Kan Xiong

UV Resonance Raman Investigations of Peptide and Protein Structure and Dynamics

A study was conducted to demonstrate ultraviolet resonance Raman (UVRR) investigations of peptide and protein structure and dynamics. The tuning of the excitation wavelengths allowed the probing of different chromophoric segments of a macromolecule. Another advantage of deep UV Raman measurements was that there was no interference from molecular relaxed fluorescence, as those chromophores that had their first transition below 260 nm were highly flexible and possessed small fluorescence quantum yields. UVRR was also used in pump-probe measurements to give kinetic information on fast biological processes. It was a powerful technique for studying static protein structure and for studying protein dynamics, such as in protein folding. The rapid development of UVRR was aided by the latest advancements in lasers, optics, and detectors.

Salt dependence of an α-helical peptide folding energy landscapes

We used CD, UV resonance Raman spectroscopy, and molecular dynamics simulation to examine the impact of salts on the conformational equilibria and the Ramachandran Psi angle (un)folding Gibbs free energy landscape coordinate of a mainly polyalanine alpha-helical peptide, AP of sequence AAAAA(AAARA)(3)A. NaClO(4) stabilizes alpha-helical-like conformations more than does NaCl, which stabilizes more than Na(2)SO(4) at identical ionic strengths. This alpha-helix stabilization ordering is the reverse of the Hofmeister series of anions in their ability to disorder water hydrogen bonding. Much of the NaClO(4) alpha-helix stabilization results from ClO(4)(-) association with the AP terminal -NH(3)(+) groups and Arg side chains. ClO(4)(-) stabilizes 3(10)-helix conformations but destabilizes turn conformations. The decreased Cl(-) and SO(4)(2-) AP alpha-helix stabilization probably results from a decreased association with the Arg and terminal -NH(3)(+) groups. Cl(-) is expected to have a smaller binding affinity and thus stabilizes alpha-helical conformations intermediately between NaClO(4) and Na(2)SO(4). Electrostatic screening stabilizes pi-bulge conformations.

UV resonance raman spectroscopy monitors polyglutamine backbone and side chain hydrogen bonding and fibrillization

We utilize 198 and 204 nm excited UV resonance Raman spectroscopy (UVRR) and circular dichroism spectroscopy (CD) to monitor the backbone conformation and the Gln side chain hydrogen bonding (HB) of a short, mainly polyGln peptide with a D(2)Q(10)K(2) sequence (Q10). We measured the UVRR spectra of valeramide to determine the dependence of the primary amide vibrations on amide HB. We observe that a nondisaggregated Q10 (NDQ10) solution (prepared by directly dissolving the original synthesized peptide in pure water) exists in a β-sheet conformation, where the Gln side chains form hydrogen bonds to either the backbone or other Gln side chains. At 60 °C, these solutions readily form amyloid fibrils. We used the polyGln disaggregation protocol of Wetzel et al. [Wetzel, R., et al. (2006) Methods Enzymol.413, 34-74] to dissolve the Q10 β-sheet aggregates. We observe that the disaggregated Q10 (DQ10) solutions adopt PPII-like and 2.5(1)-helix conformations where the Gln side chains form hydrogen bonds with water. In contrast, these samples do not form fibrils. The NDQ10 β-sheet solution structure is essentially identical to that found in the NDQ10 solid formed upon evaporation of the solution. The DQ10 PPII and 2.5(1)-helix solution structure is essentially identical to that in the DQ10 solid. Although the NDQ10 solution readily forms fibrils when heated, the DQ10 solution does not form fibrils unless seeded with the NDQ10 solution. This result demonstrates very high activation barriers between these solution conformations. The NDQ10 fibril secondary structure is essentially identical to that of the NDQ10 solution, except that the NDQ10 fibril backbone conformational distribution is narrower than in the dissolved species. The NDQ10 fibril Gln side chain geometry is more constrained than when NDQ10 is in solution. The NDQ10 fibril structure is identical to that of the DQ10 fibril seeded by the NDQ10 solution.

Direct observations of conformational distributions of intrinsically disordered p53 peptides using UV Raman and explicit solvent simulations.

