Direct UV Resonance Raman Monitoring of Protein Folding Reaction Coordinate: alpha-Helix Melting and Formation Revisited

Spectroscopic investigations of protein folding mechanism(s) require the elucidation of the relationship between spectral features and the protein folding reaction coordinate(s). This thesis describes the development and demonstrates the applications of a novel UV resonance Raman (UVRR) spectroscopic methodology, which quantitatively correlates the UVRR amide III3 (AmIII3) frequency of a peptide bond to its Psi Ramachandran angle (which is arguably the most important folding reaction coordinate). This information, then, allows us, for the first time, to obtain Psi angular population distributions of peptide bonds in peptides and proteins from their UVRR AmIII3 band profiles, as well as the Gibbs free energy landscapes along the Psi Ramachandran angle folding coordinate. Application of this methodology allows us to quantitatively characterize the ensembles of folded and unfolded states in peptides and proteins. We show that the unfolded state ensembles show no evidence of completely disordered "random coil" conformation, and are usually dominated by PPII-like conformations. In addition, we for the first time experimentally detected the extended 2.51-helix conformation in poly-L-lysine and poly-L-glutamic acid, which is stabilized by electrostatic repulsion between side-chain charges. Most importantly, we resolved and quantitatively characterized the 310-helix and pi-bulge contributions within the "alpha-helix melting" of ala-rich peptides. We for the first time obtained their individual experimental melting curves, estimated their Zimm and Bragg parameters, and estimated their individual kinetic (un)folding rates. These results challenge the classical view of protein folding and provide an important quantitative basis for future studies

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

Discriminant Analysis of Raman Spectra for Body Fluid Identification for Forensic Purposes

Detection and identification of blood, semen and saliva stains, the most common body fluids encountered at a crime scene, are very important aspects of forensic science today. This study targets the development of a nondestructive, confirmatory method for body fluid identification based on Raman spectroscopy coupled with advanced statistical analysis. Dry traces of blood, semen and saliva obtained from multiple donors were probed using a confocal Raman microscope with a 785-nm excitation wavelength under controlled laboratory conditions. Results demonstrated the capability of Raman spectroscopy to identify an unknown substance to be semen, blood or saliva with high confidence

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

Detection of glycosylation and iron-binding protein modifications using Raman spectroscopy

In this study we demonstrate the use of Raman spectroscopy to determine protein modifications as a result of glycosylation and iron binding. Most proteins undergo some modifications after translation which can directly affect protein function. Identifying these modifications is particularly important in the production of biotherapeutic agents as they can affect stability, immunogenicity and pharmacokinetics. However, post-translational modifications can often be difficult to detect with regard to the subtle structural changes they induce in proteins. From their Raman spectra apo-and holo- forms of iron-binding proteins, transferrin and ferritin, could be readily distinguished and variations in spectral features as a result of structural changes could also be determined. In particular, differences in solvent exposure of aromatic amino acids residues could be identified between the open and closed forms of the iron-binding proteins. Protein modifications as a result of glycosylation can be even more difficult to identify. Through the application of the chemometric techniques of principal component analysis and partial least squares regression variations in Raman spectral features as a result of glycosylation induced structural modifications could be identified. These were then used to distinguish between glycosylated and non-glycosylated transferrin and to measure the relative concentrations of the glycoprotein within a mixture of the native non-glycosylated protein

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