Title: Information about protein conformation is important for determining biological function
1Protein Surface Mapping by Mass Spectrometry to
Monitor pH-Dependent Structural Transitions in
Beta-lactoglobulin A Robert L. Hettich 1 Demet
Ataman1,2 Joshua Sharp 3 1Oak Ridge National
Laboratory, Oak Ridge ,TN 2 ORNL-UTK Genome
Science and Technology Graduate School, Oak
Ridge, TN, 3 NIEHS, Research Triangle Park, NC
OVERVIEW
- Information about protein conformation is
important for determining biological function - One method of probing higher order protein
structure is chemical tagging of the solvent
accessible residues using oxidation as a labeling
technique (Maleknia and Chance, Anal. Chem.
(1999) 71, 3965) - We have demonstrated a protein surface mapping
technique based on hydroxyl radical attachment
with high-resolution mass spectrometry
identification to probe the solvent accessibility
of the proteins native conformation (Sharp,
et.al. Anal. Biochem. (2003) 313, 216) - This surface mapping technique was used to
investigate the pH-dependent conformational
changes of ß-lactoglobulin A, providing specific
details of the opening of the hydrophobic cavity
RESULTS FOR ß-LACTOGLOBULIN A OXIDATION AFTER
TRYPTIC DIGESTION
RESULTS FOR ß-LACTOGLOBULIN A OXIDATION AT PH 7.5
AFTER PEPSIN DIGESTION
EXPERIMENTAL
DISCUSSION
- In this study, high-resolution mass spectrometry
and tandem mass spectrometry were used to confirm
the previous NMR data on the conformational
changes of ß-lactoglobulin A at different pH
ranges. - At pH 2, there were only a few oxidized residues,
primarily Trp 19 and Tyr 20 located near the
N-terminus, and Met 145 and Cys 160 located at
the C-terminus. None of the residues in the
hydrophobic cavity or the residues in the a-helix
were solvent accessible and oxidized. This is
consistent with fact that ß-lactoglobulin A at
this pH is in its monomer and closed
conformation. - At pH 4, the protein goes through reversible
association forming oligomeric structures. The
protein is still in its closed conformation.
Therefore the amount of labeling does not change
dramatically compared to pH 2. - At pH 6, the protein is in its closed
conformation but the E-F loop starts to expose
the opening of the cavity. In addition to the
oxidation of residues at N and C terminus, one
other residue (Ile 71) in the hydrophobic cavity
is oxidized. - At pH 8 (Tanford transition), the solvent
accessibility and therefore the exposure of
residues to oxidation increases dramatically with
the opening of the hydrophobic cavity. Apart from
residues which are oxidized at the N and C
termini, Trp61, Ile 71, Ile 72, and Ile 78
located inside the hydrophobic cavity, and
residues in the a-helix (Leu 133, Phe 136, Leu
140) are now oxidized. - In the urea-denatured sample, residues located at
different strands are oxidized in addition to all
of the oxidized residues seen at different pH
ranges, which is consistent with the fact that
ß-lactoglobulin A is now in its extended,
non-native form.
