Optimization of Fluorescence Labeling of Trace Analytes: Application to Amino Acid Biosignature Detection with Pacific Blue

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli. MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication.

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A novel amino acid derivative for fluorescence-labeling of carboxyl groups in proteins.

Agricultural and Biological Chemistry ◽

10.1271/bbb1961.53.3111 ◽

1989 ◽

Vol 53 (11) ◽

pp. 3111-3112 ◽

Cited By ~ 1

Author(s):

Nobuyuki YAMASAKI ◽

Haruhiko AOYAGI ◽

Sannamu LEE

Keyword(s):

Amino Acid ◽

Acid Derivative ◽

Fluorescence Labeling ◽

Amino Acid Derivative ◽

Carboxyl Groups

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The staining pattern of the tropomyosin sequence is displayed in a new paracrystal

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142372 ◽

1986 ◽

Vol 44 ◽

pp. 150-151

Author(s):

M.K. Lamvik ◽

L.L. Klatt

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Staining Pattern ◽

Banding Patterns ◽

Mass Thickness ◽

New Form

Tropomyosin paracrystals have been used extensively as test specimens and magnification standards due to their clear periodic banding patterns. The paracrystal type discovered by Ohtsuki1 has been of particular interest as a test of unstained specimens because of alternating bands that differ by 50% in mass thickness. While producing specimens of this type, we came across a new paracrystal form. Since this new form displays aligned tropomyosin molecules without the overlaps that are characteristic of the Ohtsuki-type paracrystal, it presents a staining pattern that corresponds to the amino acid sequence of the molecule.

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Electron probe x-ray microanalysis and electron microscopy of amino acid absorption and transport by the small intestine: inhibition by chemical poisons and correlation to the elemental composition of the epithelial cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142311 ◽

1986 ◽

Vol 44 ◽

pp. 134-135

Author(s):

A. J. Tousimis

Keyword(s):

Small Intestine ◽

Amino Acid ◽

Epithelial Cells ◽

Elemental Composition ◽

Isotope Labeling ◽

Structural Components ◽

X Ray ◽

Human Small Intestine ◽

Transport Studies

The elemental composition of amino acids is similar to that of the major structural components of the epithelial cells of the small intestine and other tissues. Therefore, their subcellular localization and concentration measurements are not possible by x-ray microanalysis. Radioactive isotope labeling: I131-tyrosine, Se75-methionine and S35-methionine have been successfully employed in numerous absorption and transport studies. The latter two have been utilized both in vitro and vivo, with similar results in the hamster and human small intestine. Non-radioactive Selenomethionine, since its absorption/transport behavior is assumed to be the same as that of Se75- methionine and S75-methionine could serve as a compound tracer for this amino acid.

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Immunoelectron microscope detection of C-type natriuretic peptide in normal human atrial tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147776 ◽

1993 ◽

Vol 51 ◽

pp. 388-389

Author(s):

Chi-Ming Wei ◽

Margaret Hukee ◽

Christopher G.A. McGregor ◽

John C. Burnett

Keyword(s):

Amino Acid ◽

Natriuretic Peptide ◽

Vascular Tone ◽

Cardiac Tissue ◽

Ring Structure ◽

Terminal Amino Acid ◽

Amino Acid Peptide ◽

Normal Human ◽

Terminal Amino ◽

The Brain

C-type natriuretic peptide (CNP) is a newly identified peptide that is structurally related to atrial (ANP) and brain natriuretic peptide (BNP). CNP exists as a 22-amino acid peptide and like ANP and BNP has a 17-amino acid ring formed by a disulfide bond. Unlike these two previously identified cardiac peptides, CNP lacks the COOH-terminal amino acid extension from the ring structure. ANP, BNP and CNP decrease cardiac preload, but unlike ANP and BNP, CNP is not natriuretic. While ANP and BNP have been localized to the heart, recent investigations have failed to detect CNP mRNA in the myocardium although small concentrations of CNP are detectable in the porcine myocardium. While originally localized to the brain, recent investigations have localized CNP to endothelial cells consistent with a paracrine role for CNP in the control of vascular tone. While CNP has been detected in cardiac tissue by radioimmunoassay, no studies have demonstrated CNP localization in normal human heart by immunoelectron microscopy.

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