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1.
bioRxiv ; 2023 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-37745461

RESUMO

The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequencing platform, based on determining the sequence positions of select amino acid types within peptides to enable their identification and quantification from a reference database. Here, we describe substantial improvements to fluorosequencing, including identifying fluorophores compatible with the sequencing chemistry, mitigating dye-dye interactions through the use of extended polyproline linkers, and developing an end-to-end workflow for sample preparation and sequencing. We demonstrate by fluorosequencing peptides in mixtures and identifying a target neoantigen from a database of decoy MHC peptides, highlighting the potential of the technology for high sensitivity clinical applications.

2.
J Am Chem Soc ; 128(38): 12404-5, 2006 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-16984176

RESUMO

The alpha-hemolysin (alphaHL) pore was used as a nanoreactor for the direct observation of the reversible photoisomerization of individual tethered azobenzene molecules in an aqueous environment. alphaHL pores, PAZO, were used that had been derivatized within the lumen at a single cysteine residue with 4-((4-(2-chloroethanoamido)phenyl)diazenyl)benzenesulfonate. Trans-cis isomerizations were monitored at the single-molecule level by observing the modulation of the current passing through PAZO by electrical recording in planar bilayers. When PAZO was irradiated at 330 nm, continuous interconversion between the trans and cis states was observed. Either the trans or the cis state was maintained in the dark, depending upon which was present when the light source was shuttered. The cis state of PAZO was surprisingly stable in the dark, and no cis --> trans transitions were seen over a total observation period of more than 8 h. Therefore, based on our findings, it might be possible to make fast digital nanoscale switches operated by light of a fixed wavelength.


Assuntos
Compostos Azo/química , Proteínas Hemolisinas/química , Água/química , Isomerismo , Bicamadas Lipídicas/química , Modelos Moleculares , Fotoquímica
3.
Chembiochem ; 4(8): 762-7, 2003 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-12898628

RESUMO

The photoregulation of the catalytic activity of butyrylcholinesterase (BChE) was investigated by treating the enzyme with a newly developed carbamylating reagent, N-methyl-N-(2-nitrophenyl)carbamoyl chloride, which has proved to be an efficient photoremovable alcohol-protecting group. BChE was efficiently inhibited in a time- and concentration-dependent manner, and the enzyme could be protected against inhibition by active-site-specific ligands (that is, tacrine). The inactivation kinetics showed a biphasic character, which can be analyzed as the result of the existence of two conformational states in solution. Pseudo-irreversible inactivation of BChE, which results from catalytic serine carbamylation, was suggested by recovery of the enzyme activity after dilution and was demonstrated by X-ray crystallography. Remarkably, the 3D structure of the carbamylated BChE conjugate showed a nonambiguous carbamylation of the catalytic serine residue as the only chemical modification on the protein. The photoreversibility of the enzyme inactivation was analyzed by irradiating the inactivated enzyme at 365 nm and was shown to reach completion in some conditions. The efficient and specific "caging" of BChE, together with the availability of carbamylated BChE crystals, will offer a unique possibility to study the catalytic properties of this enzyme by kinetic crystallography after cryophotolytic uncaging of the enzyme conjugate crystals.


Assuntos
Inibidores da Colinesterase/farmacologia , Reativadores Enzimáticos/farmacologia , Serina/química , Animais , Sítios de Ligação , Butirilcolinesterase , Células CHO , Catálise , Cricetinae , Cristalografia por Raios X , Humanos , Fotólise , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tacrina/farmacologia
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