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 AmIII 3 frequency to its Ψ 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 Ψ Ramachandran angle from measured UVRR AmIII 3 frequencies for peptide bonds (PBs) with known hydrogen bonding (HB). These relationships ignore the more modest Φ Ramachandran angle dependence and allow determination of the Ψ angle with a standard error of (8°, 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 Ψ angle by (6°. The resulting ability to measure Ψ 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 Ψ 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 Ψ reaction coordinate. Introduction The various techniques of molecular spectroscopy constitute the toolset used by scientists for investigating molecular conformations and reaction mechanisms. These various spectroscopic techniques require quantitative correlations between the spectral parameters measured and the molecular conformational parameters. NMR and especially multidimensional NMR techniques are certainly the most powerful spectroscopic methods for solution studies. 1928 J. Phys. Chem. B 2006, 110, 1928-1943 10 by C R -D. We are optimistic that these relationships will be very useful for protein conformational studies, especially in the field of protein folding. This is because any attempt to understand reaction mechanisms, such as, for example, protein folding, requires elucidation of the relevant reaction coordinate(s). The Ψ angle is precisely the reaction coordinate that determines secondary structure changes. As shown elsewhere 60 the correlation we propose can be used to experimentally determine features of the energy landscape along this Ψ reaction coordinate. Such an experimental insight into a protein conformation and energy landscape is crucially needed, since there are still a lot of unresolved questions regarding the theoretical modeling of protein folding despite remarkable recent achievments. As described elsewhere, 43 the 21-residue alanine-based peptide AAAAA(AAARA) 3 A (AP) was prepared (HPLC pure) at the Pittsburgh Peptide Facility by using the solid-state peptide synthesis method. The AP solutions in water contained 1 mg/ mL concentrations of AP, and 0.2 M concentrations of sodium perchlorate, which was used as an internal intensity and frequency standard. UV Resonance Raman Instrumentation. The Raman instrumentation has been described in detail elsewhere. Results and Discussion Dependence of AmIII 3 Frequency on Ramachandran Angles and Hydrogen Bonding. The amide III (AmIII) band region is complex. We recently examined this spectral region in detail and identified a band, which we call AmIII 3 and which is most sensitive to the peptide bond conformation. Peptide Secondary Structure Folding Reaction Coordinate J. Phys. Chem. B, Vol. 110, No. 4, 2006 1929 gives rise to strong N-H to C R -H bend coupling. In contrast, for R-helix-like Ψ and Φ Ramachandran angles the N-H and C R -H bonds are approximately trans The physical origin of this Ψ angle AmIII 3 frequency dependence is that the hydrogen van der Waals radii in the C R -H and N-H bonds are in contact for positive Ψ angles Relative Impact of the Ψ and Φ Ramachandran Angles on the AmIII 3 Frequency. Although the projections of the N-H and C R -H bending motions on each other (and as a result the degree of coupling between them) depend on both the Ψ and Φ Ramachandran angles, an examination of a model of a peptide bond Asher et al. Mirkin and Krimm 73 theoretically examined the Ψ and Φ frequency dependence of the AmIII band of "alanine dipeptide" (N-acetyl-L-alanine-N-methylamide). They concentrated on peptide bond 2, whose frequencies were close to those measured experimentally. Although Mirkin and Krimm claim in their conclusions, that AmIII frequency shows strong dependence on both Ψ and Φ Ramachandran angles, we note that the impact of changes in the Φ angle is relatively modest if we only include the allowed regions of the Ramachandran plot In the allowed regions of Ramachandran plot, Mirkin and Krimm 73 calculated a 25-40 cm -1 AmIII frequency span over the allowed Ψ angles for fixed Φ angles In addition, the largest 16 cm -1 span in the AmIII frequency with Φ angle occurs in an almost forbidden region of the Ramachandran plot between the -sheet and R-helical regions (at Φ angles of -134°and -90°and Ψ angle of 60°, Figures 47 for details). Black line (s): Fit of calculated points using the eq 3 (see text for detail). Note: Grey regions show the forbidden and/or nearly forbidden Ψ Ramachandran angles based on recent Ramachandran plots. 1930 J. Phys. Chem. B, Vol. 110, No. 4, 2006 Mikhonin et al. 3 and 4). In contrast, in the R-helical region of the Ramachandran plot the AmIII frequency of alanine dipeptide shows no more than 8 cm -1 Φ angular span, while in the -strand region of the Ramachandran plot the AmIII frequency shows no more than 6 cm -1 Φ dependence Ianoul et al.'s 59 combined experimental and theoretical studies of Ac-X-OCH 3 (X ) Val, Ile, Leu, Lys, Ala) revealed a 9 cm -1 AmIII 3 frequency shift upon an 18°increase of the Φ Ramachandran angle from -96 to -78°. In addition, Ianoul et al. also performed theoretical calculations for Ala-Ala at a fixed R-helix-like Ψ angle of -21°and calculated only a 3 cm -1 AmIII 3 frequency upshift upon the 20°increase of Φ angle from -95°to -75°. Thus, Ianoul et al. never observed more than a 9 cm -1 shift of AmIII 3 frequency due to variation of the Φ Ramachandran angle. In addition, we recently 60 measured the UVRR AmIII 3 frequencies of two different secondary structure conformations in aqueous solutions with very similar Φ angles, but very different Ψ angles. Specifically, an equimolar mixture of PLL and PGA forms an antiparallel -sheet 60 (Ψ ≈ 135°, Φ ≈ -139°), which shows an AmIII 3 frequency at 1227 cm -1 . In contrast individual PLL and PGA samples form extended 2.5 1 -helices 60 (Ψ ≈ 170°, Φ ≈ -130°), which show AmIII 3 frequencies at ∼1271 cm -1 . To summarize, the total Φ angular span of the AmIII 3 frequencies appears experimentally 59 to be no more than 9 cm -1 and no more than 16 cm -1 in the allowed regions of the Ramachandran plot from theoretical calculations. Thus, we conclude that Ψ Ramachandran angular dependence of the AmIII 3 frequency dominates the Φ angular dependence in the allowed regions of Ramachandran plot. If we totally neglect the Φ angular dependence of AmIII 3 frequency, this could enable an error in the Ψ-dependent AmIII 3 frequency of no more than (8 cm -1 (since the total Φ angular span of AmIII 3 frequencies no higher than 16 cm -1 , Formation of PB-water and PB-PB HBs upshift the AmIII 3 frequency, in part, due to the resulting increased C(O)dN double ν III3 (ψ,φ,HB P-P ,HB P-W ,T) = ν III3 (ψ,HB P-P ,HB P-W ,T) (2) ν III3 (ψ,HB P-P ,HB P-W ,T) = {ν 0 -A sin(ψ -R 0 )} + Δν III3 (HB P-P ,HB P-W ,T) (4) Peptide Secondary Structure Folding Reaction Coordinate J. Phys. Chem. B, Vol. 110, No. 4, 2006 1931 bond character. 