Disulfide Bridges Remain Intact while Native Insulin Converts into Amyloid Fibrils

Amyloid fibrils are β-sheet-rich protein aggregates commonly found in the organs and tissues of patients with various amyloid-associated diseases. Understanding the structural organization of amyloid fibrils can be beneficial for the search of drugs to successfully treat diseases associated with protein misfolding. The structure of insulin fibrils was characterized by deep ultraviolet resonance Raman (DUVRR) and Nuclear Magnetic Resonance (NMR) spectroscopy combined with hydrogen-deuterium exchange. The compositions of the fibril core and unordered parts were determined at single amino acid residue resolution. All three disulfide bonds of native insulin remained intact during the aggregation process, withstanding scrambling. Three out of four tyrosine residues were packed into the fibril core, and another aromatic amino acid, phenylalanine, was located in the unordered parts of insulin fibrils. In addition, using all-atom MD simulations, the disulfide bonds were confirmed to remain intact in the insulin dimer, which mimics the fibrillar form of insulin

Direct UV Raman monitoring of 3<inf>10</inf>-helix and π-bulge premelting during α-helix unfolding

We used UV resonance Raman (UVRR) spectroscopy exciting at ∼200 nm within the peptide bond π→π* transitions to selectively study the amide vibrations of peptide bonds during α-helix melting. The dependence of the amide frequencies on their Ψ Ramachandran angles and hydrogen bonding enables us, for the first time, to experimentally determine the temperature dependence of the peptide bond Ψ Ramachandran angle population distribution of a 21-residue mainly alanine peptide. These Ψ distributions allow us to easily discriminate between α-helix, 310-helix and α-helix/bulge conformations, obtain their individual melting curves, and estimate the corresponding Zimm and Bragg parameters. A striking finding is that α-helix melting is more cooperative and shows a higher melting temperature than previously erroneously observed. These Ψ distributions also enable the experimental determination of the Gibbs free energy landscape along the Ψ 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. © 2006 American Chemical Society

Uncoupled peptide bond vibrations in α-helical and polyproline II conformations of tolyalanine peptides

We examined the 204-nm UV resonance Raman (UVR) spectra of the polyproline II (PPII) and α-helical states of a 21-residue mainly alanine peptide (AP) in different H 2O/D 2O 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 α-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 α-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 α-helical conformations since these bands are likely to change as the α-helical length changes and the backbone conformation is perturbed. © 2005 American Chemical Society

UV Raman demonstrates that α-helical polyalanine peptides melt to polyproline II conformations

We examined the 204-nm UV Raman spectra of the peptide XAO, which was previously found by Shi et al.'s NMR study to occur in aqueous solution in a polyproline II (PPII) conformation (Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9190). The UV Raman spectra of XAO are essentially identical to the spectra of small peptides such as ala5 and to the large 21 -residue predominantly Ala peptide, AP. We conclude that the non-α-helical conformations of these peptides are dominantly PPII. Thus, AP, which is highly α-helical at room temperature, melts to a PPII conformation. There is no indication of any population of intermediate disordered conformations. We continued our development of methods to relate the Ramachandran ψ-angle to the amide III band frequency. We describe a new method to estimate the Ramachandran ψ-angular distributions from amide III band line shapes measured in 204-nm UV Raman spectra. We used this method to compare the ψ-distributions in XAO, ala5, the non-α-helical state of AP, and acid-denatured apomyoglobin. In addition, we estimated the ψ-angle distributions of peptide bonds which occur in non-α-helix and non-β-sheet conformations in a small library of proteins

UV resonance Raman determination of polyproline II, extended 2.5 <inf>1</inf>-helix, and β-sheet ψ 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-α-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 2.51-helical conformation, which is an extended β-strand-like conformation with Ψ ≈ 1170° and Φ ≈ 130°. 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 ∼60% β-sheet and contains PPII and extended 2.5 1-helix conformations. The β-sheet conformation shows little evidence of amide backbone hydrogen bonding to water. We also developed a method to estimate the distribution of Ψ Ramachandran angles for these conformations, which we used to estimate a Ψ Ramachandran angle energy landscape. We believe that these are the first experimental studies to give direct information on protein and peptide energy landscapes. © 2005 American Chemical Society

Assignments and conformational dependencies of the amide III peptide backbone UV resonance Raman bands

We investigated the assignments and the conformational dependencies of the UV resonance Raman bands of the 21-residue mainly alanine peptide (AP) and its isotopically substituted derivatives in both their α-helical and PPII states. We also examined smaller peptides to correlate conformation, hydrogen bonding, and structure. Our vibrational mode analysis confirms the complex nature of the amide III region, which contains many vibrational modes. We assign these bands by interpreting the isotopically induced frequency shifts and the conformational sensitivity of these bands and their temperature dependence. Our assignments of the amide bands in some cases agree, but in other cases challenge previous assignments by Lee and Krimm (Biopolymers 1998, 46, 283-317), Overman and Thomas (Biochemistry 1998, 37, 5654-5665), and Diem et al. (J. Phys. Chem. 1992, 96, 548-554). We see evidence for the partial dehydration of α-helices at elevated temperatures

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. © 2010 Society for Applied Spectroscopy

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 α-helix shows a broad melting curve where on average four α-helical amide bonds melt upon a temperature increase from 4 to 70 °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 °C; however, further temperature increases lead to exposure of the Trp to water. The χ2 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 °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 α-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. © 2005 American Chemical Society

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 polyL-lysine (PLL) in the presence of NaClO4 as a function of temperature. At 1 °C, in the presence of 0.83 M NaClO4, PLL shows an ∼86% α-helix-like content, which contains ́-helix and π-bulge/helix conformations. The high a-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 °C, the ́-helix and π-bulge/helix conformations melt into extended conformations (PPII and 2.51-helix). We calculate the Ψ 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 Ψ reaction coordinate. We observe a basin in the Ψ angle conformational space associated with α-helix and π-bulge/helix conformations and another basin for the extended PPII and 2.5 1-helical conformations. © 2007 American Chemical Society

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 -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 μm 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. © 2005 Society for Applied Spectroscopy