Cell-Free Synthesis of a Functional Membrane Transporter into a Tethered Bilayer Lipid Membrane

Zieleniecki, Julius L.; Nagarajan, Yagnesh; Waters, Shane; Rongala, Jay; Thompson, Vanessa C.; Hrmová, Mária; Köper, Ingo

doi:10.1021/acs.langmuir.5b04059

Cited by 26 publications

(28 citation statements)

References 32 publications

(73 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition to a functional anthrax biosensor, this study and others [ 27 , 35 , 79 , 80 , 81 ] demonstrate capabilities of a tBLM-supported biosensor using large membrane spanning proteins as the sensing element. In particular, we demonstrate that AB toxins such as the LF:PA63 from B. anthracis [ 40 ] provide a highly specific, built-in sensing scheme.…”

Section: Discussionmentioning

confidence: 86%

See 1 more Smart Citation

Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins

et al. 2016

View full text Add to dashboard Cite

Tethered lipid bilayer membranes (tBLMs) have been used in many applications, including biosensing and membrane protein structure studies. This report describes a biosensor for anthrax toxins that was fabricated through the self-assembly of a tBLM with B. anthracis protective antigen ion channels that are both the recognition element and electrochemical transducer. We characterize the sensor and its properties with electrochemical impedance spectroscopy and surface plasmon resonance. The sensor shows a sensitivity similar to ELISA and can also be used to rapidly screen for molecules that bind to the toxins and potentially inhibit their lethal effects.

show abstract

Section: Discussionmentioning

confidence: 86%

“…Solubility is, however, not a requirement. Recent work suggests that in vitro transcription and translation can also be used to synthesize, fold and insert some proteins directly into a tBLM [ 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Cell-free expression in the presence of a supported membrane has been used to study membrane-protein interactions but has been unable to provide information about protein conformation or structure 42–44 . Here, NR coupled with cell-free expression demonstrated the expression of membrane protein (p7) directly into lipid bilayers and obtained details of the protein structure and conformation with sub-nanometer resolution (Fig.…”

Section: Discussionmentioning

confidence: 99%

Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

Soranzo

Martin

Lenormand

et al. 2017

Sci Rep

View full text Add to dashboard Cite

The structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i ) high protein concentration in the membrane, (ii ) control of the protein’s isotopic constitution, and (iii ) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for both direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.

show abstract

“…The choice of anchoring group can significantly impact that quality of the resulting tethering layer. For example, changing the anchoring group from a single thiol (DPhyTT) to a disulfide DPhyTL (Schiller et al, 2003;Atanasov et al, 2006;Vockenroth et al, 2007Vockenroth et al, , 2008aZieleniecki et al, 2016) DPhySL ( (Valincius et al, 2008;McGillivray et al, 2009;Kibrom et al, 2011) TEG-DP (Basit et al, 2011) CPEO3 (also EO3-cholesterol; Boden et al, 1997;Kendall et al, 2010;Wiebalck et al, 2016;Forbrig et al, 2018) (DPhyTL) while using the same length of the spacer molecule, improved the electrical resistance of the resulting bilayer from up to 15 M ·cm 2 to 55 M ·cm 2 (Junghans and Koper, 2010). This is possibly because the size of the anchor group more closely matches that of the hydrophobic chains, resulting in improved packing density as the anchorlipid.…”

Section: Tethered Bilayer Lipid Membranesmentioning

confidence: 99%

“…Tethered membranes also offer the possibility of combining cell-free expression of a protein with a model membrane, avoiding the need for lengthy protein purification processes (Zieleniecki et al, 2016). The plant membrane transporter Bot1 was expressed in the presence of a DPhyTL-based tBLM, and spontaneously incorporated in its functional form into the lipid bilayer (Zieleniecki et al, 2016).…”

Section: Tethered Bilayer Lipid Membranesmentioning

confidence: 99%

Tethered Membrane Architectures—Design and Applications

2018

Self Cite

View full text Add to dashboard Cite

Membrane proteins perform a large number of essential biological tasks, but the understanding of these proteins has progressed much more slowly than that of globular proteins. The study of membrane proteins is hindered by the inherent complexity of the cellular membrane. Membrane proteins cannot be studied outside their native environment because their natural structure and function is compromised when the protein does not reside in the cell membrane. Model membranes have been developed to provide a controlled, membrane-like environment in which these proteins can be studied in their native form and function without interference from other membrane components. Traditionally used model membranes such as bimolecular or black lipid membranes and floating lipid membranes suffer from several disadvantages including complex assembly protocols and limited stability. Furthermore, these membranes can only be studied with a narrow range of methodologies, severely restricting their use. To increase membrane stability, simplify the assembly process and increase the number of analytical tools that can be used to study the membranes, several strategies of covalently tethering the bilayer to its solid support have been developed. This review provides an overview of the methods used to assemble various membrane architectures, the properties of the resulting membranes and the tools used to study them.

show abstract

Cell-Free Synthesis of a Functional Membrane Transporter into a Tethered Bilayer Lipid Membrane

Cited by 26 publications

References 32 publications

Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins

Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins

Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

Tethered Membrane Architectures—Design and Applications

Contact Info

Product

Resources

About