75, The relationships given below by eqs 5 (for non-HB PB in a vacuum), 6A-D (PB in aqueous solutions), and 7A-C (PB in the absence of water) are shown in Correlation between AmIII 3 Frequency and Ψ Ramachandran Angle in the Absence of HB. We measured the UVR AmIII 3 frequencies for the AP R-helix 42,89 (∼1263 cm -1 , 0°C), XAO PPII 42,43 (1247 cm -1 , 0°C), PLL and PGA 2.5 1 -helix 60 (∼1271 cm -1 , 0°C), and PLL-PGA mixture antiparallel -sheet 60 (∼1227 cm -1 , 0°C) conformations of different polypeptides in aqueous solutions. Each of these conformations has known Ramachandran angles We can calculate the AmIII 3 frequencies that would result from the above peptide conformations in the fictitious case where the PB did not partake in any HB at all. This would be done by subtracting the HB-induced AmIII 3 frequency shifts By fitting the above four "non-HB" data points to eq 3, we obtain the following semiempirical relationship, which relates 1932 J. Phys. Chem. B, Vol. 110, No. 4, 2006 Mikhonin et al. the AmIII 3 frequency to the Ψ Ramachandran angle dependent coupling between N-H and C R -H bending motions The blue curve in Correlation of AmIII 3 Frequency and Ψ Ramachandran Angle for Two-End-On PB-PB HBs: Infinite r-Helix, Interior Strands of -Sheet in Water. Each PB in infinitely long R-helices and in interior strands of multistranded -sheets in aqueous solutions (Appendix, The green curve in The magenta curve in Peptide Secondary Structure Folding Reaction Coordinate J. Phys. Chem. B, Vol. 110, No. 4, 2006 1933 cm -1 HB-induced upshift as well as the temperature-dependent term to eq 5 and write The black curve in Correlation of AmIII 3 Frequency and Ψ Angle for a PB in Water If Its HB State Is Unknown. If the HB state of a PB in aqueous solution is unknown, we suggest the use of eq 6E, which is the "average" of eqs 6A-D. This will minimize the error in determination of the Ψ Ramachandran angle and will allow the estimation of the Ψ angle with the error bounds discussed below. Correlation between AmIII 3 Frequency and Ψ Ramachandran Angle in Peptide Crystals. Figures 6 and 7 show that the crystal data appear to roughly follow the sinusoidal relationship between the AmIII 3 frequency and the Ψ Ramachandran angle (see red dashed curve in Anhydrous r-Helical and -Sheet Conformations. If we dehydrate a two-end-on PB-PB HB R-helical conformation, we will see a 5 cm -1 AmIII 3 frequency downshift due to the loss of hydrogen bonding to the normally present sheath of water. The In the case of a PB where only the CdO group is involved in PB-PB HB, we estimate that the AmIII 3 frequency is 12 cm -1 upshifted with respect to non-HB PB (Appendix, The In the case of PB, where only the NH group is PB-PB HB, we estimate the AmIII 3 frequency to be 35 cm -1 upshifted with respect to non-HB PB (Appendix, The black curve in Thus, the families of eqs 6A-D and 7A-C predict the correlation between the AmIII 3 frequency and the Ψ angle for the common conformations of peptides and proteins. If the HB is known for a particular PB, the appropriate equation can be used to determine its Ψ angle from the observed AmIII 3 frequency. In the case where the HB state of a PB in aqueous solution is unknown, one can use eq 6E. These relationships will become less accurate if the PB has an unusual Φ angle or unusual HB pattern (see below). Prediction of UVRR AmIII 3 Frequencies of Other Secondary Structures. On the basis of the known Ψ Ramachandran angle and HB patterns, we can predict the AmIII 3 frequencies of other secondary structures such as the π-helix, 3 10 -helix UVRR spectra of HEWL amyloid fibrils, 100 which are dominated by -sheet conformations contain three spectroscopic features in the AmIII 3 region: ∼1210, ∼1230, and ∼1255 cm -1 . The dominating ∼1230 cm -1 feature certainly derives from antiparallel -sheet, though a minor contribution of several turn conformations is also possible The error associated with neglecting the Φ angle also gives rise to the uncertainty in the Ψ angle determination. Ianoul et al. Additional bias can occur if we do not know the HB state of a PB in water. This could give rise to a bias of the AmIII 3 frequency of (6 cm -1 , which would lead to a Ψ angle bias of (6°in eq 6E. Thus, a typical UV Raman measurement of a typical sample would find a random error of e(8°in the Ψ angle, assuming a known HB state. However, extreme alterations in the unknown HB state of a PB in water could additionally bias the Ψ angle by (6°. Conclusions We used UV resonance Raman spectroscopy to investigate the dependence of the AmIII 3 frequency on the Ψ Ramachandran angle and on the nature of PB HBs. These results allow us to formulate relationships that allow us to estimate the Ψ Ramachandran angles from observed AmIII 3 frequencies for both aqueous solutions of peptides and proteins as well as for the anhydrous states of peptides and proteins. A typical Raman measurement of a typical sample would find a random error of e(8°in the Ψ angle, assuming a known HB state. However, if the HB state of a PB in water is unknown, extreme alterations in such a state could additionally bias the Ψ angle by (6°. We are optimistic that these relationships will be very useful for protein conformational studies, especially in the field of protein folding. This is because any attempt to understand reaction mechanisms, such as protein folding, requires elucidation of the relevant reaction coordinate(s). The Ψ angle is precisely the reaction coordinate that determines secondary structure changes. As shown elsewhere, 60 the correlation we propose can be used to determine features of the energy landscape along this Ψ reaction coordinate

