Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

doi:10.1038/ncomms8093

Cited by 25 publications

(99 citation statements)

References 69 publications

(155 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Of the eight classes shown in Fig. 2 a, five (I-V) have already been investigated in purified conditions 12,14,18,24,25 and the obtained F-d curves display the usual sawtooth-like, i.e. the piece-wise WLC behavior that is present also in our F-d curves.…”

Section: The Architecture Of Membrane Proteins and Their Unfoldingsupporting

confidence: 55%

“…We focused on native membrane proteins from hippocampal neurons, dorsal root ganglia (DRG) neurons, and the plasma and disc membrane of rod outer segments, which represent the only native sample that were approached in the past 14 . We validate the identification using the known unfolding of two proteins from rod OSs: cyclic nucleotide gated (CNG) channels 12 and rhodopsin molecules 15 .…”

Section: Introductionmentioning

confidence: 93%

“…To verify this analysis, we looked for an orthogonal validation of the proposed method, based on the results of two membrane proteins unfolded in native membranes, i.e. the cyclic nucleotide gated channel yet unfolded in semi-purified conditions 12 and hypothesized in previous experiments in the plasma membrane of rod outer segments 34 , and the rhodopsin unfolded in discs 14,34 . The unfolding pattern from the C-terminus of the CNGA1 in semi-purified conditions 12 displays 5 major unfolding barriers starting from 100 nm and with a periodicity of ~30 nm, which are features similar to those observed in cluster R4.…”

Section: Bayesian Identification Of the Unfolded Patternsmentioning

confidence: 99%

“…However, the information that is possible to extrapolate from the F-D curves (e.g. mechanical stability 9,10 , structural heterogeneity 11 ) depends on the physical and chemical properties of the cell membrane 12,13 , therefore it is desirable to unfold membrane proteins in their original membrane.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Unfolding and identification of membrane proteins in situ

Galvanetto

Maity

Ilieva

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Is the mechanical unfolding of proteins just a technological feat applicable only to synthetic preparations or is it applicable to real biological samples? Here, we describe a method providing all the necessary steps to deal with native membranes, from the isolation of the plasma membrane of single cells, to the characterization and identification of the embedded membrane proteins. We combined single-molecule force spectroscopy with an automatic pattern classification of the obtained Force-distance curves, and we provide a Bayesian identification of the unfolded proteins. The Bayesian identification is based on the crossmatching of Mass Spectrometry datasets with proteomic databases (Uniprot, PDB). We applied this method to four cell types where we classified the unfolding of 5-10% of their total content of membrane proteins. The ability to mechanically probe membrane proteins directly in their native membrane enables the phenotyping of different cell types with almost singlecell level of resolution.

show abstract

Section: The Architecture Of Membrane Proteins and Their Unfoldingsupporting

confidence: 55%

Section: Introductionmentioning

confidence: 93%

Section: Bayesian Identification Of the Unfolded Patternsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Unfolding and identification of membrane proteins in situ

Galvanetto

Maity

Ilieva

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Cyclic nucleotide-gated (CNG) ion channels are tetrameric potassium channels that are of particular interest due to their function in olfactory and visual signaling cascades (Kaupp and Seifert, 2002; Craven and Zagotta, 2006). However, there is only very limited data available on purified CNG channels under defined conditions, mostly from single molecule force spectroscopy (Higgins et al , 2002; Maity et al , 2015; Goldschen-Ohm et al , 2016; Mazzolini et al , 2018). Electrophysiological characterization of CNG channels expressed in Xenopus oocytes or mammalian cells (Biel et al , 1993 and 1994; Baumann et al , 1994; Weyand et al , 1994; Yu et al , 1996; Zagotta and Siegelbaum, 1996), shows that they are activated by micromolar concentrations of cyclic nucleotides (cNMP), but cAMP and cGMP can act differentially on different channel subtypes.…”

