The folding mechanism of the β-sheet protein CspA, the major cold shock protein of Escherichia coli, was previously reported to be a concerted, two-state process. We have reexamined the folding of CspA using multiple spectroscopic probes of the equilibrium transition and laser-induced temperature jump (T-jump) to achieve better time resolution of the kinetics. Equilibrium temperature-dependent Fourier transform infrared (1634 cm–1) and tryptophan fluorescence measurements reveal probe-dependent thermal transitions with midpoints (Tm) of 66 ± 1 and 61 ± 1 °C, respectively. Singular-value decomposition analysis with global fitting of the temperature-dependent infrared (IR) difference spectra reveals two spectral components with distinct melting transitions with different midpoints. T-Jump relaxation measurements of CspA probed by IR and fluorescence spectroscopy show probe-dependent multiexponential kinetics characteristic of non-two-state folding. The frequency-dependent IR transients all show biphasic relaxation with average time constants of 50 ± 7 and 225 ± 25 μs at a Tf of 77 °C and almost equal amplitudes. Similar biphasic kinetics are observed using Trp fluorescence of the wild-type protein and the Y42W and T68W mutants, with comparable lifetimes. All of these observations support a model for the folding of CspA through a compact intermediate state. The transient IR and fluorescence spectra are consistent with a diffuse intermediate having β-turns and substantial β-sheet structure. The loop β3–β4 structure is likely not folded in the intermediate state, allowing substantial solvent penetration into the barrel structure.
Hydration is a key determinant of the folding, dynamics, and function of proteins. In this study, temperature-dependent Fourier transform infrared (FTIR) spectroscopy combined with singular value decomposition (SVD) and global fitting were used to investigate both the interaction of water with α-helical proteins and the cooperative thermal unfolding of these proteins. This methodology has been applied to an isolated α-helix (Fs peptide) and to globular α-helical proteins including the helical subdomain and full-length villin headpiece (HP36 and HP67). The results suggest a unique IR signature for the interaction of water with the helical amide carbonyl groups of the peptide backbone. The IR spectra indicate a weakening of the net hydrogen bond strength of water to the backbone carbonyls with increasing temperature. This weakening of the backbone solvation occurs as a discrete transition near the maximum of the temperature-dependent hydrophobic effect, not a continuous change with increasing temperature. Possible molecular origins of this effect are discussed with respect to previous molecular dynamics simulations of the temperature-dependent solvation of the helix backbone.
The route of O 2 to and from the high-spin heme in heme-copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to Cu B+ on the pathway to and from the high-spin heme. The presence of CO on Cu B+ suggests that O 2 binding may be compromised in CO flow-flash experiments. Time-resolved optical absorption studies show that the rate of O 2 and NO binding in the bovine enzyme (1 × 10 8 M - 1 s - 1 ) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t 1/2 = 1.5 μs) of CO from Cu B+ . In contrast, in Thermus thermophilus (Tt) cytochrome ba 3 the O 2 and NO binding to heme a 3 slows by an order of magnitude in the presence of CO (from 1 × 10 9 to 1 × 10 8 M - 1 s - 1 ), but is still considerably faster (∼ 10 μs at 1 atm O 2 ) than the CO off-rate from Cu B in the absence of O 2 (milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O 2 and NO in Tt ba 3 , namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba 3 the presence of CO on Cu B+ impedes the binding of O 2 to Cu B+ or, if O 2 does not bind to Cu B+ prior to heme a 3 , whether the Cu B+ -CO complex sterically restricts access of O 2 to the heme. Both possibilities are discussed, and we argue that O 2 binds directly to heme a 3 in Tt ba 3 , causing CO to dissociate from Cu B+ in a concerted manner through steric and/or electronic effects. This would allow Cu B+ to function as an electron donor during the fast (5 μs) breaking of the OO bond. These results suggest that the binding of CO to Cu B+ on the path to and from heme a 3 may not be applicable to O 2 and NO in all heme-copper oxidases. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.
The timescale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and side chains is particularly important for the formation of β-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed α/β structure. We have developed a general approach for introducing site-specific IR probes for the side chains (azide) and backbone (13C=18O) using recombinant protein expression. T-jump, time-resolved IR spectroscopy combined with site-specific labeling enables independent measurement of the respective backbone and side chain dynamics with single residue resolution. We find that side chain ordering in a key region of the β-sheet structure occurs on a slower time scale than ordering of the backbone during the folding of NTL9, likely due to the transient formation of nonnative side chain interactions.
The dynamics of the gel to fluid phase transformation in 100 nm large unilamellar vesicles (LUV) of 1,2-dipalmitoyl(d62)-sn-glycero-3-phosphocholine (d62-DPPC), has been studied by laser-induced temperature-jump initiation coupled with time-resolved infrared spectroscopy and by MD simulations. The infrared transients that characterize the temperature dependent phase transformation are complex, extending from the nanosecond to the millisecond time scales. An initial fast (submicrosecond) component can be modeled by partial melting of the gel domains, initiated at pre-existing defects at the edges of the faceted structure of the gel phase. Molecular dynamics simulations support the model of fast melting from edge defects. The extent of melting during the fast phase is limited by the area expansion on melting, which leads to a surface pressure that raises the effective melting temperature. Subsequent melting is observed to follow highly stretched exponential kinetics, consistent with collective relaxation of the surface pressure through a hierarchy of surface undulations with different relaxation times. The slowest step is water diffusion through the bilayer to allow the vesicle volume to grow along with its expanded surface area. The results demonstrate that the dominant relaxation in the gel to fluid phase transformation in response to a large T-jump perturbation (compared to the transition width) is fast (submicrosecond), which has important practical and fundamental consequences.
