. 85 (18), 3854-3857 (2000).</li><li>Yan, E. Y., Fu, L., Wang, Z., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Macromolecules+at+Interfaces+Probed+by+Chiral+Vibrational+Sum+Frequency+Generation+Spectroscopy.">Biological Macromolecules at Interfaces Probed by Chiral Vibrational Sum Frequency Generation Spectroscopy.</a> Chemical Reviews. 114, 8471-8498 (2014).</li><li>Belkin, M. A., Kulakov, T. A., Ernst, K. H., Yan, L., Shen, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum-Frequency+Vibrational+Spectroscopy+on+Chiral+Liquids:+A+Novel+Technique+to+Probe+Molecular+Chirality.">Sum-Frequency Vibrational Spectroscopy on Chiral Liquids: A Novel Technique to Probe Molecular Chirality.</a> Physical Review Letters. 85, 4474 (2000).</li><li>Rockwood, D. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+fabrication+from+Bombyx+mori+silk+fibroin.">Materials fabrication from Bombyx mori silk fibroin.</a> Nature Protocols. 6, 1612-1631 (2011).</li></ol>

We have nothing to disclose.

To investigate the structural information from a molecular level, SFG has its inherent advantages (i.e., monolayer or sub-monolayer sensitivity and interfacial selectivity), which can be applied to study various interfaces, such as the solid/solid, solid/liquid, solid/gas, liquid/gas, liquid/liquid interfaces. Although the equipment maintenance and the optical alignment are still time-consuming, the payoff is significant in that the detailed molecular-level information at the surfaces and interfaces can be obtained.
...

Development of sum frequency generation (SFG) vibrational spectroscopy can be dated back to the work done by Shen et al. thirty years ago1,2. The uniqueness of the interfacial selectivity and sub-monolayer sensitivity makes SFG vibrational spectroscopy appreciated by a large number of researchers in the fields of physics, chemistry, biology, and materials science, etc3,4,5. Currently, a broad range of scientific issues related to surfaces and interfaces are being investigated using SFG, especially for complex interfaces with respect to polymers and biomacromolecules, such as the chain structures and structural relaxation at the buried polymer interfaces, the protein secondary structures, and the interfacial water structures9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26.
For polymer surfaces and interfaces, thin-film samples are generally prepared by spin-coating to obtain the desired surfaces or interfaces. The problem arises due to the signal interference from the two interfaces of the as-prepared films, which leads to inconvenience for analyzing the collected SFG spectra27,28,29. In most cases, the vibrational signal only from one single interface, either film/substrate or film/the other medium, is desirable. Actually, the solution to this problem is quite easy, namely, to experimentally maximize the light fields at the desirable interface and minimize the light fields at the other interface. Hence, the Fresnel coefficients or the local field coefficients need to be calculated via the thin film model and to be validated with respect to the experimental results3,9,10,11,12,13,14,15,30.
With the above background in mind, some polymer and biological interfaces could be investigated in order to understand fundamental science from the molecular level. In the following, taking three interfacial issues as examples: probing poly(2-hydroxyethyl methacrylate) (PHEMA) surface and buried interface with substrate9, formation of silk fibroin (SF) secondary structures on the polystyrene (PS) surface and water structures surrounding model short-chain oligonucleotide duplex16,21, we will show how the SFG vibrational spectroscopy helps to reveal the interfacial molecular-level structures in connection to the underlying science.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)&#160;</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>850355P-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>100092680</td>
			<td>&#8805;99.7%</td>
		</tr>
		<tr>
			<td>CaF2 prism</td>
			<td>Chengdu YaSi Optoelectronics Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride anhydrous</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10005817</td>
			<td>&#8805;96.0%</td>
		</tr>
		<tr>
			<td>deuterated DPPC (d-DPPC)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>860345P-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Electromagnetic oven</td>
			<td>Zhejiang Supor Co., Ltd</td>
			<td>C21-SDHCB37</td>
			<td></td>
		</tr>
		<tr>
			<td>Langmuir-Blodgett (LB) trough</td>
			<td>KSV NIMA Co., Ltd.</td>
			<td>KN 2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium bromide anhydrous</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>20056926</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q synthesis system</td>
			<td>Millipore</td>
			<td></td>
			<td>Ultrapure water</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Chengdu Mingheng Science&#38;Technology Co., Ltd</td>
			<td>PDC-MG</td>
			<td>Oxygen plasma cleaning</td>
		</tr>
		<tr>
			<td>Poly(2-hydroxyethyl methacrylate) (PHEMA)</td>
			<td>Sigma-Aldrich Co., LLC.</td>
			<td>192066 MSDS</td>
			<td>Mw = 300 000</td>
		</tr>
		<tr>
			<td>Polystyrene</td>
			<td>Sigma-Aldrich Co., LLC.</td>
			<td>330345 MSDS</td>
			<td>Mw = 48 kDa and Mn = 47 kDa</td>
		</tr>
		<tr>
			<td>Silk cocoons</td>
			<td></td>
			<td></td>
			<td>From Bombyx mori</td>
		</tr>
		<tr>
			<td>Single complementary strand of oligonucleotide</td>
			<td>Nanjing Genscript Biotechnology Co., Ltd.</td>
			<td>H03596</td>
			<td>5&#39;-CGAAGGCTTCCAGCT-3&#39;</td>
		</tr>
		<tr>
			<td>Single strand of oligonucleotide</td>
			<td>Nanjing Genscript Biotechnology Co., Ltd.</td>
			<td>H04936</td>
			<td>&#160;3&#162;-end modified by cholesterol-triethylene glycol(Chol-TEG) (5&#162;-GCTTCCGAAGGTCGA-3&#162;)</td>
		</tr>
		<tr>
			<td>Sodium carbonate anhydrous</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10019260</td>
			<td>&#8805;99.8%</td>
		</tr>
		<tr>
			<td>Spin-coater</td>
			<td>Institute of Microelectronics of the Chinese Academy of Sciences</td>
			<td>KW-4A</td>
			<td>For the prepartion of ploymer films&#160;</td>
		</tr>
		<tr>
			<td>Step profiler</td>
			<td>Veeco</td>
			<td>DEKTAK 150</td>
			<td>For the measurement of film thickness</td>
		</tr>
		<tr>
			<td>Sum frequency generation (SFG) vibrational spectroscopy system</td>
			<td>EKSPLA</td>
			<td></td>
			<td>A commercial picosecond SFG system</td>
		</tr>
	</tbody>
</table>

