Name: ensemble force spectroscopy by shear forces
Uploaded: 2022-07-26T13:00:00
Description: Watch this Scientific Journal Video about Ensemble Force Spectroscopy by Shear Forces at JoVE.com

Este trabalho de pesquisa foi apoiado pela National Science Foundation [CBET-1904921] e pelos Institutos Nacionais de Saúde [NIH R01CA236350] para H. M.

NOTA: Todos os tampões e os reagentes químicos utilizados neste protocolo estão listados na Tabela de Materiais.
1. Preparação do microscópio de força de cisalhamento
NOTA: O microscópio de força de cisalhamento contém duas partes, uma unidade de reação (homogeneizador) e uma unidade de detecção (microscópio de fluorescência). A ampliação da ocular é de 10x, e a ampliação da lente objetiva (ar) é de 4x.
<ol><...

<ol>
	<li>Neuman, K. C., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+force+spectroscopy:+Optical+tweezers,+magnetic+tweezers+and+atomic+force+microscopy.">Single-molecule force spectroscopy: Optical tweezers, magnetic tweezers and atomic force microscopy.</a> Nature Methods. 5 (6), 491-505 (2008).</li><li>Woodside, M. 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=Nanomechanical+measurements+of+the+sequence-dependent+folding+landscapes+of+single+nucleic+acid+hairpins.">Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (16), 6190-6195 (2006).</li><li>Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+strong+is+a+covalent+bond.">How strong is a covalent bond.</a> Science. 283 (5408), 1727-1730 (1999).</li><li>Strick, T. R., Allemand, J. F., Bensimon, D., Croquette, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+supercoiled+DNA.">Behavior of supercoiled DNA.</a> Biophysical Journal. 74 (4), 2016-2028 (1998).</li><li>Yang, D., Ward, A., Halvorsen, K., Wong, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+single-molecule+force+spectroscopy+using+a+centrifuge.">Multiplexed single-molecule force spectroscopy using a centrifuge.</a> Nature Communications. 7, 11026 (2016).</li><li>Su, H., 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=Light-responsive+polymer+particles+as+force+clamps+for+the+mechanical+unfolding+of+target+molecules.">Light-responsive polymer particles as force clamps for the mechanical unfolding of target molecules.</a> Nano Letters. 18 (4), 2630-2636 (2018).</li><li>Kirkness, M. W. H., Forde, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+assay+for+proteolytic+susceptibility:+Force-induced+collagen+destabilization.">Single-molecule assay for proteolytic susceptibility: Force-induced collagen destabilization.</a> Biophysical Journal. 114 (3), 570-576 (2018).</li><li>Astumian, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+and+kinetics+of+molecular+motors.">Thermodynamics and kinetics of molecular motors.</a> Biophysical Journal. 98 (11), 2401-2409 (2010).</li><li>Bekard, I. B., Asimakis, P., Bertolini, J., Dunstan, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+shear+flow+on+protein+structure+and+function.">The effects of shear flow on protein structure and function.</a> Biopolymers. 95 (11), 733-745 (2011).</li><li>Chistiakov, D. A., Orekhov, A. N., Bobryshev, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+shear+stress+on+endothelial+cells:+go+with+the+flow.">Effects of shear stress on endothelial cells: go with the flow.</a> Acta Physiologica. 219 (2), 382-408 (2017).</li><li>Hu, C., Jonchhe, S., Pokhrel, P., Karna, D., Mao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+unfolding+of+ensemble+biomolecular+structures+by+shear+force.">Mechanical unfolding of ensemble biomolecular structures by shear force.</a> Chemical Science. 12 (30), 10159-10164 (2021).</li><li>Sedghi Masoud, 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=Analysis+of+interactions+between+telomeric+i-motif+DNA+and+a+cyclic+tetraoxazole+compound.">Analysis of interactions between telomeric i-motif DNA and a cyclic tetraoxazole compound.</a> ChemBioChem. 19 (21), 2268-2272 (2018).</li><li>Abraham Punnoose, 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=Adaptive+and+specific+recognition+of+telomeric+G-quadruplexes+via+polyvalency+induced+unstacking+of+binding+units.">Adaptive and specific recognition of telomeric G-quadruplexes via polyvalency induced unstacking of binding units.</a> Journal of the American Chemical Society. 139 (22), 7476-7484 (2017).</li><li>Dhakal, 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=Coexistence+of+an+ILPR+i-motif+and+a+partially+folded+structure+with+comparable+mechanical+stability+revealed+at+the+single-molecule+level.">Coexistence of an ILPR i-motif and a partially folded structure with comparable mechanical stability revealed at the single-molecule level.</a> Journal of the American Chemical Society. 132 (26), 8991-8997 (2010).</li><li>Hu, C., Tahir, R., Mao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+mechanochemical+sensing.">Single-molecule mechanochemical sensing.</a> Accounts of Chemical Research. 55 (9), 1214-1225 (2022).</li></ol>

O protocolo descrito neste manuscrito permite a investigação em tempo real do desdobramento de um conjunto de estruturas biomoleculares por força de cisalhamento. Os resultados aqui apresentados ressaltam que as estruturas do i-motivo do DNA podem ser desdobradas pela força de cisalhamento. O desdobramento do i-motivo ligado ao ligante e as reações de clique acionadas por força de cisalhamento foram aplicações de prova de conceito para este método de espectroscopia de força de conjunto.
<p class="jove_cont...