pH 2 pH 4 pH 6 pH 8 Urea-denatured
Met 7 O (N-terminus) O,2O (N-terminus) O,2O (N-terminus) O(N-terminus) O(N-terminus)
Leu 10 O (N-terminus) O(N-terminus)
Trp 19 O (Strand A ) O (Strand A )
Tyr 20 O (Strand A )
Met 24 O (Strand A ) O (Strand A ) O (Strand A ) O (Strand A ) O (Strand A )
Ile 29 O (A-B loop)
Leu 54 O (Strand C)
Leu 57 O (Strand C)
Trp 61 O (Strand C) O (Strand C)
Cys 66 3O (C-D loop) 3O (C-D loop) 3O (C-D loop) 3O (C-D loop)
Ile 71 O (Strand D) O (Strand D) O (Strand D)
Ile 72 O (Strand D) O (Strand D) O (Strand D)
Ile 78 O (Strand D)
Phe 82 O(D-E loop)
Cys 106 O (Strand G)
Met 107 O (Strand G) O (Strand G) O (Strand G) O (Strand G) O (Strand G)
Leu 117 O (G-H loop)
Cys 121 3O (Strand H) 3O (Strand H) 3O (Strand H) 3O (Strand H) 3O (Strand H)
Leu 122 O (Strand H)
Pro 126 O (H- a-helix)
Leu 133 O (a-helix) O (a-helix)
Phe 136 O (a-helix) O (a-helix)
Leu 140 O (a-helix)
Met 145 O (a-strand I) O (a-strand I) O (a-strand I) O,2O (a-strand I) O,2O (a-strand I)
Phe 151 O (strand I) O (strand I) O (strand I)
Leu 156 O (strand I) O (strand I)
Cys 160 2O,3O (310 helical) 2O,3O(310 helical) 3O(310 helical) 3O (310 helical) 3O (310 helical)
- Stock solutions of 0.5 mg/ml ß-lactoglobulin A
were prepared in 10 mM NaCl. - Sample solutions were prepared by adjusting the
pH of the stock solution with HCl or NaOH to 2,
4, 6 or 8, respectively, in order to obtain
different structural states of ß-lactoglobulin A. - A urea-denaturation sample was prepared by
adding urea to the pH7.5 sample solution. - Each sample had hydrogen peroxide added (15),
and then was exposed to UV irradiation in a
Stratalinker 2400 for 5 minutes to generate
hydroxyl radicals (Sharp, Anal. Chem. 2004, 76,
672) - The intact oxidized protein samples were analyzed
by ES-FTMS in order to determine the extent of
oxidation. - The oxidized samples were digested with either
trypsin or pepsin and analyzed by LC-MS/MS
(quadrupole ion trap, QIT) in order to determine
the site(s) of the oxidized residues. - Turbo SEQUEST was used to determine the putative
sites of oxidation, which were verified manually.
ESI-FTMS
INTRODUCTION
- ß-lactoglobulin is a major whey protein found in
ruminants. In addition to its nutritional
function, it also is thought to have a
transportation function due to the pH-dependent
conformational changes of its structure, as shown
below. - During the transport of the hydrophobic molecules
in the acidic environment of the stomach, the
cavity of ß-lactoglobulin A is in its closed
conformation. In the basic environment of the
intestine, this protein adjusts its conformation
to an open position, thereby releasing its cargo. - In this study, a surface mapping technique
involving high resolution mass spectrometry is
demonstrated for probing the conformational
changes of native ß-lactoglobulin A in the pH
range of 2-8. A urea-denaturated sample of the
protein was used as a control to provide the most
extended conformation and investigate the most
oxidized version of this protein. - The solvent accessibility, and therefore the
extent of oxidation, should increase as
ß-lactoglobulin A adopts to its open conformation
in response to the pH increase.
Electrospray-Fourier Transform Ion Cyclotron
Resonance Mass Spectrometry (9.4 T)
- As seen in the above table, the level of
oxidation is pH dependent, and increases with pH.
- The urea denatured sample was used as a control
to observe the most extensive labeling. - There were residues (Met 7, Met 24, Met 107, Met
145,Cys 121 and Cys 160) which were oxidized
regardless of the pH. - The residues in the a-helix region were solvent
accessible and labeled at pH 8 and urea
denaturation, whereas these residues were buried
and not oxidized at lower pHs. - The degree of residue labeling appears to
correlate with the different structural changes
ß-lactoglobulin A goes through at different pHs.
