Isotope-edited FTIR Research Articles

Hydrogen Bond Strengthens Acceptor Group: The Curious Case of the C-H···O=C Bond.

An H-bond involves the sharing of a hydrogen atom between an electronegative atom to which it is covalently bound (the donor) and another electronegative atom serving as an acceptor. Such bonds represent a critically important geometrical force in biological macromolecules and, as such, have been characterized extensively. H-bond formation invariably leads to a weakening within the acceptor moiety due to the pulling exerted by the donor hydrogen. This phenomenon can be compared to a spring connecting two masses; pulling one mass stretches the spring, similarly affecting the bond between the two masses. Herein, we describe the opposite phenomenon when investigating the energetics of the C-H···O=C bond. This bond underpins the most prevalent protein transmembrane dimerization motif (GxxxG) in which a glycine Cα-H on one helix forms a hydrogen bond with a carbonyl in a nearby helix. We use isotope-edited FT-IR spectroscopy and corroborating computational approaches to demonstrate a surprising strengthening of the acceptor C=O bond upon binding with the glycine Cα-H. We show that electronic factors associated with the Cα-H bond strengthen the C=O oscillator by increasing the s-character of the σ-bond, lowering the hyperconjugative disruption of the π-bond. In addition, a reduction of the acceptor C=O bond's polarity is observed upon the formation of the C-H···O=C bond. Our findings challenge the conventional understanding of H-bond dynamics and provide new insights into the structural stability of inter-helical protein interactions.

CATCH Peptides Coassemble into Structurally Heterogeneous β-Sheet Nanofibers with Little Preference to β-Strand Alignment.

Coassembling peptides offer an additional degree of freedom in the design of nanostructured biomaterials when compared to analogous self-assembling peptides. Yet, our understanding of how amino acid sequences encodes coassembled nanofiber structure is limited. Prior work on a charge-complementary pair, CATCH+ and CATCH- peptides, detected like-peptide nearest neighbors (CATCH+:CATCH+ and CATCH-:CATCH-) within coassembled β-sheet nanofibers; these self-associated peptide pairs marked a departure from an "ideal" coassembled structure. In this work, we employ solid-state NMR, isotope-edited FTIR, and coarse-grained molecular dynamics simulations to evaluate the alignment of β-strands within CATCH peptide nanofibers. Both experimental and computational results suggest that CATCH molecules coassemble into structurally heterogeneous nanofibers, which is consistent with our observations in another coassembling system, the King-Webb peptides. Within β-sheet nanofibers, β-strands were found to have nearest neighbors aligned in-register parallel, in-register antiparallel, and out-of-register. In comparison to the King-Webb peptides, CATCH nanofibers exhibit a greater degree of structural heterogeneity. By comparing the amino acid sequences of CATCH and King-Webb peptides, we can begin to unravel sequence-to-structure relationships, which may encode more precise coassembled β-sheet nanostructures.

Quantitative Analysis of Multiplex H-Bonds.

H-bonding is the predominant geometrical determinant of biomolecular structure and interactions. As such, considerable analyses have been undertaken to study its detailed energetics. The focus, however, has been mostly reserved for H-bonds comprising a single donor and a single acceptor. Herein, we measure the prevalence and energetics of multiplex H-bonds that are formed between three or more groups. We show that 92% of all transmembrane helices have at least one non-canonical H-bond formed by a serine or threonine residue whose hydroxyl side chain H-bonds to an over-coordinated carbonyl oxygen at position i–4, i–3, or i in the sequence. Isotope-edited FTIR spectroscopy, coupled with DFT calculations, enables us to determine the bond enthalpies, pointing to values that are up to 127% higher than that of a single canonical H-bond. We propose that these strong H-bonds serve to stabilize serine and threonine residues in hydrophobic environments while concomitantly providing them flexibility between different configurations, which may be necessary for function.

Isotope-edited FTIR reveals distinct aggregation and structural behaviors of unmodified and pyroglutamylated amyloid β peptides.

