6 research outputs found
His26 Protonation in Cytochrome c Triggers Microsecond β‑Sheet Formation and Heme Exposure: Implications for Apoptosis
Cytochrome c unfolds locally and reversibly upon heating
at pH
3. UV resonance Raman (UVRR) spectra reveal that instead of producing
unordered structure, unfolding converts turns and some helical elements
to β-sheet. It also disrupts the Met80–heme bond, and
has been previously shown to induce peroxidase activity. Aromatic
residues that are H-bonded to a heme propionate (Trp59 and Tyr48)
alter their orientation, indicating heme displacement. T-jump/UVRR
measurements give time constants of 0.2, 3.9, and 67 μs for
successive phases of β-sheet formation and concomitant reorientation
of Trp59. UVRR spectra reveal protonation of histidines, and specifically
of His26, whose H-bond to Pro44 anchors the 40s Ω loop; this
loop is known to be the least stable ‘foldon’ in the
protein. His26 protonation is proposed to disrupt its H-bond with
Pro44, triggering the extension of a short β-sheet segment at
the ‘neck’ of the 40s Ω loop into the loop itself
and back into the 60s and 70s helices. The secondary structure change
displaces the heme via H-bonds from residues in the growing β-sheet,
thereby exposing it to exogenous ligands, and inducing peroxidase
activity. This unfolding mechanism may play a role in cardiolipin
peroxidation by cyt c during apoptosis
Heme Reactivity is Uncoupled from Quaternary Structure in Gel-Encapsulated Hemoglobin: A Resonance Raman Spectroscopic Study
Encapsulation of hemoglobin (Hb) in silica gel preserves
structure
and function but greatly slows protein motion, thereby providing access
to intermediates along the allosteric pathway that are inaccessible
in solution. Resonance Raman (RR) spectroscopy with visible and ultraviolet
laser excitation provides probes of heme reactivity and of key tertiary
and quaternary contacts. These probes were monitored in gels after
deoxygenation of oxyHb and after CO binding to deoxyHb, which initiate
conformational change in the R–T and T–R directions,
respectively. The spectra establish that quaternary structure change
in the gel takes a week or more but that the evolution of heme reactivity,
as monitored by the Fe–histidine stretching vibration, ν<sub>FeHis</sub>, is completed within two days, and is therefore uncoupled
from the quaternary structure. Within each quaternary structure, the
evolving ν<sub>FeHis</sub> frequencies span the full range of
values between those previously associated with the high- and low-affinity
end states, R and T. This result supports the tertiary two-state (TTS)
model, in which the Hb subunits can adopt high- and low-affinity tertiary
structures, <i>r</i> and <i>t,</i> within each
quaternary state. The spectra also reveal different tertiary pathways,
involving the breaking and reformation of E and F interhelical contacts
in the R–T direction but not the T–R direction. In the
latter, tertiary motions are restricted by the T quaternary contacts
Ultrafast Charge Transfer in Nickel Phthalocyanine Probed by Femtosecond Raman-Induced Kerr Effect Spectroscopy
The
recently developed technique of femtosecond stimulated Raman
spectroscopy, and its variant, femtosecond Raman-induced Kerr effect
spectroscopy (FRIKES), offer access to ultrafast excited-state dynamics
via structurally specific vibrational spectra. We have used FRIKES
to study the photoexcitation dynamics of nickelÂ(II) phthalocyanine
with eight butoxy substituents, NiPcÂ(OBu)<sub>8</sub>. NiPcÂ(OBu)<sub>8</sub> is reported to have a relatively long-lived ligand-to-metal
charge-transfer (LMCT) state, an essential characteristic for efficient
electron transfer in photocatalysis. Following photoexcitation, vibrational
transitions in the FRIKES spectra, assignable to phthalocyanine ring
modes, evolve on the femtosecond to picosecond time scales. Correlation
of ring core size with the frequency of the ν<sub>10</sub> (asymmetric
C–N stretching) mode confirms the identity of the LMCT state,
which has a ∼500 ps lifetime, as well as that of a precursor
d-d excited state. An even earlier (∼0.2 ps) transient is observed
and tentatively assigned to a higher-lying Jahn–Teller-active
LMCT state. This study illustrates the power of FRIKES spectroscopy
in elucidating ultrafast molecular dynamics
Detection and Identification of the Vibrational Markers for the Quantification of Methionine Oxidation in Therapeutic Proteins
Methionine
oxidation is a major degradation pathway in therapeutic
proteins which can impact the structure and function of proteins as
well as risk to drug product quality. Detecting Met oxidation in proteins
by peptide mapping followed by liquid chromatography with mass spectrometry
(LC–MS) is the industry standard but is also labor intensive
and susceptible to artifacts. In this work, vibrational difference
spectroscopy in combination with <sup>18</sup>O isotopic shift enabled
us to demonstrate the application of Raman and FTIR techniques for
the detection and quantification of Met oxidation in various therapeutic
proteins, including mAbs, fusion proteins, and antibody drug conjugate.
