Do Amino Acid Substitutions Alter the Structure of Gramicidin Channels? Chemistry at the Single Molecule Level

1988 ◽  
pp. 115-132 ◽  
Author(s):  
Olaf S. Andersen ◽  
John T. Durkin ◽  
Roger E. Koeppe
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Amino Acid Substitutions ◽  
Single Molecule Level

scholarly journals Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing

2021 ◽  
Author(s):  
Henry Brinkerhoff ◽  
Albert C. W. Kang ◽  
Jingqian Liu ◽  
Aleksei Aksimentiev ◽  
Cees Dekker
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Cell Biology ◽  
Dna Helicase ◽  
Peptide Sequence ◽  
Size Exclusion ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Protein Sequencer ◽  
Dynamics Simulations

As identifying proteins is of paramount importance for cell biology and applications, it is of interest to develop a protein sequencer with the ultimate sensitivity of decoding individual proteins. Here, we demonstrate a nanopore-based single-molecule sequencing approach capable of reliably detecting single amino-acid substitutions within individual peptides. A peptide is linked to a DNA molecule that is pulled through the biological nanopore MspA by a DNA helicase in single amino-acid steps. The peptide sequence yields clear stepping ion current signals which allows to discriminate single-amino-acid substitutions in single reads. Molecular dynamics simulations show these signals to result from size exclusion and pore binding. Notably, we demonstrate the capability to 'rewind' peptide reads, obtaining indefinitely many independent reads of the same individual molecule, yielding virtually 100% read accuracy in variant identification, with an error rate less than 10-6. These proof-of-concept experiments constitute a promising basis for developing a single-molecule protein sequencer.

Simultaneous single-molecule discrimination of cysteine and homocysteine with a protein nanopore

2019 ◽  
Vol 55 (63) ◽  
pp. 9311-9314 ◽  
Author(s):  
Yao Lu ◽  
Xue-Yuan Wu ◽  
Yi-Lun Ying ◽  
Yi-Tao Long
Keyword(s):  
Amino Acid ◽  
Spatial Resolution ◽  
Single Molecule ◽  
High Spatial Resolution ◽  
Amino Acid Difference ◽  
Confined Space ◽  
Single Molecule Level

Discrimination between cysteine and homocysteine at the single-molecule level is achieved within a K238Q mutant aerolysin nanopore, which provides a confined space for high spatial resolution to identify the amino acid difference.

scholarly journals Semisynthetic protein nanoreactor for single-molecule chemistry

2015 ◽  
Vol 112 (45) ◽  
pp. 13768-13773 ◽  
Author(s):  
Joongoo Lee ◽  
Hagan Bayley
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Ionic Current ◽  
Unnatural Amino Acid ◽  
Chemical Ligation ◽  
Single Molecule Level ◽  
Flow Through ◽  
Recombinant Polypeptides

The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach.

Probing Conformers and Adsorption Footprints at the Single-Molecule Level in a Highly Organized Amino Acid Assembly of (S)-Proline on Cu(110)

2009 ◽  
Vol 131 (29) ◽  
pp. 10173-10181 ◽  
Author(s):  
Matthew Forster ◽  
Matthew S. Dyer ◽  
Mats Persson ◽  
Rasmita Raval
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Single Molecule Level

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

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