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

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

UV resonance Raman study of the spatial dependence of α-helix unfolding

We used ultraviolet resonance Raman (UVRR) spectra to examine the spatial dependence and the thermodynamics of α-helix melting of an isotopically labeled α-helical, 21-residue, mainly alanine peptide. The peptide was synthesized with six natural abundance amino acids at the center and mainly perdeuterated residues elsewhere. Cα deuteration of a peptide bond decouples Cα-H bending from N-H bending, which significantly shifts the random coil conformation amide III band; this shift clearly resolves it from the amide III band of the nondeuterated peptide bonds. Analysis of the isotopically spectrally resolved amide III bands from the external and central peptide amide bonds show that the six central amide bonds have a higher α-helix melting temperature (∼32°C) than that of the exterior amide bonds (∼5°C)

Nanogel Nanosecond Photonic Crystal Optical Switching

We developed a robust nanosecond photonic crystal switching material by using poly(N-isopropylacrylamide) (PNIPAM) nanogel colloidal particles that self-assemble into crystalline colloidal arrays (CCAs). The CCA was polymerized into a loose-knit hydrogel which permits the individual embedded nanogel PNIPAM particles to coherently and synchronously undergo their thermally induced volume phase transitions. A laser T-jump from 30 to 35°C actuates the nanogel particle shrinkage; the resulting increased diffraction decreases light transmission within 900 ns. Additional transmission decreases occur with characteristic times of 19 and 130 ns. Individual NIPAM sphere volume switching occurs in the ∼100 ns time regime. These nanogel nanosecond phenomena may be useful in the design of fast photonic crystal switches and optical limiting materials. Smaller nanogels will show even faster volume phase transitions