Vibrational markers of Met oxidation products, such as sulfoxide and
sulfone, corresponding to SO and C–SO stretching
frequencies were unequivocally identified based <sup>18</sup>O isotoptic
shifts. The intensity of the isolated νC–S Raman band
at 702 cm<sup>–1</sup> was successfully applied to quantify
the average Met oxidation level in multiple proteins. These results
are further corroborated by oxidation levels measured by tryptic peptide
mapping, and thus the confirmed Met oxidation levels derived from
Raman and mass spectrometry are indeed consistent with each other.
Thus, we demonstrate the broader application of vibrational spectroscopy
to detect the subtle spectral changes associated with various chemical
or physical degradation of proteins, including Met oxidation as well
as higher order structural changes
Mode Recognition in UV Resonance Raman Spectra of Imidazole: Histidine Monitoring in Proteins
The imidazole side-chains of histidine residues perform
key roles
in proteins, and spectroscopic markers are of great interest. The
imidazole Raman spectrum is subject to resonance enhancement at UV
wavelengths, and a number of UVRR markers of structure have been investigated.
We report a systematic experimental and computational study of imidazole
UVRR spectra, which elucidates the band pattern, and the effects of
protonation and deprotonation, of H/D exchange, of metal complexation,
and of addition of a methyl substituent, modeling histidine itself.
A consistent assignment scheme is proposed, which permits tracking
of the bands through these chemical variations. The intensities are
dominated by normal mode contributions from stretching of the strongest
ring bonds, C<sub>2</sub>N and C<sub>4</sub>C<sub>5</sub>, consistent
with enhancement via resonance with a dominant imidazole π–π*
transition
Differential Control of Heme Reactivity in Alpha and Beta Subunits of Hemoglobin: A Combined Raman Spectroscopic and Computational Study
The
use of hybrid hemoglobin (Hb), with mesoheme substituted for
protoheme, allows separate monitoring of the α or β hemes
along the allosteric pathway. Using resonance Raman (rR) spectroscopy
in silica gel, which greatly slows protein motions, we have observed
that the Fe–histidine stretching frequency, νFeHis, which
is a monitor of heme reactivity, evolves between frequencies characteristic
of the R and T states, for both α or β chains, prior to
the quaternary R–T and T–R shifts. Computation of νFeHis,
using QM/MM and the conformational search program PELE, produced remarkable
agreement with experiment. Analysis of the PELE structures showed
that the νFeHis shifts resulted from heme distortion and, in
the α chain, Fe–His bond tilting. These results support
the tertiary two-state model of ligand binding (Henry et al., <i>Biophys. Chem.</i> <b>2002</b>, <i>98</i>, 149).
Experimentally, the νFeHis evolution is faster for β than
for α chains, and pump–probe rR spectroscopy in solution
reveals an inflection in the νFeHis time course at 3 μs
for β but not for α hemes, an interval previously shown
to be the first step in the R–T transition. In the α
chain νFeHis dropped sharply at 20 μs, the final step
in the R–T transition. The time courses are fully consistent
with recent computational mapping of the R–T transition via
conjugate peak refinement by Karplus and co-workers (Fischer et al., <i>Proc. Natl. Acad. Sci. U. S. A.</i> <b>2011</b>, <i>108</i>, 5608). The effector molecule IHP was found to lower
νFeHis selectively for α chains within the R state, and
a binding site in the α<sub>1</sub>α<sub>2</sub> cleft
is suggested