Collagen molecules are structural in nature and primarily found in eukaryotic, multicellular orga... more Collagen molecules are structural in nature and primarily found in eukaryotic, multicellular organisms. Recently, a collagen-like protein, TrpA, was identified and characterized in the marine cyanobacterium Trichodesmium erythraeum IMS 101, and it was shown to be involved in maintaining the structural integrity of the trichomes. The TrpA protein contains one glycine interruption in the otherwise perfectly uninterrupted collagenous domain. In this study, we used phylogenetic analysis to determine that the TrpA protein sequence is most closely associated with non-fibril-forming collagen proteins. Structural modelling and circular dichroism data suggest that the glycine insertion decreases the stability of TrpA compared to uninterrupted collagen sequences. Additionally, scanning electron microscopy revealed that TrpA is expressed entirely on the surface of the trichomes, with no specific pattern of localization. These data indicate that the TrpA protein is part of the outer sheath of t...
Methods in molecular biology (Clifton, N.J.), 2012
Raman spectroscopy has positioned itself as an invaluable tool in the study of complex biological... more Raman spectroscopy has positioned itself as an invaluable tool in the study of complex biological systems, consistently being used to obtain information illustrating a vast array of fundamental properties. Of primary interest, with respect to the focus of this chapter, are conformational changes of peptide backbones. For short peptides to larger biological systems this understanding can be extended to local hydrogen bonding interactions and the probing of other structural or organizational properties. With regard to unfolded peptides Raman spectroscopy can be used as a technique complementary to infrared (IR) and vibrational circular dichroism (VCD) spectroscopy. This chapter describes how high quality polarized Raman spectra of peptide can be recorded with a Raman microspectrometer and how the structure sensitive amide I band profiles of isotropic and anisotropic Raman scattering can be analyzed in conjunction with the respective IR and VCD profiles to obtain conformational distrib...
Physical chemistry chemical physics : PCCP, Jan 7, 2014
As established by several groups over the last 20 years, amino acid residues in unfolded peptides... more As established by several groups over the last 20 years, amino acid residues in unfolded peptides and proteins do not exhibit the unspecific random distribution as assumed by the classical random coil model. Individual amino acid residues in small peptides were found to exhibit different conformational preferences. Here, we utilize recently obtained conformational distributions of guest amino acid residues in GxG peptides to estimate their conformational entropy, which we find to be significantly lower than the entropy of an assumed random coil like distribution. Only at high temperature do backbone entropies approach random coil like values. We utilized the obtained backbone entropies of the investigated amino acid residues to estimate the loss of conformational entropy caused by a coil → helix transition and identified two subsets of amino acid residues for which the thus calculated entropy losses correlate well with the respective Gibbs energy of helix formation obtained for alan...
To explore the influence of nearest neighbors on conformational biases in unfolded peptides, we c... more To explore the influence of nearest neighbors on conformational biases in unfolded peptides, we combined vibrational and 2D NMR spectroscopy to obtain the conformational distributions of selected "GxyG" host-guest peptides in aqueous solution: GDyG, GSyG, GxLG, GxVG, where x/y=A, K, L, V. Large changes of conformational propensities were observed due to nearest-neighbor interactions, at variance with the isolated pair hypothesis. We found that protonated aspartic acid and serine lose their above-the-average preference for turn-like structures in favor of polyproline II (pPII) populations in the presence of neighbors with bulky side chains. Such residues also decrease the above-the-average pPII preference of alanine. These observations suggest that the underlying mechanism involves a disruption of the hydration shell. Thermodynamic analysis of (3) J(H(N) ,H(α) ) (T) data for each x,y residue reveals that modest changes in the conformational ensemble masks larger changes of enthalpy and entropy governing the pPII↔β equilibrium indicating a significant residue dependent temperature dependence of the peptides' conformational ensembles. These results suggest that nearest-neighbor interactions between unlike residues act as conformational randomizers close to the enthalpy-entropy compensation temperature, eliminating intrinsic biases in favor of largely balanced pPII/β dominated ensembles at physiological temperatures.
