UV Resonance Raman-Selective Amide Vibrational Enhancement:  Quantitative Methodology for Determining Protein Secondary Structure

Chi, Zhenhuan; Chen, X. G.; Holtz, J.; Asher, Sanford A.

doi:10.1021/bi971160z

Cited by 301 publications

(480 citation statements)

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“…The C R -H bending band intensity is derived from non-R-helical peptide bond conformations (61,66). A weak AmIII 2 band is located at ∼1320 cm -1 .…”

Section: Resultsmentioning

confidence: 99%

UV Resonance Raman Investigation of a 3₁₀-Helical Peptide Reveals a Rough Energy Landscape

Ahmed

Asher

2006

Biochemistry

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We used UVRRS at 194 and 204 nm excitation to examine the backbone conformation of a 13-residue polypeptide (gp41 [659][660][661][662][663][664][665][666][667][668][669][670][671] ) that has been shown by NMR to predominantly fold into a 3 10 -helix. Examination of the conformation sensitive AmIII 3 region indicates the peptide has significant populations of -turn, PPII, 3 10 -helix, and π-helix-like conformations but little R-helix. We estimate that at 1°C on average six of the 12 peptide bonds are in folded conformations (predominantly 3 10 -and π-helix), while the other six are in unfolded ( -turn/PPII) conformations. The folded and unfolded populations do not change significantly as the temperature is increased from 1 to 60°C, suggesting a unique energy landscape where the folded and unfolded conformations are essentially degenerate in energy and exhibit identical temperature dependences.An understanding of the mechanisms of protein folding will enable the de novo design of proteins with profound commercial and medical applications (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Over the past 50 years, significant effort to elucidate protein folding and unfolding mechanisms has been expended. Part of this effort has examined the thermodynamics and kinetics of small model systems such as -hairpins (20)(21)(22) and alanine-based R-helices (23-33). These studies have provided detailed microscopic knowledge of the folding energy landscape of these isolated motifs. Theoretical simulations have suggested that 3 10 -helices and/or type III -turns may serve as intermediates in the R-helix folding pathway (34-39). The formation of kinetic intermediates such as -turn or 3 10 -helix lowers the entropic cost of nucleating the first helical residue, thus facilitating the helix folding transition (34,40,41).The 3 10 -helix is the fourth most common secondary structural motif in proteins (34-59). On average, in proteins, 3-4% of peptide residues occur in a 3 10 -helix conformation (48). The 3 10 -helix differs from an R-helix in that the tighter packing of the backbone forces the CdO‚‚‚H-N hydrogen bonds to point outward, away from the helical axis, resulting in decreased stability of the 3 10 -helix compared to that of the R-helix (25,48).Recent ESR (39, 42) and NMR (43) work by Millhauser et al. (41) suggests the presence of 3 10 -helix at the terminus of short alanine-based helical peptides. The authors proposed that 3 10 -helices are a relic of the folding process. They reasoned that nascent helices contain mixtures of unfolded and turn conformations, specifically type III turns. As folding conditions begin to favor helical structures, the type III turn conformation (a single 3 10 -helix unit) propagates; i.e., the chain adopts a 3 10 -helix conformation. As the helical domain lengthens, the R-helix conformation competes with the 3 10 -helix conformation. Finally, at longer helical lengths, the R-helix conformation dominates. Some 3 10 -helix/type III turn-like conformations may survive...

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“…The C R -H bending band intensity is derived from non-R-helical peptide bond conformations (61,66). A weak AmIII 2 band is located at ∼1320 cm -1 .…”

Section: Resultsmentioning

confidence: 99%

UV Resonance Raman Investigation of a 3₁₀-Helical Peptide Reveals a Rough Energy Landscape

Ahmed

Asher

2006

Biochemistry

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“…Quantitative determination of secondary structure composition was performed using the PSSRS methodology recently developed by Chi et al (34). This technique models the experimentally obtained spectra by least-squares fitting of the basis spectra for the R-helix, -sheet, and random coil secondary structures to the measured peptide spectra.…”