Amyloid β peptide (Aβ) is causatively associated with Alzheimer's disease (AD), and N-terminally truncated and pyroglutamylated Aβ peptides (AβpE) exert hypertoxic effect by an unknown mechanism. Recent evidence has identified the prefibrillar oligomers of Aβ, not the fibrils, as the prevalent cytotoxic species. Structural characterization of Aβ and AβpE oligomers is therefore important for better understanding of their toxic effect. Here we have used isotope-edited Fourier transform infrared (FTIR) spectroscopy to identify the conformational changes in Aβ(1-42) and AβpE(3-42) upon aggregation, individually and in 1 : 1 molar combination. During the first two hours of exposure to aqueous buffer, the peptides undergo transition from mostly α-helical to mostly β-sheet structure. Data on peptides (13)C,(15)N-labeled at K(16)L(17)V(18) or V(36)G(37)G(38)V(39) allowed construction of structural models for the monomer and early oligomers. The peptide monomer comprises a β-hairpin that involves residues upstream of the K(16)L(17)V(18) sequence and an N-terminal α-helix. The oligomers form by non-H-bonding interactions between the β-strands of neighboring β-hairpins, in lateral or staggered manner, with the strands running parallel or antiparallel. Relative α-helical and β-sheet propensities of Aβ(1-42) and AβpE(3-42) depend on the ionic strength of the buffer, emphasizing the importance of ionic interactions in Aβ peptide structure and aggregation. It is inferred that N-terminal modification of AβpE(3-42) affects the helix stability and thereby modulates β-sheet oligomer formation. The data thus provide new insight into the molecular mechanism of Aβ oligomerization by emphasizing the role of the N-terminal transient α-helical structure and by identifying structural constraints for molecular organization of the oligomers.

Site-Specific Structural Changes in Unmodified and Pyroglutamylated Amyloid Beta Peptide by Isotope-Edited Ftir

Amyloid beta-peptide (Abeta) forms cytotoxic assemblies that contribute to Alzheimer's disease. Recent evidence indicates that prefibrillar aggregates and not the fibrillar deposits exert the main toxic effect. In addition, naturally occurring N-terminally truncated and pyroglutamylated peptide (pE-Abeta) displays augmented cytotoxicity by an unknown mechanism. This study examines the conformational changes in both unmodified Abeta and pE-Abeta upon exposure to an aqueous environment. FTIR and circular dichroism were used to identify alpha-helix-to-beta-sheet conformational transitions of both peptides during aggregation. To gain site-specific structural information, the peptides were 13C,15N-labeled at residues 16-18 (KLV) or 36-39 (VGGV), followed by FTIR analysis. The peptides dried from hexafluoroisopropanol were alpha-helical (amide I peak at 1660-1657 cm-1) and showed negligible intensity of the labeled segments around 1615 cm-1, suggesting that in alpha-helical conformation the labeled amide groups behave like isolated oscillators. Upon addition of aqueous buffer (pH 7.2) both peptides rapidly adopted beta-sheet structure (amide I peak at 1637-1629 cm-1), with disproportionally prominent components around 1604-1597 cm-1 generated by the labeled segments. The intensity and the frequency of the amide I mode of the isotope-labeled segments suggest 12C-13C vibrational coupling, consistent with formation of antiparallel beta-sheet structures. Moreover, the amide I contours of the peptides under near-physiological and low ionic strength conditions were significantly different; both peptides exhibited an increased alpha-helical and decreased beta-sheet propensity under low ionic strength conditions, indicating a strong influence of the ionic strength on the aggregation kinetics and accompanying structural changes. Ongoing studies focus on structural differences between the unmodified Abeta and pE-Abeta peptides as well as their mutual structural effects when combined at various molar ratios, in an attempt to understand the structural basis of the elevated cytotoxicity of pE-Abeta.

Use of Isotope-Edited FTIR to Derive a Backbone Structure of a Transmembrane Protein.

Solving structures of membrane proteins has always been a formidable challenge, yet even upon success, the results are normally obtained in a mimetic environment that can be substantially different from a biological membrane. Herein, we use noninvasive isotope-edited FTIR spectroscopy to derive a structural model for the SARS coronavirus E protein transmembrane domain in lipid bilayers. Molecular-dynamics-based structural refinement, incorporating the IR-derived orientational restraints points to the formation of a helical hairpin structure. Disulfide cross-linking and X-ray reflectivity depth profiling provide independent support of the results. The unusually short helical hairpin structure of the protein might explain its ability to deform bilayers and is reminiscent of other peptides with membrane disrupting functionalities. Taken together, we show that isotope-edited FTIR is a powerful tool to analyze small membrane proteins in their native environment, enabling us to relate the unusual structure of the SARS E protein to its function.

Pyroglutamylated amyloid-β peptide reverses cross β-sheets by a prion-like mechanism.

The amyloid hypothesis causatively relates the fibrillar deposits of amyloid β peptide (Aβ) to Alzheimer’s disease (AD). More recent data, however, identify the soluble oligomers as the major cytotoxic entities. Pyroglutamylated Aβ (pE-Aβ) is present in AD brains and exerts augmented neurotoxicity, which is believed to result from its higher β-sheet propensity and faster fibrillization. While this concept is based on a set of experimental results, others have reported similar β-sheet contents in unmodified and pyroglutamylated Aβ, and slower aggregation of pE-Aβ as compared to unmodified Aβ, leaving the issue unresolved. Here, we assess the structural differences between Aβ and pE-Aβ peptides that may underlie their distinct cytotoxicities. Transmission electron microscopy identifies a larger number of prefibrillar aggregates of pE-Aβ at early stages of aggregation and suggests that pE-Aβ affects the fibrillogenesis even at low molar fractions. Circular dichroism and FTIR data indicate that while the unmodified Aβ readily forms β-sheet fibrils in aqueous media, pE-Aβ displays increased α-helical and decreased β-sheet propensity. Moreover, isotope-edited FTIR spectroscopy shows that pE-Aβ reverses β-sheet formation and hence fibrillogenesis of the unmodified Aβ peptide via a prion-like mechanism. These data provide a novel structural mechanism for pE-Aβ hypertoxicity; pE-Aβ undergoes faster formation of prefibrillar aggregates due to its increased hydrophobicity, thus shifting the initial stages of fibrillogenesis toward smaller, hypertoxic oligomers of partial α-helical structure.

