Using mass spectrometry to study copper-protein binding under native and non-native conditions: β-2-microglobulin

Jilyeon Lim, Richard W. Vachet

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

A method based on metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) has been used to determine the Cu(II) binding sites in both native and unfolded conformations of β-2-microglobulin (β2m). Recent studies have shown that β2m is destabilized and can form amyloid fibers in the presence of Cu(II). An increased affinity for Cu in unfolded states compared to that of the native state is suspected to facilitate overall protein destabilization. Cu-binding site information for native β2m is difficult to obtain using traditional techniques because of its propensity to form amyloid fibers at relatively high protein concentrations in the presence of Cu and because of the nonspecific paramagnetic peak broadening observed in NMR analyses. In addition, Cu-binding information of unfolded β2m is complicated by the high concentrations of denaturants (e.g., 8 M urea) needed to ensure protein unfolding. The MCO/MS approach has been successfully employed in this work to overcome these difficulties. The sensitivity of MS allowed the Cu-binding site of the native protein to be determined at the low concentrations of β2m necessary to avoid amyloid fiber formation. Results indicate that the N-terminus of the protein and His31 are responsible for Cu(II) coordination in the native state. The MCO/MS method was also successful at determining the Cu-binding site in the presence of 8 M urea with the N-terminus, His31, His51, and His81 found to be Cu-bound in the unfolded state. This result supports the existence of a well-defined but different coordination structure in the unfolded state, which leads to the greater affinity for Cu(II) observed in the unfolded state of the protein. In general, it appears that the MCO/MS method is capable of providing Cu-binding site information for proteins that are difficult to study by traditional means.

Original languageEnglish (US)
Pages (from-to)3498-3504
Number of pages7
JournalAnalytical Chemistry
Volume76
Issue number13
DOIs
StatePublished - Jul 1 2004
Externally publishedYes

Fingerprint

Mass spectrometry
Binding Sites
Metals
Amyloid
Proteins
Oxidation
Urea
Fibers
copper-binding protein
Conformations
Nuclear magnetic resonance

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Using mass spectrometry to study copper-protein binding under native and non-native conditions : β-2-microglobulin. / Lim, Jilyeon; Vachet, Richard W.

In: Analytical Chemistry, Vol. 76, No. 13, 01.07.2004, p. 3498-3504.

Research output: Contribution to journalArticle

@article{de785bf9a7124c4ebc0b5d6a7345403b,
title = "Using mass spectrometry to study copper-protein binding under native and non-native conditions: β-2-microglobulin",
abstract = "A method based on metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) has been used to determine the Cu(II) binding sites in both native and unfolded conformations of β-2-microglobulin (β2m). Recent studies have shown that β2m is destabilized and can form amyloid fibers in the presence of Cu(II). An increased affinity for Cu in unfolded states compared to that of the native state is suspected to facilitate overall protein destabilization. Cu-binding site information for native β2m is difficult to obtain using traditional techniques because of its propensity to form amyloid fibers at relatively high protein concentrations in the presence of Cu and because of the nonspecific paramagnetic peak broadening observed in NMR analyses. In addition, Cu-binding information of unfolded β2m is complicated by the high concentrations of denaturants (e.g., 8 M urea) needed to ensure protein unfolding. The MCO/MS approach has been successfully employed in this work to overcome these difficulties. The sensitivity of MS allowed the Cu-binding site of the native protein to be determined at the low concentrations of β2m necessary to avoid amyloid fiber formation. Results indicate that the N-terminus of the protein and His31 are responsible for Cu(II) coordination in the native state. The MCO/MS method was also successful at determining the Cu-binding site in the presence of 8 M urea with the N-terminus, His31, His51, and His81 found to be Cu-bound in the unfolded state. This result supports the existence of a well-defined but different coordination structure in the unfolded state, which leads to the greater affinity for Cu(II) observed in the unfolded state of the protein. In general, it appears that the MCO/MS method is capable of providing Cu-binding site information for proteins that are difficult to study by traditional means.",
author = "Jilyeon Lim and Vachet, {Richard W.}",
year = "2004",
month = "7",
day = "1",
doi = "10.1021/ac049716t",
language = "English (US)",
volume = "76",
pages = "3498--3504",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "13",

}

TY - JOUR

T1 - Using mass spectrometry to study copper-protein binding under native and non-native conditions

T2 - β-2-microglobulin

AU - Lim, Jilyeon

AU - Vachet, Richard W.