ABSTRACT In order to enable structural predictions of intrinsically disordered proteins (IDPs) th... more ABSTRACT In order to enable structural predictions of intrinsically disordered proteins (IDPs) the intrinsic conformational propensities of amino acids must be complimented by information on nearest-neighbor interactions. To explore the influence of nearest-neighbors on conformational distributions, we preformed a joint vibrational (Infrared, Vibrational Circular Dichroism (VCD), polarized Raman) and 2D-NMR study of selected GxyG host-guest peptides: GDyG, GSyG, GxLG, GxVG, where x/y=A,K,LV. D and S (L and V) were chosen at the x (y) position due to their observance to drastically change the distribution of alanine in xAy tripeptide sequences in truncated coil libraries. The conformationally sensitive amide' profiles of the respective spectra were analyzed in terms of a statistical ensemble described as a superposition of 2D-Gaussian functions in Ramachandran space representing sub-ensembles of pPII-,β-strand-, helical-, and turn-like conformations. Our analysis and simulation of the amide I^' band profiles exploits excitonic coupling between the local amide I^' vibrational modes in the tetra-peptides. The resulting distributions reveal that D and S, which themselves have high propensities for turn-structures, strongly affect the conformational distribution of their downstream neighbor. Taken together, our results indicate that Dx and Sx motifs might act as conformational randomizers in proteins, attenuating intrinsic propensities of neighboring residues. Overall, our results show that nearest neighbor interactions contribute significantly to the Gibbs energy landscape of disordered peptides and proteins.
The driving forces governing the unique and restricted conformational preferences of amino acid r... more The driving forces governing the unique and restricted conformational preferences of amino acid residues in the unfolded state are still not well understood. In this study, we experimentally determine the individual thermodynamic components underlying intrinsic conformational propensities of these residues. Thermodynamic analysis of ultraviolet-circular dichroism (UV-CD) and (1)H NMR data for a series of glycine capped amino acid residues (i.e., G-x-G peptides) reveals the existence of a nearly exact enthalpy-entropy compensation for the polyproline II-β strand equilibrium for all investigated residues. The respective ΔHβ, ΔSβ values exhibit a nearly perfect linear relationship with an apparent compensation temperature of 295 ± 2 K. Moreover, we identified iso-equilibrium points for two subsets of residues at 297 and 305 K. Thus, our data suggest that within this temperature regime, which is only slightly below physiological temperatures, the conformational ensembles of amino acid residues in the unfolded state differ solely with respect to their capability to adopt turn-like conformations. Such iso-equilibria are rarely observed, and their existence herein indicates a common physical origin behind conformational preferences, which we are able to assign to side-chain dependent backbone solvation. Conformational effects such as differences between the number of sterically allowed side chain rotamers can contribute to enthalpy and entropy but not to the Gibbs energy associated with conformational preferences. Interestingly, we found that alanine, aspartic acid, and threonine are the only residues which do not share these iso-equilbiria. The enthalpy-entropy compensation discovered as well as the iso-equilbrium and thermodynamics obtained for each amino acid residue provide a new and informative way of identifying the determinants of amino acid propensities in unfolded and disordered states.
It is now well-established that different amino acid residues can exhibit different conformationa... more It is now well-established that different amino acid residues can exhibit different conformational distributions in the unfolded state of peptides and proteins. These conformational propensities can be modulated by nearest neighbors. In the current study, we combined vibrational and NMR spectroscopy to determine the conformational distributions of the central and C-terminal residues in trilysine peptides in aqueous solution. The study was motivated by earlier observations suggesting that interactions between ionized nearest neighbor residues can substantially change conformational propensities. We found that the central lysine residue predominantly adopts conformations that are located at the upper border of the upper left quadrant of the Ramachandran plot and the left border of the polyproline II region. We term this type of conformation deformed polyproline II (pPII(d)). The structures of less populated subensembles of trilysine resemble are comparable with structures at the i + 1 position of type I and type II β-turns. For the C-terminal residue, however, we obtained a mixture of polyproline II, β-strand, and right-handed helical conformations, which is typical for lysine residues in alanine- and glycine-based peptides. Our data thus indicate that the terminal lysines modify and restrict the conformational distribution of the central lysine residue. DFT calculations for ionized trilysine and lysyllysyllysylglycine in vacuo indicate that the pPII(d) is stabilized by a rather strong hydrogen bond between the NH3(+) group of the central lysine and the carbonyl group of the C-terminal peptide. This intramolecular hydrogen bonding induces optical activity in the C-terminal CO stretching vibration, which leads to an unusual and relatively intense positive Cotton band. Additionally, we analyzed the amide I' band profile of ionized triornithine in water. Ornithine is structurally similar to lysine in that its side chain is terminated with an amino group; however, the side chain of ornithine is shorter than lysine's side chain by one methylene group. We found that the conformational distribution of the central ornithine in this peptide must be very similar to that of the central lysine residue in trilysine. This suggests that the ionized ammonium group, which lysine and ornithine side chains have in common, is the main determinant of their conformational propensities at the central position in the respective tripeptides. The results of a DFT-based geometry optimization confirm this notion. In principle, our results suggest that lysine-rich segments in unfolded/disordered proteins and peptides can switch between different types of local order, i.e., an extended pPII(d)-like conformation and transient turns. However, for longer polylysine segments nonlocal interactions between side chains might impede the formation of turns, thus enabling the formation of pPII(d)-helix segments.