Discriminating trpzip2 and trpzip4 peptides’ folding landscape using the two-dimensional infrared spectroscopy: A simulation study

We analyzed, based on the theoretical spectroscopic modeling, how the differences in the folding landscapes of two β-hairpin peptides trpzip2 and trpzip4 are reflected in their thermal unfolding infrared measurements. The isotope-edited equilibrium FTIR and two dimensional infrared spectra of the two peptides were calculated, using the nonlinear exciton propagation method, at a series of temperatures. The spectra calculations were based on the configuration distributions generated using the GB(OBC) implicit solvent MD simulation and the integrated tempering sampling technique. Conformational analysis revealed the different local thermal stabilities for these two peptides, which suggested the different folding landscapes. Our study further suggested that the ellipticities of the isotope peaks in the coherent IR signals are more sensitive to these local stability differences compared with other spectral features such as the peak intensities. Our technique can thus be combined with the relevant experimental measurements to achieve a better understanding of the peptide folding behaviors.

Isotope-edited FTIR in H2O: determination of the conformation of specific residues in a model α-helix peptide by 13C labeled carbonyls

Isotope-edited FTIR spectroscopy has been shown to be able to determine peptide's structure in the residue level in D2O, which is not a physiological solvent. Here, attenuated total reflection technique was utilized to successfully apply isotope-edited FTIR spectroscopy in H2O to determine the conformation of specific residues in a model peptide.

FT-IR Spectroscopy and Density Functional Theory Calculations of Carbon-13 Isotopologues of the Helical Peptide Z-AIB(6)-OTBU

Isotope-edited FT-IR spectroscopy is a combined synthetic and spectroscopic method used to characterize local (e.g., residue-level) vibrational environments of biomolecules. We have prepared the 31...

FT-IR Spectroscopy and Density Functional Theory Calculations of 13C Isotopologues of the Helical Peptide Z-Aib6-OtBu

Isotope-edited FT-IR spectroscopy is a combined synthetic and spectroscopic method used to characterize local (e.g., residue-level) vibrational environments of biomolecules. We have prepared the 3(10) helical peptide Z-Aib6-OtBu and seven (13)C-enriched analogues that vary only in the number and position(s) of (13)C═O isotopic enrichment. FT-IR spectra of these eight peptides solvated in the nonpolar aprotic solvent dichloromethane have been collected and compared to frequency, intensity, and normal mode results of DFT calculations. Single (13)C enrichment of amide functional groups tends to localize amide I vibrational eigenmodes, providing residue-specific information regarding the local environment (e.g., hydrogen bonding or solvent exposure) of the peptide bond. Double (13)C enrichment of Z-Aib6-OtBu allows for examination of interamide coupling between two labeled amide functional groups, providing experimental evidence of interamide coupling in the context of 3(10) helical structure. Although the calculated and observed interamide couplings of Z-Aib6-OtBu are a few cm(-1) and less, the eight peptides exhibit distinct infrared spectra, revealing details of interamide coupling and residue level vibrational environments.

Modeling the Thermal Unfolding 2DIR Spectra of a β-Hairpin Peptide Based on the Implicit Solvent MD Simulation

We simulated the equilibrium isotope-edited FTIR and 2DIR spectra of a β-hairpin peptide trpzip2 at a series of temperatures. The simulation was based on the configuration distributions generated using the GB(OBC) implicit solvent model and the integrated tempering sampling (ITS) technique. A soaking procedure was adapted to generate the peptide in explicit solvent configurations for the spectroscopy calculations. The nonlinear exciton propagation (NEP) method was then used to calculate the spectra. Agreeing with the experiments, the intensities and ellipticities of the isotope-shifted peaks in our simulated signals have the site-specific temperature dependences, which suggest the inhomogeneous local thermal stabilities along the peptide chain. Our simulation thus proposes a cost-effective means to understand a peptide's conformational change and related IR spectra across its thermal unfolding transition.