CONCLUSIONS
- Surface mapping is a useful tool for probing
pH-dependent conformational changes of
ß-lactoglobulin A. - Consistent with the published NMR and XRC
results, as the pH increases, the number of
solvent accessible residues and therefore the
oxidation level increases as the hydrophobic
cavity opens. - At pH 8, as the E-F loop exposes the hydrophobic
cavity, the residues within the cavity were
oxidized. - The highest level of oxidation was seen in
urea-denatured sample as a control, and the
lowest level of oxidation was seen at pH 2
(closed conformation). - In order to better validate and detect more
oxidized residues in the peptides which are
created, both pepsin and trypsin were used for
digestion of this protein. The protease pepsin
gave smaller peptides because of its non-specific
cleavage. The verification of the spectra with
more peptides will be useful in determining the
other residues which were not observable with the
trypsin digestion.
pH-dependent structural states of ß-lactoglobulin
A
ß-lactoglobulin A structure N-terminus (310
helical turn) (residues 1-15) Strand A (residues
16-27) A-B loop (310 helical turn) (residues
28-45) Strand B (residues 46-49) B-C loop
(residues 50-52) Strand C (residues 53-63) C-D
loop (residues 64-69) Strand D (residues
70-78) D-E loop (residues 79-83) Strand E
(residues 84-85) E-F loop (residues 86-89) Strand
F (residues 90-98) F-G loop (residues
99-100) Strand G (residues 101-109) G-H loop (310
helical turn) (residues 110-118) Strand H
(residues 119-124) H-a helix loop (residues
125-130) a helix (residues 131-142 ) a-strand I
(residues 143-150) Strand I (residues
151-157) 310 helical turn (residues 158-162)
Red font indicates regions where oxidized
residues were found
LC-MS/MS (QIT) Base peak
chromatogram of oxidized ß-lactoglobulin A at pH
6.65
- pH 2 monomer- closed conformation ?hydrophobic
cavity is closed - pH 3.5(monomer?dimer equilibrium)
- pH gt 3.5(shift to dimer conformation)
- pH 4 - 5.2 (dimer?octamer)
- association into aggregates starts around pH 4.6
(octamer) - Maximal association is at the pH range of
4.40-4.65 - pH 5.2(octamer?dimer)
- pH 5.2 - 6 (N-to-Q (Native to Acidic)
transition) - pH 6dimer ?hydrophobic cavity begins to open
- pH gt 7 (Tanford Transition? N-to-R (Native to
New conformation) - dimer conformation-open conformation?hydrophobic
cavity is completely exposed - pH 10irreversible base-induced unfolding
ß-lactoglobulin A sequence (Urea-denaturation
oxidation coverage)
LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVYVEELKPTP
EGDLEILLQKWENDECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDY
K KYLLFCMENSAEPEQSLVCQCLVRTPEVDDEALEKFDKALKALPMHIR
LS FNPTQLEEQCHI Blue label - Verified
residues Red label - Unverified residues
Verified residues 4/5 Cysteine 4/4
Methionine 0/2 Histidine 0/4 Tyrosine
1/2 Tryptophan 2/4 Phenylalanine 6/22
Leucine 3/10 Isoleucine 1/8 Proline
ACKNOWLEDGEMENTS
Oxidized Peptides Identified by Pepsin
Digestion Measured Mass Residues
Peptide Sequence 943.442 Da
20-28 YSLAMAASD
(strand A) 699.704
46-51 LKPTPE
(strand B) 756.288
94-99 VLDTDY
(strand F) 1233.658
1-11 LIVTQTMKGLD (N-terminus) 1755.052
137-151 KALKALPMHIRLSF (alpha
strand I)
- Every color-coded residue is a potentially
taggable amino acid. - Most of the highly-reactive residues (C, M,
aromatics) were found to be oxidized. - The ability to detect and identify the labeled
residues depends on the efficiency of the
chemical reaction, sample digestion and
purification, and the mass spectrometry
detection.
- DA acknowledge support from the ORNL-UTK Genome
Science and Technology Graduate Program - Nathan VerBerkmoes and David L. Tabb are
acknowledged for their technical input. - Research was sponsored by the U.S. Department of
Energy, Office of Biological and Environmental
Research. Oak Ridge National Laboratory is
managed by UT-Battelle, LLC for the U.S.
Department of Energy under Contract No.
DE-AC05-00OR22725.