Section: Methodsmentioning

confidence: 99%

UV Raman Studies of Peptide Conformation Demonstrate That Betanova Does Not Cooperatively Unfold

Boyden

Asher

2001

Biochemistry

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We used UV resonance Raman spectroscopy (UVRR) excited within the peptide bond π f π* electronic transitions and within the aromatic amino acid π f π* electronic transitions to examine the temperature dependence of the solution conformation of betanova, a 20-residue -sheet polypeptide [Kortemme, T., Ramirez-Alvarado, M., and Serrano, L. (1998) Science 281, 253-256]. The 206.5 nm excited UVRR enhances the amide vibrations and demonstrates that betanova has a predominantly -sheet structure between 5 and 82°C. The 229 nm excited UVRR, which probes the tyrosine and tryptophan side chain vibrations, shows an increase in the solvent exposure of the tryptophan side chains as the temperature is increased. Our results are consistent with the existence of an intermediate state similar to that calculated by Bursulaya and Brooks [Bursulaya, B. D., and Brooks, C. L. (1999) J. Am. Chem. Soc. 121, 9947-9951] and exclude the previously proposed two-state cooperative folding mechanism. Betanova's structure appears to be molten globule over the 3-82°C temperature range of our study.The completion of the human genome sequence makes available the primary sequence of all proteins synthesized by the human body. This represents only the first stage in the development of an understanding of the protein structural basis of disease. The next stage in this development requires an ability to predict the structure and function of proteins from their primary sequences. At present this is impossible, since not enough is known about the dynamics and thermodynamics of protein folding (1-5).The understanding of protein folding mechanisms is advancing as experimentalists develop better strategies for the de novo design of model peptides and better methodologies for characterizing equilibrium secondary structures, as well as the dynamics of folding. To date, synthetic schemes have been successfully developed for the synthesis of R-helix-forming peptides, and much has been uncovered about their folding dynamics (6-13). In contrast, only limited progress has been made toward understanding the synthesis of and the dynamics of -sheet peptides. Primarily, studies have focused on -hairpins rather than complete sheet structures (14-18). Successful synthesis of three-stranded -sheet structures has been accomplished only recently. However, these model peptides only form complete -sheet structures under limited conditions, in the presence of organic solvents and with the use of unnatural amino acids (19)(20)(21)(22). Thus, there remains much to learn about -sheet folding.Significant advances have also occurred in the theoretical understanding of protein folding (23-28). Such work includes molecular dynamics simulations of energy landscapes that describe folding thermodynamics. Currently, some of these models accurately predict experimental results. Thus, theory can now impact how experimental data are interpreted. It seems likely that the most powerful insights into the rules of protein folding will result from combined experimental and theoreti...

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“…Because of its structural plasticity and ease of manipulation, poly(K) is a good candidate system for modeling the dynamics of the initial protein conformation changes involved in α-helix to β-sheet transitions. At high pH, poly(K) is α-helical and readily converts to β-sheet structure at elevated temperatures(19).The major protein conformations, α-helix and β-sheet, can be distinguished by their UVRR spectra (20)(21)(22). The protein conformational sensitivity of UVRR arises from the selective excitation of the π*←π electronic transition of the amide bonds.…”

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Intermediacy of Poly(l-proline) II and β-Strand Conformations in Poly(l-lysine) β-Sheet Formation Probed by Temperature-Jump/UV Resonance Raman Spectroscopy