Local structure in unfolded proteins, especially turn segments, has been suggested to initiate th... more Local structure in unfolded proteins, especially turn segments, has been suggested to initiate the hierarchical protein-folding process. To determine the intrinsic propensity to form such turn structures, amide I' band profiles of the Raman, IR, and vibrational circular dichroism (VCD) spectra, and several structure-sensitive NMR J-coupling constants, have been measured for a series of GxG (x=D, N, T, C) peptides, in which the central x residues are abundant in various turn motifs in folded proteins. In addition, we revisited earlier measured GSG experimental data. To check whether this relatively high propensity for these residues to sample turns reflects an intrinsic propensity, the experimental data were analyzed in terms of conformational distributions that can be described as a superposition of two-dimensional Gaussian distributions associated with different so-called mesostates. The analysis reveals that the investigated residues sample dihedral angles similar to those found in the corner residues of various turns, namely, type I/I', II/II', and IV β-turns. Aspartic acid (D) was found to predominantly sample regions attributed to turns, including distributions at the upper border of the upper-right quadrant of the Ramachandran plot, which bear some resemblance to asx-turns observed in proteins. This conformation enables hydrogen bonding between the side-chain carboxylate and the C-terminal amide group. Altogether, the study shows that the high propensity for T, S, C, N, and D to be located in turn motifs reflects, to a substantial degree, an intrinsic property and supports the role of these residues as initiation sites for hierarchical folding processes that can lead to compact structures in the unfolded state of peptides and proteins.
Collagen molecules are structural in nature and primarily found in eukaryotic, multicellular orga... more Collagen molecules are structural in nature and primarily found in eukaryotic, multicellular organisms. Recently, a collagen-like protein, TrpA, was identified and characterized in the marine cyanobacterium Trichodesmium erythraeum IMS 101, and it was shown to be involved in maintaining the structural integrity of the trichomes. The TrpA protein contains one glycine interruption in the otherwise perfectly uninterrupted collagenous domain. In this study, we used phylogenetic analysis to determine that the TrpA protein sequence is most closely associated with non-fibril-forming collagen proteins. Structural modelling and circular dichroism data suggest that the glycine insertion decreases the stability of TrpA compared to uninterrupted collagen sequences. Additionally, scanning electron microscopy revealed that TrpA is expressed entirely on the surface of the trichomes, with no specific pattern of localization. These data indicate that the TrpA protein is part of the outer sheath of t...
Methods in molecular biology (Clifton, N.J.), 2012
Raman spectroscopy has positioned itself as an invaluable tool in the study of complex biological... more Raman spectroscopy has positioned itself as an invaluable tool in the study of complex biological systems, consistently being used to obtain information illustrating a vast array of fundamental properties. Of primary interest, with respect to the focus of this chapter, are conformational changes of peptide backbones. For short peptides to larger biological systems this understanding can be extended to local hydrogen bonding interactions and the probing of other structural or organizational properties. With regard to unfolded peptides Raman spectroscopy can be used as a technique complementary to infrared (IR) and vibrational circular dichroism (VCD) spectroscopy. This chapter describes how high quality polarized Raman spectra of peptide can be recorded with a Raman microspectrometer and how the structure sensitive amide I band profiles of isotropic and anisotropic Raman scattering can be analyzed in conjunction with the respective IR and VCD profiles to obtain conformational distrib...