Photochemically Controlled Photonic Crystals

We have developed photochemically controlled photonic crystals that may be useful in novel recordable and erasable memories and/or display devices. These materials can operate in the UV, visible, or near-IR spectral regions. Information is recorded and erased by exciting the photonic crystal with ∼360 nm UV light or ∼480 nm visible light. The information recorded is read out by measuring the photonic crystal diffraction wavelength. The active element of the device is an azobenzene-functionalized hydrogel, which contains an embedded crystalline colloidal array. UV excitation forms cis-azobenzene while visible excitation forms trans-azobenzene. The more favorable free energy of mixing of cis-azobenzene causes the hydrogel to swell and to red-shift the photonic crystal diffraction. We also observe fast nanosecond, microsecond, and millisecond transient dynamics associated with fast heating lattice constant changes, refractive index changes, and thermal relaxations

UV resonance Raman elucidation of the terminal and internal peptide bond conformations of crystalline and solution oligoglycines

Spectroscopic investigations of macromolecules generally attempt to interpret the measured spectra in terms of the summed contributions of the different molecular fragments. This is the basis of the local-mode approximation in vibrational spectroscopy. In the case of resonance Raman spectroscopy, independent contributions of molecular fragments require both a local-mode-like behavior and the uncoupled electronic transitions. Here, we show that the deep-UV resonance Raman spectra of aqueous solution-phase oligoglycines show independent peptide bond molecular fragment contributions, indicating that peptide bond electronic transitions and vibrational modes are uncoupled. We utilize this result to separately determine the conformational distributions of the internal and penultimate peptide bonds of oligoglycines. Our data indicate that in aqueous solution, the oligoglycine terminal residues populate conformations similar to those found in crystals (31-helices and β-strands) but with a broader distribution, while the internal peptide bond conformations are centered around the 31-helix Ramachandran angles. © 2009 American Chemical Society

UV resonance Raman investigation of the conformations and lowest energy allowed electronic excited states of tri- and tetraalanine: Charge transfer transitions

UV resonance Raman excitation profiles and Raman depolarization ratios were measured for trialanine and tetraalanine between 198 and 210 nm. Excitation within the π → π* electronic transitions of the peptide bond results in UVRR spectra dominated by amide peptide bond vibrations. In addition to the resonance enhancement of the normal amide vibrations, we find enhancement of the symmetric terminal COO- vibration. The Ala3 UVRR AmIII3 band frequencies indicate that poly-proline II and 2.5 1 helix conformations and type II turns are present in solution. We also find that the conformation of the interior peptide bond of Ala4 is predominantly poly-proline-II-like. The Raman excitation profiles of both Ala3 and Ala4 reveal a charge transfer electronic transition at 202 nm, where electron transfer occurs from the terminal nonbonding carboxylate orbital to the adjacent peptide bond π* orbital. Raman depolarization ratio measurements support this assignment. An additional electronic transition is found in Ala4 at 206 nm. © 2010 American Chemical Society

Steady-state and transient ultraviolet resonance Raman spectrometer for the 193-270 nm spectral region

We describe a state-of-the-art tunable ultraviolet (UV) Raman spectrometer for the 193-270 nm spectral region. This instrument allows for steady-state and transient UV Raman measurements. We utilize a 5 kHz Ti-sapphire continuously tunable laser (∼20 ns pulse width) between 193 nm and 240 nm for steady-state measurements. For transient Raman measurements we utilize one Coherent Infinity YAG laser to generate nanosecond infrared (IR) pump laser pulses to generate a temperature jump (T-jump) and a second Coherent Infinity YAG laser that is frequency tripled and Raman shifted into the deep UV (204 nm) for transient UV Raman excitation. Numerous other UV excitation frequencies can be utilized for selective excitation of chromophoric groups for transient Raman measurements. We constructed a subtractive dispersion double monochromator to minimize stray light. We utilize a new charge-coupled device (CCD) camera that responds efficiently to UV light, as opposed to the previous CCD and photodiode detectors, which required intensifiers for detecting UV light. For the T-jump measurements we use a second camera to simultaneously acquire the Raman spectra of the water stretching bands (2500-4000 cm -1) whose band-shape and frequency report the sample temperature. © 2005 Society for Applied Spectroscopy