et al. 2005

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Ultraviolet resonance Raman spectroscopy (UVRR) in combination with a nanosecond temperaturejump (T-jump) was used to investigate early steps in the temperature induced α-helix to β-sheet conformational transition of poly(L-lysine) (poly(K)). Excitation at 197 nm from a tunable frequency-quadrupled Ti:Sapphire laser provided high-quality UVRR spectra, containing multiple conformation-sensitive amide bands. Although unionized poly(K) (pH 11.6) is mainly α-helical below 30°C, there is a detectable fraction (∼15 %) of unfolded polypeptide, which is mainly in the polyproline II (PPII) conformation. However, deviations from the expected amide I and II signals indicate an additional conformation, suggested to be β-strand. Above 30°C unionized poly(K) forms β-sheet at a rate (minutes) which increases with increasing temperature. A 22 -44°C T-jump is accompanied by prompt amide I and II difference signals suggested to arise from a rapid shift in the PPII/β-strand equilibrium. These signals are superimposed on a subsequently evolving difference spectrum which is characteristic of PPII, although the extent of conversion is low, ∼ 2 % at the 3 μs time limit of the experiment. The rise time of the PPII signals is ∼250 ns, consistent with melting of short α-helical segments. A model is proposed in which the melted PPII segments interconvert with β-strand conformation, whose association through inter-strand H-bonding nucleates the formation of β-sheet. The intrinsic propensity for β-strand formation could be a determinant of β-sheet induction time, with implications for the onset of amyloid diseases. KeywordsUVRR; resonance Raman; protein folding; poly(L-lysine); PPII; β-strand; α-helix; β-sheet; temperature-jump; T-jump Several degenerative diseases, including Alzheimer's, Huntington's, Parkinson's, and transmissible spongiform encephalopathies (TSE), are associated with misfolding and aggregation of proteins (1). The misfolded proteins form fibrillous aggregates, termed amyloid fibrils, which have a cross β-sheet structure in which the polypeptide backbone is perpendicular to the fibril axis(2). This allows the fibril to grow lengthwise with the addition of subsequent misfolded protein molecules. It is interesting to note that conditions which promote partial folding of proteins, appear to promote fibril formation in vitro (3)(4)(5). † This work was supported by NIH Grants R01 GM 025158 to T.G.S. and NIH F32 AG 022770 to R.D.J. * To whom correspondence should be addressed: Princeton University, Department of Chemistry, Princeton, NJ, 08544. Telephone: (609) Certain proteins with abundant α-helical structure, including the prion protein (PrP C ) and apomyoglobin, convert to β-sheet rich structures under denaturing conditions (4-6). What is equally provocative is the correlation between metastable α-helices and β-sheet amyloid formation. The β-amyloid (Aβ) peptide involved in Alzheimer's disease and its constituent fragments contain no stable secondary structure in aqueous solutions (7,8), but when the nascent helic...

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UV Resonance Raman-Selective Amide Vibrational Enhancement: Quantitative Methodology for Determining Protein Secondary Structure

Cited by 301 publications

References 54 publications

UV Resonance Raman Investigation of a 3₁₀-Helical Peptide Reveals a Rough Energy Landscape

UV Resonance Raman Investigation of a 3₁₀-Helical Peptide Reveals a Rough Energy Landscape

UV Raman Studies of Peptide Conformation Demonstrate That Betanova Does Not Cooperatively Unfold

Intermediacy of Poly(l-proline) II and β-Strand Conformations in Poly(l-lysine) β-Sheet Formation Probed by Temperature-Jump/UV Resonance Raman Spectroscopy

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UV Resonance Raman-Selective Amide Vibrational Enhancement: Quantitative Methodology for Determining Protein Secondary Structure

Cited by 301 publications

References 54 publications

UV Resonance Raman Investigation of a 310-Helical Peptide Reveals a Rough Energy Landscape

UV Resonance Raman Investigation of a 310-Helical Peptide Reveals a Rough Energy Landscape

UV Raman Studies of Peptide Conformation Demonstrate That Betanova Does Not Cooperatively Unfold

Intermediacy of Poly(l-proline) II and β-Strand Conformations in Poly(l-lysine) β-Sheet Formation Probed by Temperature-Jump/UV Resonance Raman Spectroscopy

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UV Resonance Raman Investigation of a 3₁₀-Helical Peptide Reveals a Rough Energy Landscape

UV Resonance Raman Investigation of a 3₁₀-Helical Peptide Reveals a Rough Energy Landscape