UV resonance Raman measurements of poly-L-lysine's conformational energy landscapes: dependence on perchlorate concentration and temperature.

UV resonance Raman spectroscopy has been used to determine the conformational energy landscape of poly-L-lysine (PLL) in the presence of NaClO4 as a function of temperature. At 1 degree C, in the presence of 0.83 M NaClO4, PLL shows an approximately 86% alpha-helix-like content, which contains alpha-helix and pi-bulge/helix conformations. The high alpha-helix-like content of PLL occurs because of charge screening due to strong ion-pair formation between ClO4- and the lysine side chain -NH3+. As the temperature increases from 1 to 60 degrees C, the alpha-helix and pi-bulge/helix conformations melt into extended conformations (PPII and 2.51-helix). We calculate the Psi Ramachandran angle distribution of the PLL peptide bonds from the UV Raman spectra which allows us to calculate the PLL (un)folding energy landscapes along the Psi reaction coordinate. We observe a basin in the Psi angle conformational space associated with alpha-helix and pi-bulge/helix conformations and another basin for the extended PPII and 2.51-helical conformations.

UV Raman spatially resolved melting dynamics of isotopically labeled polyalanyl peptide: slow alpha-helix melting follows 3(10)-helices and pi-bulges premelting.

We used UV resonance Raman (UVRR) to examine the spatial dependence of the T-jump secondary structure relaxation of an isotopically labeled 21-residue mainly Ala peptide, AdP. The AdP penultimate Ala residues were perdeuterated, leaving the central residues hydrogenated, to allow separate monitoring of melting of the middle versus the end peptide bonds. For 5 to 30 degrees C T-jumps, the central peptide bonds show a approximately 2-fold slower relaxation time (189 +/- 31 ns) than do the exterior peptide bonds (97 +/- 15 ns). In contrast, for a 20 to 40 degrees C T-jump, the central peptide bond relaxation appears to be faster (56 +/- 6 ns) than that of the penultimate peptide bonds (131 +/- 46 ns). We show that, if the data are modeled as a two-state transition, we find that only exterior peptide bonds show anti-Arrhenius folding behavior; the middle peptide bonds show both normal Arrhenius-like folding and unfolding. This anti-Arrhenius behavior results from the involvement of pi-bulges/helices and 3(10)-helix states in the melting. The unusual temperature dependence of the (un)folding rates of the interior and exterior peptide bonds is due to the different relative (un)folding rates of 3(10)-helices, alpha-helices, and pi-bulges/helices. Pure alpha-helix unfolding rates are approximately 12-fold slower (approximately 1 micros) than that of pi-bulges and 3(10)-helices. In addition, we also find that the alpha-helix is most stable at the AdP N-terminus where eight consecutive Ala occur, whereas the three hydrophilic Arg located in the middle and at the C-terminus destabilize the alpha-helix in these regions and induce defects such as pi-bulges and 3(10)-helices.

Peptide secondary structure folding reaction coordinate: correlation between uv raman amide III frequency, Psi Ramachandran angle, and hydrogen bonding.

We used UV resonance Raman (UVRR) spectroscopy to quantitatively correlate the peptide bond AmIII3 frequency to its Psi Ramachandran angle and to the number and types of amide hydrogen bonds at different temperatures. This information allows us to develop a family of relationships to directly estimate the Psi Ramachandran angle from measured UVRR AmIII3 frequencies for peptide bonds (PBs) with known hydrogen bonding (HB). These relationships ignore the more modest Phi Ramachandran angle dependence and allow determination of the Psi angle with a standard error of +/-8 degrees , if the HB state of a PB is known. This is normally the case if a known secondary structure motif is studied. Further, if the HB state of a PB in water is unknown, the extreme alterations in such a state could additionally bias the Psi angle by +/-6 degrees . The resulting ability to measure Psi spectroscopically will enable new incisive protein conformational studies, especially in the field of protein folding. This is because any attempt to understand reaction mechanisms requires elucidation of the relevant reaction coordinate(s). The Psi angle is precisely the reaction coordinate that determines secondary structure changes. As shown elsewhere (Mikhonin et al. J. Am. Chem. Soc. 2005, 127, 7712), this correlation can be used to determine portions of the energy landscape along the Psi reaction coordinate.