PY - 2004/7/1

Y1 - 2004/7/1

N2 - A method based on metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) has been used to determine the Cu(II) binding sites in both native and unfolded conformations of β-2-microglobulin (β2m). Recent studies have shown that β2m is destabilized and can form amyloid fibers in the presence of Cu(II). An increased affinity for Cu in unfolded states compared to that of the native state is suspected to facilitate overall protein destabilization. Cu-binding site information for native β2m is difficult to obtain using traditional techniques because of its propensity to form amyloid fibers at relatively high protein concentrations in the presence of Cu and because of the nonspecific paramagnetic peak broadening observed in NMR analyses. In addition, Cu-binding information of unfolded β2m is complicated by the high concentrations of denaturants (e.g., 8 M urea) needed to ensure protein unfolding. The MCO/MS approach has been successfully employed in this work to overcome these difficulties. The sensitivity of MS allowed the Cu-binding site of the native protein to be determined at the low concentrations of β2m necessary to avoid amyloid fiber formation. Results indicate that the N-terminus of the protein and His31 are responsible for Cu(II) coordination in the native state. The MCO/MS method was also successful at determining the Cu-binding site in the presence of 8 M urea with the N-terminus, His31, His51, and His81 found to be Cu-bound in the unfolded state. This result supports the existence of a well-defined but different coordination structure in the unfolded state, which leads to the greater affinity for Cu(II) observed in the unfolded state of the protein. In general, it appears that the MCO/MS method is capable of providing Cu-binding site information for proteins that are difficult to study by traditional means.

AB - A method based on metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) has been used to determine the Cu(II) binding sites in both native and unfolded conformations of β-2-microglobulin (β2m). Recent studies have shown that β2m is destabilized and can form amyloid fibers in the presence of Cu(II). An increased affinity for Cu in unfolded states compared to that of the native state is suspected to facilitate overall protein destabilization. Cu-binding site information for native β2m is difficult to obtain using traditional techniques because of its propensity to form amyloid fibers at relatively high protein concentrations in the presence of Cu and because of the nonspecific paramagnetic peak broadening observed in NMR analyses. In addition, Cu-binding information of unfolded β2m is complicated by the high concentrations of denaturants (e.g., 8 M urea) needed to ensure protein unfolding. The MCO/MS approach has been successfully employed in this work to overcome these difficulties. The sensitivity of MS allowed the Cu-binding site of the native protein to be determined at the low concentrations of β2m necessary to avoid amyloid fiber formation. Results indicate that the N-terminus of the protein and His31 are responsible for Cu(II) coordination in the native state. The MCO/MS method was also successful at determining the Cu-binding site in the presence of 8 M urea with the N-terminus, His31, His51, and His81 found to be Cu-bound in the unfolded state. This result supports the existence of a well-defined but different coordination structure in the unfolded state, which leads to the greater affinity for Cu(II) observed in the unfolded state of the protein. In general, it appears that the MCO/MS method is capable of providing Cu-binding site information for proteins that are difficult to study by traditional means.

UR - http://www.scopus.com/inward/record.url?scp=3042782808&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3042782808&partnerID=8YFLogxK

U2 - 10.1021/ac049716t

DO - 10.1021/ac049716t

M3 - Article

C2 - 15228316

AN - SCOPUS:3042782808

VL - 76

SP - 3498

EP - 3504

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 13

ER -