Na espectroscopia de força de molécula única1 (SMFS), as propriedades mecânicas de estruturas moleculares individuais têm sido estudadas por instrumentos sofisticados como o microscópio de força atômica, pinças ópticas e pinças magnéticas 2,3,4. Restrito pelo mesmo requisito de direcionalidade das moléculas nas configurações de geração/detecção de força ou pelo pequeno campo de visão em pinças magnéticas e no microscópio de força de centrífuga em miniatura (MCF)5,6,7,8, apenas um número limitado de moléculas pode ser investigado simultaneamente usando SMFS. O baixo rendimento do SMFS impede sua ampla aplicação no campo do reconhecimento molecular, o que requer o envolvimento de um grande conjunto de moléculas.
O fluxo de cisalhamento fornece uma solução potencial para aplicar forças a um conjunto maciço de moléculas9. Em um fluxo de líquido dentro de um canal, quanto mais próximo da superfície do canal, mais lenta a taxa de fluxo10. Tal gradiente de velocidade de fluxo causa tensão de cisalhamento paralela à superfície limite. Quando uma molécula é colocada nesse fluxo de cisalhamento, a molécula se reorienta para que seu eixo longo se alinhe com a direção do fluxo, à medida que a força de cisalhamento é aplicada ao eixo longo11. Como resultado dessa reorientação, espera-se que todas as moléculas do mesmo tipo (tamanho e comprimento das alças) se alinhem na mesma direção enquanto experimentam a mesma força de cisalhamento.
Este trabalho descreve um protocolo para usar tal fluxo de cisalhamento para exercer força de cisalhamento em um conjunto maciço de estruturas moleculares, como exemplificado pelo i-motivo do DNA. Neste protocolo, um fluxo de cisalhamento é gerado entre o rotor e o estator em uma ponta do homogeneizador. O presente estudo descobriu que a estrutura dobrada do i-motivo do DNA poderia ser desdobrada por taxas de cisalhamento de 9724-97245 s−1. Além disso, uma constante de dissociação de 36 μM foi encontrada entre o ligante L2H2-4OTD e o motivo i. Este valor é consistente com o de 31 μM medido pelo ensaio de deslocamento de gel12. Além disso, a técnica atual é usada para desdobrar o i-motivo, que pode expor o cobre quelado (I) para catalisar uma reação de clique. Este protocolo permite, assim, desdobrar um grande conjunto de estruturas i-motif com instrumentos de baixo custo em um tempo razoável (menor que 30 min). Dado que a técnica de força de cisalhamento aumenta drasticamente o rendimento da espectroscopia de força, chamamos essa técnica de espectroscopia de força de conjunto (EFS). Este protocolo tem como objetivo fornecer diretrizes experimentais para facilitar a aplicação deste EFS baseado em força de cisalhamento.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3K MWCO Amicon</td>
			<td>Millipore Sigma</td>
			<td>ufc900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>VWR</td>
			<td>VWRC0143-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calfluor 488 azide</td>
			<td>Click Chemistry Tools</td>
			<td>1369-1</td>
			<td></td>
		</tr>
		<tr>
			<td>CuCl</td>
			<td>Thermo&#160;</td>
			<td>ACRO270525000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispersion tip</td>
			<td>Switzerland</td>
			<td>PT-DA07/2EC-B101</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA oligos</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dye</td>
			<td>IDT</td>
			<td>/5Cy5/</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Janpan</td>
			<td>Nikon TE2000-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>Switzerland</td>
			<td>PT 3100D</td>
			<td></td>
		</tr>
		<tr>
			<td>HPG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>cs-295271</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR</td>
			<td>VWRC26760.295</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>VWR</td>
			<td>VWRCE169-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Quencher</td>
			<td>IDT</td>
			<td>/3IAbRQSp/</td>
			<td></td>
		</tr>
		<tr>
			<td>TBTA</td>
			<td>Tokyo Chemical Industry</td>
			<td>T2993</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>VWR</td>
			<td>VWRCE133-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