Femtosecond time-resolved infrared spectroscopy was employed to study intramolecular charge transfer in triphenylmethane dyes, including malachite green (MG), malachite green carbinol base (MGCB), and leucomalachite green (LMG). A local excited state (LE) and a twisted intramolecular charge-transfer (TICT) state have been observed directly in MG. Furthermore, solvent-controlled TICT measurements in a series of linear alcohols indicate that the transition time (4–11 ps) from LE to TICT is strongly dependent on alcohol viscosity, which is due to rotational hindrance of dimethylaniline in high-viscosity solvents. For LMG, no TICT is observed due to steric hindrance caused by the sp3-hybridized central carbon atom. However, for MGCB, TICT is rescued by the addition of the electron-donating hydroxyl group to the bridge. These results for MG and its analogues provide new insight regarding the dynamics and mechanism of twisted intramolecular charge transfer (TICT) in triphenylmethane dyes.
Understanding the folding of the β-hairpin is a crucial step in studying how β-rich proteins fold. We have studied CLN025, an optimized ten residue synthetic peptide, which adopts a compact, well-structured β-hairpin conformation. Formation of the component β-sheet and β-turn structures of CLN025 was probed independently using a combination of equilibrium Fourier transform infrared spectroscopy and laser-induced temperature jump coupled with time-resolved infrared and fluorescence spectroscopies. We find that CLN025 is an ultrafast folder due to its small free energy barrier to folding and that it exceeds the predicted speed limit for β-hairpin formation by an order of magnitude. We also find that the folding mechanism cannot be described by a simple two-state model, but rather is a heterogeneous process involving two independent parallel processes. Formation of stabilizing cross-strand hydrophobic interactions and turn alignment occur competitively, with relaxation lifetimes of 82 ± 10 and 124 ± 10 ns, respectively, at the highest probed temperature. The ultrafast and heterogeneous folding kinetics observed for CLN025 provide evidence for folding on a nearly barrierless free energy landscape, and recalibrate the speed limit for the formation of a β-hairpin.
Conformational control of excited-state intramolecular electron transfer (ET) in folic acid (FA) has been investigated using femtosecond time-resolved infrared (TRIR) spectroscopy. Ultrafast excited-state ET between the pterin and the 4-aminobenzoyl subunits of FA is observed for the anionic form (at pH 10.0). An ET lifetime of 2.5 ps is estimated from Marcus theory for FA in the “U” conformation, in close agreement with the observed lifetime of 2.0 ps. Return to the ground state through the reverse ET reaction happens almost as rapidly, within 5 ps, resulting in rapid quenching of the singlet excited state. In mixed water:dimethyl sulfoxide solvent, ET becomes more unfavorable as FA adopts a more open conformation, thereby increasing the effective donor–acceptor distance and reducing the coupling energy. In contrast, no ET is observed for the cationic form of FA at low pH (6.0). In this case, the initial singlet excited state is localized on the pterin moiety of FA, and the excited-state charge distribution evolves with time. The charge redistribution in the pterin that occurs with intersystem crossing to the triplet state is characterized by changes in the transient IR spectrum. The excited-state lifetime is much longer in the absence of an ET quenching pathway. These results provide new insight into the mechanism of photodegradation and toxicity of FA. Ultrafast intramolecular ET in closed conformations of FA rapidly quenches the excited state and prevents efficient triplet state formation. Thus, conformations of FA that allow ultrafast intra-ET and rapid quenching of the singlet excited state play a key role in inhibiting pathological pathways following photoexcitation of FA.
Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) via an enediol(ate) intermediate. The active-site residue Glu165 serves as the catalytic base during catalysis. It abstracts a proton from C1 carbon of DHAP to form the reaction intermediate and donates a proton to C2 carbon of the intermediate to form product GAP. Our difference Fourier transform infrared spectroscopy studies on the yeast TIM (YeTIM)/phosphate complex revealed a C=O stretch band at 1706 cm -1 from the protonated Glu165 carboxyl group at pH 7.5, indicating that the pK a of the catalytic base is increased by >3.0 pH units upon phosphate binding, and that the Glu165 carboxyl environment in the complex is still hydrophilic in spite of the increased pK a . Hence, the results show that the binding of the phosphodianion group is part of the activation mechanism which involves the pK a elevation of the catalytic base Glu165. The deprotonation kinetics of Glu165 in the μs to ms time range were determined via infrared (IR) T-jump studies on the YeTIM/phosphate and ("heavy enzyme") [U- 13 C,- 15 N]YeTIM/phosphate complexes. The slower deprotonation kinetics in the ms time scale is due to phosphate dissociation modulated by the loop motion, which slows down by enzyme mass increase to show a normal heavy enzyme kinetic isotope effect (KIE) ∼1.2 (i.e., slower rate in the heavy enzyme). The faster deprotonation kinetics in the tens of μs time scale is assigned to temperature-induced pK a decrease, while phosphate is still bound, and it shows an inverse heavy enzyme KIE ∼0.89 (faster rate in the heavy enzyme). The IR static and T-jump spectroscopy provides atomic-level resolution of the catalytic mechanism because of its ability to directly observe the bond breaking/forming process.