Uncoupled peptide bond vibrations in alpha-helical and polyproline II conformations of polyalanine peptides.

We examined the 204-nm UV resonance Raman (UVR) spectra of the polyproline II (PPII) and alpha-helical states of a 21-residue mainly alanine peptide (AP) in different H2O/D2O mixtures. Our hypothesis is that if the amide backbone vibrations are coupled, then partial deuteration of the amide N will perturb the amide frequencies and Raman cross sections since the coupling will be interrupted; the spectra of the partially deuterated derivatives will not simply be the sum of the fully protonated and deuterated peptides. We find that the UVR spectra of the AmIII and AmII' bands of both the PPII conformation and the alpha-helical conformation (and also the PPII AmI, AmI', and AmII bands) can be exactly modeled as the linear sum of the fully N-H protonated and N-D deuterated peptides. Negligible coupling occurs for these vibrations between adjacent peptide bonds. Thus, we conclude that these peptide bond Raman bands can be considered as being independently Raman scattered by the individual peptide bonds. This dramatically simplifies the use of these vibrational bands in IR and Raman studies of peptide and protein structure. In contrast, the AmI and AmI' bands of the alpha-helical conformation cannot be well modeled as a linear sum of the fully N-H protonated and N-D deuterated derivatives. These bands show evidence of coupling between adjacent peptide bond vibrations. Care must be taken in utilizing the AmI and AmI' bands for monitoring alpha-helical conformations since these bands are likely to change as the alpha-helical length changes and the backbone conformation is perturbed.

Simple nanosecond to minutes transient absorption spectrophotometer.

We built a transient absorption spectrophotometer that can determine transient absorption spectral changes that occur at times as fast as approximately 200 ns and as slow as a minute. The transient absorption can be induced by a temperature-jump (T-jump) or by optical pumping from the deep ultraviolet (UV) to the infrared (IR) by use of single ns Nd:YAG laser pulses. Our use of a fiber-optic spectrometer coupled to a XeF flashlamp makes the collection of transient spectra easy and convenient in the spectral range from the near IR (1700 nm) down to the deep UV (200 nm), with high signal-to-noise (S/N) ratios. The spectral resolution is determined by the specific configuration of the fiber-optic spectrometer (grating groove density, fiber diameter, slit width) and varies between 0.3 and 10 nm. The utility of this spectrometer was demonstrated by measuring the rate at which a polymerized crystalline colloidal array (PCCA) of poly(N-isopropylacrylamide) nanogel particles optically switch light due to a T-jump induced by nanosecond 1.9 microm laser pulses. In addition, we measured the rate of optical switching induced by a 3 ns 355 nm pump pulse in PCCA functionalized with azobenzene.

Deep ultraviolet resonance Raman excitation enables explosives detection.

We measured the 229 nm absolute ultraviolet (UV) Raman cross-sections of the explosives trinitrotoluene (TNT), pentaerythritol tetranitrate (PETN), cyclotrimethylene-trinitramine (RDX), the chemically related nitroamine explosive HMX, and ammonium nitrate in solution. The 229 nm Raman cross-sections are 1000-fold greater than those excited in the near-infrared and visible spectral regions. Deep UV resonance Raman spectroscopy enables detection of explosives at parts-per-billion (ppb) concentrations and may prove useful for stand-off spectroscopic detection of explosives.

Computational and experimental determination of the alpha-helix unfolding reaction coordinate.

We demonstrate a calculated alpha-helix peptide folding energy landscape which accurately simulates the first experimentally measured alpha-helix melting energy landscape. We examine a 21-amino acid, mainly polyalanine peptide and calculate the free energy along the Psi Ramachandran angle secondary folding coordinate. The experimental free energy landscape was determined using UV resonance Raman spectroscopy. The relative free energy values are very close as are the equilibrium peptide conformations. We find 2.3 kcal/mol activation barriers between the alpha-helix-like and PPII-like basins. We also find that the alpha-helix-like conformations are quite defective and the alpha-helix-like structure dynamically samples 310-helix and pi-bulges.

Direct UV Raman monitoring of 3(10)-helix and pi-bulge premelting during alpha-helix unfolding.