We report the first experimental measurements of Ramachandran Ψ-angle distributions for intrinsically disordered peptides: the N-terminal peptide fragment of tumor suppressor p53 and its P27S mutant form. To provide atomically detailed views of the conformational distributions, we performed classical, explicit-solvent molecular dynamics simulations on the microsecond time scale. Upon binding its partner protein, MDM2, wild-type p53 peptide adopts an α-helical conformation. Mutation of Pro27 to serine results in the highest affinity yet observed for MDM2-binding of the p53 peptide. Both UV resonance Raman spectroscopy (UVRR) and simulations reveal that the P27S mutation decreases the extent of PPII helical content and increases the probability for conformations that are similar to the α-helical MDM2-bound conformation. In addition, UVRR measurements were performed on peptides that were isotopically labeled at the Leu26 residue preceding the Pro27 in order to determine the conformational distributions of Leu26 in the wild-type and mutant peptides. The UVRR and simulation results are in quantitative agreement in terms of the change in the population of non-PPII conformations involving Leu26 upon mutation of Pro27 to serine. Finally, our simulations reveal that the MDM2-bound conformation of the peptide is significantly populated in both the wild-type and mutant isolated peptide ensembles in their unbound states, suggesting that MDM2 binding of the p53 peptides may involve conformational selection.

Circular dichroism and UV resonance Raman study of the impact of alcohols on the Gibbs free energy landscape of an α-helical peptide

We used CD and UV resonance Raman spectroscopy to study the impact of alcohols on the conformational equilibria and relative Gibbs free energy landscapes along the Ramachandran Psi-coordinate of a mainly poly-Ala peptide, AP with an AAAAA(AAARA)(3)A sequence. 2,2,2-Trifluoroethanol (TFE) most stabilizes the alpha-helix-like conformations, followed by ethanol, methanol, and pure water. The pi-bulge conformation is stabilized more than the alpha-helix, while the 3(10)-helix is destabilized due to the alcohol-increased hydrophobicity. Turns are also stabilized by alcohols. We also found that while TFE induces more alpha-helices, it favors multiple, shorter helix segments.

Lowest Energy Electronic Transition in Aqueous Cl− Salts: Cl− → (H2O)6 Charge Transfer Transition

We use UV resonance Raman spectroscopy to probe the lowest energy allowed electronic transitions of aqueous solutions containing Cl(-) salts. We show that the waters hydrating the Cl(-) are involved in charge transfer transitions that transfer electron density from Cl(-) to the water molecules. These charge transfer transitions cause significant change in the H-O-H bond angle in the excited state, which results in a strong enhancement of the preresonance Raman intensity of the water bending modes. Our work gives the first insight into the lowest allowed electronic transition of hydrated Cl(-).

Conformation of poly-l-glutamate is independent of ionic strength

CD and UV resonance Raman measurements surprisingly find that the charge screening of even 2 M concentrations of NaCl and KCl does not alter the unfolded PPII and 2.5(1)-helix conformations of poly-L-glutamate. These salts appear to be excluded from the region between the side chain charges and the peptide backbone. Furthermore, no direct ion pairing occurs between these salts and the side chain carboxylates.

Impact of Ion Binding on Poly-l-Lysine (Un)folding Energy Landscape and Kinetics

We utilize T-jump UV resonance Raman spectroscopy (UVRR) to study the impact of ion binding on the equilibrium energy landscape and on (un)folding kinetics of poly-L-lysine (PLL). We observe that the relaxation rates of the folded conformations (including π-helix (bulge), pure α-helix, and turns) of PLL are slower than those of short alanine-based peptides. The PLL pure α-helix folding time is similar to that of short alanine-based peptides. We for the first time have directly observed that turn conformations are α-helix and π-helix (bulge) unfolding intermediates. ClO(4)(-) binding to the Lys side chain -NH(3)(+) groups and the peptide backbone slows the α-helix unfolding rate compared to that in pure water, but little impacts the folding rate, resulting in an increased α-helix stability. ClO(4)(-) binding significantly increases the PLL unfolding activation barrier but little impacts the folding barrier. Thus, the PLL folding coordinate(s) differs from the unfolding coordinate(s). The-π helix (bulge) unfolding and folding coordinates do not directly go through the α-helix energy well. Our results clearly demonstrate that PLL (un)folding is not a two-state process.

Circular Dichroism and UV Resonance Raman Study of the Impact of Salts and Alcohols on the Gibbs Free Energy Landscape of an α‐helical Peptide

We used CD and UV resonance Raman spectroscopy to study the impact of alcohols on the conformational equilibria and relative Gibbs free energy landscapes along the Ramachandran Ψ-coordinate of a mainly poly-Ala peptide, AP with an AAAAA(AAARA)3A sequence. 2,2,2-Trifluoroethanol (TFE) most stabilizes the α-helix-like conformations, followed by ethanol, methanol, and pure water. The π-bulge conformation is stabilized more than the α-helix, while the 310-helix is destabilized due to the alcohol-increased hydrophobicity. Turns are also stabilized by alcohols. We also found that while TFE induces more α-helices, it favors multiple, shorter helix segments.