ensemble force spectroscopy by shear forces

Técnicas de molécula única baseadas em princípios de fluorescência e mecanoquímicos fornecem sensibilidade superior em sensoriamento biológico. No entanto, devido à falta de recursos de alto rendimento, a aplicação dessas técnicas é limitada em biofísica. A espectroscopia de força de conjunto (EFS) demonstrou alto rendimento na investigação de um conjunto maciço de estruturas moleculares, convertendo estudos mecanoquímicos de moléculas individuais em conjuntos moleculares. Neste protocolo, as estruturas secundárias do DNA (i-motivos) foram desdobradas no fluxo de cisalhamento entre o rotor e o estator de uma ponta do homogeneizador a taxas de cisalhamento de até 77796/s. Os efeitos das taxas de fluxo e tamanhos moleculares sobre as forças de cisalhamento experimentadas pelo motivo i foram demonstrados. A técnica EFS também revelou a afinidade de ligação entre i-motivos de DNA e ligantes. Além disso, demonstramos uma reação química de clique que pode ser acionada por força de cisalhamento (ou seja, química de clique mecano). Esses resultados estabelecem a eficácia do uso da força de cisalhamento para controlar a conformação de estruturas moleculares.

A Figura 1 descreve o desdobramento mecânico e a detecção em tempo real das moléculas do conjunto no EFS. Na Figura 1B, observou-se que a intensidade de fluorescência do DNA i-motif aumentou com a taxa de cisalhamento variando de 9.724 s−1 a 97.245 s−1 em um tampão MES de pH 5,5. Como controle, a intensidade da fluorescência não foi aumentada quando o mesmo DNA i-motif foi cortado a uma taxa de 63.209 s−1...

Watch this Scientific Journal Video about Ensemble Force Spectroscopy by Shear Forces at JoVE.com

Ensemble Force Spectroscopy by Shear Forces

A espectroscopia de força de conjunto (EFS) é uma técnica robusta para desdobramento mecânico e detecção em tempo real de um conjunto de estruturas biomoleculares em campos biofísicos e de biossensoriamento.

Espectroscopia de Força de Ensemble por Forças de Cisalhamento

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the ...