Physical chemistry chemical physics : PCCP, Jan 7, 2014
As established by several groups over the last 20 years, amino acid residues in unfolded peptides... more As established by several groups over the last 20 years, amino acid residues in unfolded peptides and proteins do not exhibit the unspecific random distribution as assumed by the classical random coil model. Individual amino acid residues in small peptides were found to exhibit different conformational preferences. Here, we utilize recently obtained conformational distributions of guest amino acid residues in GxG peptides to estimate their conformational entropy, which we find to be significantly lower than the entropy of an assumed random coil like distribution. Only at high temperature do backbone entropies approach random coil like values. We utilized the obtained backbone entropies of the investigated amino acid residues to estimate the loss of conformational entropy caused by a coil → helix transition and identified two subsets of amino acid residues for which the thus calculated entropy losses correlate well with the respective Gibbs energy of helix formation obtained for alan...
To explore the influence of nearest neighbors on conformational biases in unfolded peptides, we c... more To explore the influence of nearest neighbors on conformational biases in unfolded peptides, we combined vibrational and 2D NMR spectroscopy to obtain the conformational distributions of selected "GxyG" host-guest peptides in aqueous solution: GDyG, GSyG, GxLG, GxVG, where x/y=A, K, L, V. Large changes of conformational propensities were observed due to nearest-neighbor interactions, at variance with the isolated pair hypothesis. We found that protonated aspartic acid and serine lose their above-the-average preference for turn-like structures in favor of polyproline II (pPII) populations in the presence of neighbors with bulky side chains. Such residues also decrease the above-the-average pPII preference of alanine. These observations suggest that the underlying mechanism involves a disruption of the hydration shell. Thermodynamic analysis of (3) J(H(N) ,H(α) ) (T) data for each x,y residue reveals that modest changes in the conformational ensemble masks larger changes of enthalpy and entropy governing the pPII↔β equilibrium indicating a significant residue dependent temperature dependence of the peptides' conformational ensembles. These results suggest that nearest-neighbor interactions between unlike residues act as conformational randomizers close to the enthalpy-entropy compensation temperature, eliminating intrinsic biases in favor of largely balanced pPII/β dominated ensembles at physiological temperatures.
ABSTRACT In order to enable structural predictions of intrinsically disordered proteins (IDPs) th... more ABSTRACT In order to enable structural predictions of intrinsically disordered proteins (IDPs) the intrinsic conformational propensities of amino acids must be complimented by information on nearest-neighbor interactions. To explore the influence of nearest-neighbors on conformational distributions, we preformed a joint vibrational (Infrared, Vibrational Circular Dichroism (VCD), polarized Raman) and 2D-NMR study of selected GxyG host-guest peptides: GDyG, GSyG, GxLG, GxVG, where x/y=A,K,LV. D and S (L and V) were chosen at the x (y) position due to their observance to drastically change the distribution of alanine in xAy tripeptide sequences in truncated coil libraries. The conformationally sensitive amide' profiles of the respective spectra were analyzed in terms of a statistical ensemble described as a superposition of 2D-Gaussian functions in Ramachandran space representing sub-ensembles of pPII-,β-strand-, helical-, and turn-like conformations. Our analysis and simulation of the amide I^' band profiles exploits excitonic coupling between the local amide I^' vibrational modes in the tetra-peptides. The resulting distributions reveal that D and S, which themselves have high propensities for turn-structures, strongly affect the conformational distribution of their downstream neighbor. Taken together, our results indicate that Dx and Sx motifs might act as conformational randomizers in proteins, attenuating intrinsic propensities of neighboring residues. Overall, our results show that nearest neighbor interactions contribute significantly to the Gibbs energy landscape of disordered peptides and proteins.
The driving forces governing the unique and restricted conformational preferences of amino acid r... more The driving forces governing the unique and restricted conformational preferences of amino acid residues in the unfolded state are still not well understood. In this study, we experimentally determine the individual thermodynamic components underlying intrinsic conformational propensities of these residues. Thermodynamic analysis of ultraviolet-circular dichroism (UV-CD) and (1)H NMR data for a series of glycine capped amino acid residues (i.e., G-x-G peptides) reveals the existence of a nearly exact enthalpy-entropy compensation for the polyproline II-β strand equilibrium for all investigated residues. The respective ΔHβ, ΔSβ values exhibit a nearly perfect linear relationship with an apparent compensation temperature of 295 ± 2 K. Moreover, we identified iso-equilibrium points for two subsets of residues at 297 and 305 K. Thus, our data suggest that within this temperature regime, which is only slightly below physiological temperatures, the conformational ensembles of amino acid residues in the unfolded state differ solely with respect to their capability to adopt turn-like conformations. Such iso-equilibria are rarely observed, and their existence herein indicates a common physical origin behind conformational preferences, which we are able to assign to side-chain dependent backbone solvation. Conformational effects such as differences between the number of sterically allowed side chain rotamers can contribute to enthalpy and entropy but not to the Gibbs energy associated with conformational preferences. Interestingly, we found that alanine, aspartic acid, and threonine are the only residues which do not share these iso-equilbiria. The enthalpy-entropy compensation discovered as well as the iso-equilbrium and thermodynamics obtained for each amino acid residue provide a new and informative way of identifying the determinants of amino acid propensities in unfolded and disordered states.