We used UV resonance Raman (UVRR) spectroscopy exciting at approximately 200 nm within the peptide bond pi --> pi* transitions to selectively study the amide vibrations of peptide bonds during alpha-helix melting. The dependence of the amide frequencies on their Psi Ramachandran angles and hydrogen bonding enables us, for the first time, to experimentally determine the temperature dependence of the peptide bond Psi Ramachandran angle population distribution of a 21-residue mainly alanine peptide. These Psi distributions allow us to easily discriminate between alpha-helix, 3(10)-helix and pi-helix/bulge conformations, obtain their individual melting curves, and estimate the corresponding Zimm and Bragg parameters. A striking finding is that alpha-helix melting is more cooperative and shows a higher melting temperature than previously erroneously observed. These Psi distributions also enable the experimental determination of the Gibbs free energy landscape along the Psi reaction coordinate, which further allows us to estimate the free energy barriers along the AP melting pathway. These results will serve as a benchmark for the numerous untested theoretical studies of protein and peptide folding.

UV raman examination of alpha-helical peptide water hydrogen bonding.

UV resonance Raman spectra (UVRS) of an alpha-helical, 21 residue, mainly Ala peptide (AP) in the dehydrated solid state were compared to those in aqueous solution at different temperatures. The UVRS amide band frequencies of a dehydrated solid alpha-helix peptide show frequency shifts compared to those in aqueous solution due to the loss of amide backbone hydrogen bonding to water; the amide II and amide III bands of the solid alpha-helix downshift, while the amide I band upshifts. The shifts are identical in direction but smaller than those that occur for alpha-helices in aqueous solution as the temperature increases; water hydrogen bonding strengths decrease as the temperature increases. The UV Raman amide band frequency shifts can be used to monitor alpha-helix hydrogen bonding.

UV-resonance raman thermal unfolding study of Trp-cage shows that it is not a simple two-state miniprotein.

Trp-cage, a synthetic 20 residue polypeptide, is proposed to be an ultrafast folding synthetic miniprotein which utilizes tertiary contacts to define its native conformation. We utilized UV resonance Raman spectroscopy (UVRS) with 204 and 229 nm excitation to follow its thermal melting. Our results indicate that Trp-cage melting is complex, and it is not a simple two-state process. Using 204 nm excitation we probe the peptide secondary structure and find the Trp-cage's alpha-helix shows a broad melting curve where on average four alpha-helical amide bonds melt upon a temperature increase from 4 to 70 degrees C. Using 229 nm excitation we probe the environment of the Trp side chain and find that its immediate environment becomes more compact as the temperature is increased from 4 to 20 degrees C; however, further temperature increases lead to exposure of the Trp to water. The chi(2) angle of the Trp side chain remains invariant throughout the entire temperature range. Previous kinetic results indicated a single-exponential decay in the 4-70 degrees C temperature range, suggesting that Trp-cage behaves as a two-state folder. However, this miniprotein does not show clear two-state behavior in our steady-state studies. Rather it shows a continuous distribution of steady-state spectral parameters. Only the alpha-helix melting curve even hints of a cooperative transition. Possibly, the previous kinetic results monitor only a small region of the Trp-cage which locally appears two-state. This would then argue for spatially decoupled folding even for this small peptide.

UV resonance Raman determination of polyproline II, extended 2.5(1)-helix, and beta-sheet Psi angle energy landscape in poly-L-lysine and poly-L-glutamic acid.

UV resonance Raman (UVR) spectroscopy was used to examine the solution conformation of poly-l-lysine (PLL) and poly-l-glutamic acid (PGA) in their non-alpha-helical states. UVR measurements indicate that PLL (at pH = 2) and PGA (at pH = 9) exist mainly in a mixture of polyproline II (PPII) and a novel left-handed 2.5(1)-helical conformation, which is an extended beta-strand-like conformation with Psi approximately +170 degrees and Phi approximately -130 degrees . Both of these conformations are highly exposed to water. The energies of these conformations are very similar. We see no evidence of any disordered "random coil" states. In addition, we find that a PLL and PGA mixture at neutral pH is approximately 60% beta-sheet and contains PPII and extended 2.5(1)-helix conformations. The beta-sheet conformation shows little evidence of amide backbone hydrogen bonding to water. We also developed a method to estimate the distribution of Psi Ramachandran angles for these conformations, which we used to estimate a Psi Ramachandran angle energy landscape. We believe that these are the first experimental studies to give direct information on protein and peptide energy landscapes.