interfacial molecular-level structures of polymers and biomacromolecules revealed via sum frequency generation vibrational spectroscopy

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various surfaces and interfaces. This non-invasive optical technique can provide the local molecular-level information with monolayer or submonolayer sensitivity. We here are providing experimental methodology on how to selectively detect the buried interface for both macromolecules and biomacromolecules. With this in mind, interfacial secondary structures of silk fibroin and water structures around model short-chain oligonucleotide duplex are discussed. The former shows a chain-chain overlap or spatial confinement effect and the latter shows a protection function against the Ca2+ ions resulting from the chiral spine superstructure of water.

In the Fresnel coefficient part of Protocol Section, we have shown that, theoretically, it is feasible to selectively detect only one single interface at one time. Here, experimentally, we confirmed that this methodology is basically correct, as shown in Figure 5 and Figure 6.&#160;
Figure 5 shows the buried interfacial PHEMA structure after water intrusion with a ~150 nm PHEMA hydrogel film and <strong c...

Watch this Scientific Journal Video about Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy at JoVE.com

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

Being comprehensively utilized, sum frequency generation (SFG) vibrational spectroscopy can help to reveal chain conformational order and secondary structural change happening at polymer and biomacromolecule interfaces.

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various ...

interfacial-molecular-level-structures-polymers-biomacromolecules

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JoVE Journal

Biochemistry

9.3K Views.  Southeast University. Being comprehensively utilized, sum frequency generation (SFG) vibrational spectroscopy can help to reveal chain conformational order and secondary structural change happening at polymer and biomacromolecule interfaces.

Video: Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

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 ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

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

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

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

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

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

Analysis of SEC-SAXS data via EFA deconvolution and Scatter

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces&#8212;chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW.

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ...