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

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

Circular dichroism and ultraviolet resonance raman indicate little Arg-Glu side chain α-helix peptide stabilization

Electrostatic interactions between side chains can control the conformation and folding of peptides and proteins. We used circular dichroism (CD) and ultraviolet (UV) resonance Raman spectroscopy (UVRR) to examine the impact of side chain charge on the conformations of two 21 residue mainly polyala peptides with a few Arg and Glu residues. We expected that attractions between Arg-10 and Glu-14 side chains would stabilize the α-helix conformation compared to a peptide with an Arg-14. Surprisingly, CD suggests that the peptide with the Glu-14 is less helical. In contrast, the UVRR show that these two peptides have similar α-helix content. We conclude that the peptide with Glu-14 has the same net α-helix content as the peptide with the Arg but has two α-helices of shorter length. Thus, side chain interactions between Arg-10 and Glu-14 have a minor impact on α-helix stability. The thermal melting of these two peptides is similar. However the Glu-14 peptide pH induced melting forms type III turn structures that form α-helix-turn-α-helix conformations. © 2011 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

Elucidating peptide and protein structure and dynamics: UV resonance raman spectroscopy

UV resonance Raman spectroscopy (UVRR) is a powerful method that has the requisite selectivity and sensitivity to incisively monitor biomolecular structure and dynamics in solution. In this Perspective, we highlight applications of UVRR for studying peptide and protein structure and the dynamics of protein and peptide folding. UVRR spectral monitors of protein secondary structure, such as the amide III3 band and the Cα-H band frequencies and intensities, can be used to determine Ramachandran ψ angle distributions for peptide bonds. These incisive, quantitative glimpses into conformation can be combined with kinetic T-jump methodologies to monitor the dynamics of biomolecular conformational transitions. The resulting UVRR structural insight is impressive in that it allows differentiation of, for example, different α-helix-like states that enable differentiating π and 310 states from pure α-helices. These approaches can be used to determine the Gibbs free-energy landscape of individual peptide bonds along the most important protein (un)folding coordinate. Future work will find spectral monitors that probe peptide bond activation barriers that control protein (un)folding mechanisms. In addition, UVRR studies of side chain vibrations will probe the role of side chains in determining protein secondary, tertiary, and quaternary structures. © 2011 American Chemical Society

UV resonance raman investigation of electronic transitions in α-helical and polyproline II-like conformations

UV resonance Raman (UVRR) excitation profiles and Raman depolarization ratios were measured for a 21-residue predominantly alanine peptide, AAAAA(AAARA)3A (AP), excited between 194 and 218 nm. Excitation within the π→π* electronic transitions of the amide group results in UVRR spectra dominated by amide vibrations. The Raman cross sections and excitation profiles provide information about the nature of the electronic transitions of the α-helix and polyproline II (PPII)-like peptide conformations. AP is known to be predominantly a-helical at low temperatures and to take on a PPII helix-like conformation at high temperatures. The PPII-like and a-helix conformations show distinctly different Raman excitation profiles. The PPII-like conformation cross sections are approximately twice those of the a-helix. This is due to hypochromism that results from excitonic interactions between the NV1 transition of one amide group with higher energy electronic transitions of other amide groups, which decreases the α-helical NV1 (π→π*) oscillator strengths. Excitation profiles of the α-helix and PPII-like conformations indicate that the highest signal-to-noise Raman spectra of a-helix and PPII-like conformations are obtained at excitation wavelengths of 194 and 198 nm, respectively. We also see evidence of at least two electronic transitions underlying the Raman excitation profiles of both the a-helical and the PPII-like conformations. In addition to the well-known ∼190 nm π→π* transitions, the Raman excitation profiles and Raman depolarization ratio measurements show features between 205-207 nm, which in the a-helix likely results from the parallel excitonic component. The PPII-like helix appears to also undergo excitonic splitting of its π→π* transition which leads to a 207 nm feature. © 2008 American Chemical Society

To switch or not to switch : the effects of potassium and sodium ions on alpha-poly-L-glutamate conformations in aqueous solutions

Molecular dynamics simulations demonstrate that differences in the interaction of sodium and potassium with the carboxylate side chains of alpha-Poly-L-glutamate (alpha-PGA) have a dramatic effect on the conformational properties of the polypeptide. Potassium ions cluster mainly in the second and third solvation shells of alpha-PGA because their low charge density makes the electrostatic interactions between them and alpha-PGA too weak for K* to compete with water for the first solvation shell of the alpha-PGA glutamic acid residuals. Unlike sodium ions, they do not switch the conformation of alpha-PGA from extended to alpha-helical. Potentials of mean force for pure water, sodium ion solutions, and potassium ion solutions show marked differences in ion association behavior. This supports the idea that Hofmeister effects depend upon direct ion-macromolecule interactions as well as interactions with water molecules in the first solvation shell rather than bulk water structuring