Ratiometric pulsed-alkylation mass spectrometry : method development and application for the study of protein folding and of chelate stability in metalloproteins /
Ratiometric Pulsed-Alkylation Mass Spectrometry (PA/MS) has been developed as a new technique in bioanalytical chemistry, which measures the reactivity of cysteine residues as a site-selective probe of proteins in different states. Pulsed-alkylation involves a pulse of modification of reactive thio...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2001.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=726102551&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Ratiometric Pulsed-Alkylation Mass Spectrometry (PA/MS) has been developed as a new technique in bioanalytical chemistry, which measures the reactivity of cysteine residues as a site-selective probe of proteins in different states. Pulsed-alkylation involves a pulse of modification of reactive thiolates with N-d₅-ethylmaleimide (d₅-NEM) at a variable pulsed-time, t, followed by a chase with a 10-fold molar excess of H₅-N-ethylmaleimide (H₅-NEM) to fully derivatized the cysteine thiolates. During the chase, the fully alkylated samples are proteolyzed with suitable protease and the resulting peptides subjected to high-resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Identification of the mass peaks corresponding to d₅-NEM and H₅-NEM derivatized peptides and peak area integration allows quantitation of the molar fractions of d₅-NEM and H₅-NEM differentiated mass peaks as a function of pulse time, t, with the resulting data analyzed with an appropriate kinetic model to extract the apparent constant for the reactivity of a thiolate[ (kiapp)]. The method has been applied to study the structure of a stable unfolding intermediate of native bacterial luciferase, an αβ heterodimer, as well as the conformation of the β₂ homodimer, a kinetically trapped off-pathway folding intermediate. In addition, ratiometric PA/MS has been used to extract the pseudo-first order rate constants for the reactivity of cysteine pairs in individual Cys₂-His₂ zinc fingers of human MTF-1, a zinc metalloregulatory protein involved in zinc homeostasis, and in S. aureus CadC, a Cd/Pb/Bi heavy metal sensor protein. These results provide new functional insights into metal chelate stability and in coordination chemistry in distinct metalloregulatory metal complexes. Ratiometric PA/MS has been demonstrated to be a rapid and sensitive technique to implement and relatively adaptable to a wide variety of solution conditions required by different protein systems. More importantly, it has the potential to gain wide-ranging application to the study of enzyme catalysis, pertinent to those that employ the cysteine thiolate nucleophile in their reaction chemistry, protein-protein, and protein-DNA interactions, in addition to the area of protein folding and coordination of metal ions. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xvi, 176 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 162-175). |