Section: [Background]mentioning

confidence: 99%

Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels

Schmidpeter

Nimigean

2018

BIO-PROTOCOL

View full text Add to dashboard Cite

The cyclic-nucleotide modulated ion channel family includes cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels, which play essential roles in visual and olfactory signaling and the heart pacemaking activity. Functionally, these channels have been extensively characterized by electrophysiological techniques from protein heterologously expressed in Xenopus oocytes and mammalian cells. On the other hand, expression and purification of these proteins for biophysical and structural analyses in vitro is problematic and expensive and, accordingly, only limited information on the purified channels is available in the literature. Here we describe a protocol for binding studies of fluorescently labeled cyclic nucleotides to a homologue of eukaryotic CNG channels. Furthermore, we describe how to directly probe binding of unlabeled cyclic nucleotides in a competition assay. The use of fluorescence as a sensitive probe for ligand binding reduces the amount of protein needed and enables fast and easy measurements using standard laboratory equipment.

show abstract

Discrimination of Protein Amino Acid or Its Protonated State at Single‐Residue Resolution by Graphene Nanopores

Zhang

et al. 2019

Small

View full text Add to dashboard Cite

cellular processes at a single-molecule level by taking advantage of the electrochemical nanoconfinement of a nanopore. Thus nanopore is a powerful sensor for investigation of fundamental physical, chemical or biological issues, such as molecule gating [5] and cation-induced current changing, [6] at single-molecule resolution [8] compared to other traditional technologies. To date, nanopores have been exploited for expanded applications, including detection of nanoparticles, [9][10][11] biopolymers, [1,12] proteins, [13][14][15] DNA-origami, [16] DNA-protein complexes, [17][18][19][20] and so on. One of the most potential applications of nanopore technology is nucleic acid sequencing, which is characterized as low cost and high throughput. Most recently, a lot of works have shown that each type of DNA nucleotide can induce correlated modulation of ionic current as they translocate through a nanopore. [12,[21][22][23] With the help of enzyme [24] or polymerase, [25] some biological nanopores [26,27] have shown the ability to determine the nucleotide sequence of a singlestranded DNA at single-base resolution.Different from DNA molecules, proteins consist of more than 20 amino acids (AAs) and are irregularly and slightly charged. Besides, proteins usually keep in folded state in natural environment. Thus, investigation of proteins is much more challenging than DNA. Unraveling the mechanisms of protein folding and unfolding and even realizing protein sequencing are essential for cellular behaviors and diseases. [28] Measurement of tunneling current [29,30] was reported to recognize single amino acids, however to make the tunneling device be a protein sequencing tool it should be coupled with a means that can thread the peptide chain through the gap in a controllable way, which could be possibly realized if the tunneling device is coupled to a nanopore. In the past two decades, most interests in nanopores have focused on DNA sequencing. Only in recent years, nanopore technology has been extended to protein sequencing as nanopores could sequence full-length proteins with high temporal and spatial resolution while traditional sequencing methods such as mass spectrometry [31] and Edman degradation [32] only rely on digestion of proteins into short peptides. Many experiments have shown that protein molecules can be electrophoretically driven through nanopores. [33,34] Some experiments showed that proteins can be unfolded using nanopores by adjusting pH values of the solution, [35] The function of a protein is determined by the composition of amino acids and is essential to proteomics. However, protein sequencing remains challenging due to the protein's irregular charge state and its high-order structure. Here, a proof of principle study on the capability of protein sequencing by graphene nanopores integrated with atomic force microscopy is performed using molecular dynamics simulations. It is found that nanopores can discriminate a protein sequence and even its protonation state at single-residue resolution. Both the pull...

show abstract

Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

Cited by 25 publications

References 69 publications

Unfolding and identification of membrane proteins in situ

Unfolding and identification of membrane proteins in situ

Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels

Discrimination of Protein Amino Acid or Its Protonated State at Single‐Residue Resolution by Graphene Nanopores

Contact Info

Product

Resources

About