Este protocolo fornece uma técnica de alto rendimento para estudar a ligação intermolecular entre o DNA e os ligantes. Esta técnica demonstrou alto rendimento na investigação de um conjunto maciço de estruturas biomoleculares. Ele faz isso convertendo as propriedades químicas mecânicas de moléculas individuais em propriedades de conjuntos moleculares.Acho que é um experimento muito avançado, mas há poucos passos críticos. Por exemplo, o alinhamento do microscópio, homogeneizador e do tubo de reação. Essas três coisas devem estar perfeitamente alinhadas para rastrear com precisão a mudança na fluorescência.Comece montando o homogeneizador e o microscópio em uma mesa de montagem. Depois de usar os óculos, ligue o microscópio de fluorescência e, em seguida, ajuste o homogeneizador para se certificar de que o feixe de luz de excitação passe pelo centro da ponta de dispersão do homogeneizador. Em seguida, prepare uma câmara de reação de fundo plano com cinco centímetros de altura e 1,5 por 1,5 centímetros quadrados de seção transversal, garantindo que o material da câmara selecionado não fluoresce para reduzir o fundo.Depois, prenda a câmara de reação no estágio de amostra do microscópio de fluorescência e, em seguida, ajuste a câmara para segurar a ponta de dispersão do homogeneizador, garantindo que a ponta esteja ligeiramente acima da superfície inferior da câmara de reação. Em seguida, ajuste a posição vertical do homogeneizador e da câmara juntos para garantir que o foco do microscópio esteja na superfície da ponta de dispersão. Em seguida, ajuste a posição horizontal da ponta de dispersão para garantir que a área de detecção esteja definida entre o rotor e o stater.Ligue os canais fluorescentes, de acordo com o corante fluorescente usado no experimento. Antes do experimento de cisalhamento de alta velocidade, usava-se água deionizada para testar a cisalhamento com uma baixa velocidade de cisalhamento. Primeiro, prepare um DNA telomeric i-motif humano marcado com um corante e um quencher em suas duas extremidades, respectivamente, em água deionizada, conforme descrito no manuscrito.Em seguida, dilua o DNA para cinco micromolares em tampão MES de 30 milimolares a pH 5,5 ou pH 7,4. À solução de ADN, adicionar o ligante L2H24OTD" num intervalo de concentração de zero a 60 micromolares e misturar a solução suavemente durante 10 minutos para dobrar as estruturas i-motif sem luz. Verifique e minimize a intensidade de fluorescência de fundo da câmara de reação que é preenchida com água deionizada usando o microscópio fluorescente sem cisalhamento.Em seguida, defina os parâmetros da câmera CCD usando o software com tempo de exposição como 0,5 segundos, sensibilidade CCD de 1, 600 e tempo de gravação igual a 20 minutos. Usando uma pipeta longa, adicione a solução de DNA na câmara de reação vazia e limpa e cubra a câmara de reação com uma caixa preta. Em seguida, inicie o homogeneizador para realizar a cisalhamento a uma taxa de cisalhamento selecionada variando de 9, 724 por segundo a 97, 245 por segundo por 20 minutos, com a câmera CCD ligada para gravar os dados.Após o experimento, remova a câmara e lave-a com água deionizada. Prepare o DNA i-motif e a água deionizada. Em seguida, incubar 10 micromolares de DNA i-motif e 300 microlitros de tampão tris milimolar suplementado com 150 cloreto cúprico micromolar e 300 ácido ascórbico micromolar por 10 minutos para dobrar as estruturas i-motif.Em seguida, ultrafiltre a solução com um dispositivo de ultrafiltração a uma força centrífuga de 14, 300 vezes G.Reabasteça a solução para aproximadamente 500 microlitros com 30 tampões tris milimolares suplementados com 300 ácidos ascórbicos micromolares após cada filtração. Após a coleta da solução residual, faça um volume final de 300 microlitros adicionando 30 tris milimolares suplementados com 300 ácido ascórbico micromolar juntamente com 20 CalFluor 488 Azida micromolar, 20 HPG micromolares e 10 TBTA micromolar. Uma vez que os reagentes são adicionados, mova a solução para a sala escura.Em seguida, verifique e minimize a intensidade de fluorescência de fundo da câmara de reação preenchida com água deionizada usando o microscópio antes dos experimentos de cisalhamento. Em seguida, adicione a solução de DNA na câmara de reação vazia com uma pipeta longa e, em seguida, inicie o cisalhamento do homogeneizador a uma taxa de cisalhamento de 63, 209 por segundo por 20 minutos com a câmera CCD ligada. Após o experimento, remova a câmara e lave-a com água deionizada.Observou-se que a intensidade de fluorescência do DNA i-motif aumentou com a taxa de puro variando de 9, 724 por segundo a 97, 245 por segundo em um tampão MES de pH 5,5. No entanto, a intensidade da fluorescência não foi aumentada quando o mesmo DNA i-motif foi cortado a uma taxa de 63, 209 por segundo, pois não se dobra em pH 7,4 no tampão MES. A ligação do ligante às moléculas de i-motivo de DNA submetidas ao fluxo puro foi avaliada, o que mostrou que o incremento da intensidade de fluorescência tornou-se menos óbvio com o aumento da concentração de ligantes.A constante de dissociação foi determinada para a interação entre o ligante e o motivo i, sendo 34 micromoles. O i-motivo quelado de cobre foi desdobrado na taxa de cisalhamento de 63, 209 por segundo, e o de cobre liberado desencadeou a reação de clique fluorgênico, resultando em aumento da intensidade de fluorescência ao longo do tempo. Em primeiro lugar, certifique-se de que a área de detecção esteja definida entre o rotor e o stater.Em segundo lugar, verifique e minimize o fundo de fluorescência. E, por último, certifique-se de filtrar a reação antes de executar a reação de clique. Este método abre uma porta de entrada para explorar a resistência mecânica, dobrar e desdobrar todos os tipos de polímeros.Podem ser outras estruturas de DNA, proteínas ou outros polímeros, e sua interação com outros tipos de moléculas. Como dito, várias outras macromoléculas poderiam ser estudadas usando essa abordagem.

ensemble-force-spectroscopy-by-shear-forces

Research

JoVE Journal

Bioengineering

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Termodinâmica de enovelamento de proteínas de membrana Medido por espectroscopia de fluorescência

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Bioengenharia

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

A imobilização de bactérias for Imaging por Microscopia de Força Atômica

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Investigando Sistemas receptor-ligante do Celulossoma com base-AFM única molécula Força Espectroscopia

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

Geração de cisalhamento Adesão Mapa Usando SynVivo Networks microvasculares sintético

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Fabricação de concentrado, oxigênio à base de microbolhas Lipid Emulsões pelo Alto cisalhamento homogeneização e Concentração de série

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Medição do isométrica máxima força gerada pelo permeabilizadas fibras musculares esqueléticas

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Projeção de imagem atômica e força Espectroscopia de Bicamadas Lipídicas suportados

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A Protocol for Using F&amp;#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

Um protocolo para usar a transferência de Förster Ressonância Energia (FRET) Biossensores -force medir forças mecânicas em todo o Complexo LINC Nuclear

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Deteção da endotoxina em Nano-formulações usando Limulus Amoebocyte Lysate (LAL) ensaios

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Impressão 3D de Microcarriers de hidrogel in vitro alternando jatos de força viscosa-inercial

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...