It is now well-established that different amino acid residues can exhibit different conformationa... more It is now well-established that different amino acid residues can exhibit different conformational distributions in the unfolded state of peptides and proteins. These conformational propensities can be modulated by nearest neighbors. In the current study, we combined vibrational and NMR spectroscopy to determine the conformational distributions of the central and C-terminal residues in trilysine peptides in aqueous solution. The study was motivated by earlier observations suggesting that interactions between ionized nearest neighbor residues can substantially change conformational propensities. We found that the central lysine residue predominantly adopts conformations that are located at the upper border of the upper left quadrant of the Ramachandran plot and the left border of the polyproline II region. We term this type of conformation deformed polyproline II (pPII(d)). The structures of less populated subensembles of trilysine resemble are comparable with structures at the i + 1 position of type I and type II β-turns. For the C-terminal residue, however, we obtained a mixture of polyproline II, β-strand, and right-handed helical conformations, which is typical for lysine residues in alanine- and glycine-based peptides. Our data thus indicate that the terminal lysines modify and restrict the conformational distribution of the central lysine residue. DFT calculations for ionized trilysine and lysyllysyllysylglycine in vacuo indicate that the pPII(d) is stabilized by a rather strong hydrogen bond between the NH3(+) group of the central lysine and the carbonyl group of the C-terminal peptide. This intramolecular hydrogen bonding induces optical activity in the C-terminal CO stretching vibration, which leads to an unusual and relatively intense positive Cotton band. Additionally, we analyzed the amide I' band profile of ionized triornithine in water. Ornithine is structurally similar to lysine in that its side chain is terminated with an amino group; however, the side chain of ornithine is shorter than lysine's side chain by one methylene group. We found that the conformational distribution of the central ornithine in this peptide must be very similar to that of the central lysine residue in trilysine. This suggests that the ionized ammonium group, which lysine and ornithine side chains have in common, is the main determinant of their conformational propensities at the central position in the respective tripeptides. The results of a DFT-based geometry optimization confirm this notion. In principle, our results suggest that lysine-rich segments in unfolded/disordered proteins and peptides can switch between different types of local order, i.e., an extended pPII(d)-like conformation and transient turns. However, for longer polylysine segments nonlocal interactions between side chains might impede the formation of turns, thus enabling the formation of pPII(d)-helix segments.
Local structure in unfolded proteins, especially turn segments, has been suggested to initiate th... more Local structure in unfolded proteins, especially turn segments, has been suggested to initiate the hierarchical protein-folding process. To determine the intrinsic propensity to form such turn structures, amide I' band profiles of the Raman, IR, and vibrational circular dichroism (VCD) spectra, and several structure-sensitive NMR J-coupling constants, have been measured for a series of GxG (x=D, N, T, C) peptides, in which the central x residues are abundant in various turn motifs in folded proteins. In addition, we revisited earlier measured GSG experimental data. To check whether this relatively high propensity for these residues to sample turns reflects an intrinsic propensity, the experimental data were analyzed in terms of conformational distributions that can be described as a superposition of two-dimensional Gaussian distributions associated with different so-called mesostates. The analysis reveals that the investigated residues sample dihedral angles similar to those found in the corner residues of various turns, namely, type I/I', II/II', and IV β-turns. Aspartic acid (D) was found to predominantly sample regions attributed to turns, including distributions at the upper border of the upper-right quadrant of the Ramachandran plot, which bear some resemblance to asx-turns observed in proteins. This conformation enables hydrogen bonding between the side-chain carboxylate and the C-terminal amide group. Altogether, the study shows that the high propensity for T, S, C, N, and D to be located in turn motifs reflects, to a substantial degree, an intrinsic property and supports the role of these residues as initiation sites for hierarchical folding processes that can lead to compact structures in the unfolded state of peptides and proteins.
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Papers by Siobhan Toal