Side-chain specific isotopic labeling of proteins for infrared structural biology: The case of ring-D4-tyrosine isotope labeling of photoactive yellow protein

An important bottleneck in the use of infrared spectroscopy as a powerful tool for obtaining detailed information on protein structure is the assignment of vibrational modes to specific amino acid residues. Side-chain specific isotopic labeling is a general approach towards obtaining such assignments. We report a method for high yield isotope editing of the bacterial blue light sensor photoactive yellow protein (PYP) containing ring-D4-Tyr. PYP was heterologously overproduced in Escherichia coli in minimal media containing ring-D4-Tyr in the presence of glyphosate, which inhibits endogenous biosynthesis of aromatic amino acids (Phe, Trp, and Tyr). Mass spectrometry of the intact protein and of tryptic peptides unambiguously demonstrated highly specific labeling of all five Tyr residues in PYP with 98% incorporation and undetectable isotopic scrambling. FTIR spectroscopy of the protein reveals a characteristic Tyr ring vibrational mode at 1515cm−1 that is shifted to 1436cm−1, consistent with that from ab initio calculations. PYP is a model system for protein structural dynamics and for receptor activation in biological signaling. The results described here open the way to the analysis of PYP using isotope-edited FTIR spectroscopy with side-chain specific labeling.

Isotope-Edited FT-IR Spectra of Isotopmers of Helical Hexamers of Alpha-Aminoisobutyric Acid

Isotope-edited FT-infrared spectra of the Amide I region of hexamers of alpha-aminoisobutyric acid (Z-Aib6-OtBu) have been collected in order to explore the effects of 13C=O enrichment on the FT-IR spectra in the conformational context of 310 helices. Oligomers of Aib are known to adopt predominantly 310 helical structures, even at short peptide lengths. The Amide I band is sensitive to the details of peptide secondary structure, but the competency of this band to distinguish between alpha- and 310 helical secondary structure remains an open question. The 310 helix is shorter than an alpha-helix of the same number of residues and exhibits an i to i+3 hydrogen bonding pattern, instead of an i to i +4 pattern of the alpha-helix. These differences bring amide oscillators of a 310 helix slightly closer in space and in shorter periodicity of hydrogen-bonding partnership as compared to an alpha-helix. We have collected infrared spectra of isotopomers of hexamers of Aib (e.g., Z-Aib-Aib-Aib-Aib-Aib-Aib-OtBu, Z-Aib∗-Aib-Aib∗-Aib-Aib-Aib-OtBu, and Z-Aib∗-Aib∗-Aib-Aib-Aib-Aib-OtBu, Aib∗ = 13C enrichment at ∗C=O) in dichloromethane (a nonpolar aprotic solvent) and methanol (a polar, protic solvent) to examine the effects of carbon-13 enrichment on the spectra. Differences among the spectra of peptide isotopomers enriched with one or two 13C labels will be examined, detailing the effects of coupling between pairs of 13C-labeled Amide I oscillators.

High Pressure FTIR Studies on Model α-Helical Peptides

High-pressure conditions force water into internal cavities of proteins; the presence of water in a hydrophobic interior can destabilize the tertiary structure resulting in protein unfolding. This effect has been predicted in molecular dynamics simulations for model alpha-helical peptides at high pressures, where water is forced into closer contact with backbone carbonyls (as measured by an increased in backbone hydration). According to simulations, this increase hydration depends on local sequence and the impact that specific side chains have on backbone conformation. We have investigated the effects of pressure on a series of 20-residue peptides. Model peptides based on the alanine and lysine repeats form water soluble, stable alpha helices (1). The sequence (AAAAK)3-AAAAY is a well-characterized, synthetic polypeptide, ideal for the study of helix properties(2). Using the model peptides, we experimentally confirm that the helical content is conserved under pressure. Perturbations were monitored using a probe of secondary structure, the amide I’ band, with infrared (IR) spectroscopy and a diamond anvil cell. Amide I’ mode shifts to a lower frequency with the increase of pressure. Local information is obtained by measuring amide I'bands in 13C labeled peptides, where 13C alanines are placed at different positions relative to the lysines within the peptide. We examined the shielding effects of lysine at the various positions and confirm that shielding of backbone carbonyls occurs.1. Starzyk, A., Barber-Armstrong, W., Sridharan, M., and Decatur, S. M. (2005) Spectroscopic evidence for backbone desolvation of helical peptides by 2,2,2-trifluoroethanol: an isotope-edited FTIR study, Biochemistry 44, 369-376.2. Paschek, D., Gnanakaran, S., and Garcia, A. E. (2005) Simulations of the pressure and temperature unfolding of an alpha-helical peptide, Proc Natl Acad Sci U S A 102, 6765-6770.

Isotope-edited FT-IR Spectra of Hexamers of α−Aminoisobutyric Acid

Isotope-edited FT-infrared spectra of the Amide I region of hexamers of alpha-aminoisobutyric acid (Z-Aib6-OtBu) have been collected in order to explore the effects of 13C=O enrichment on the FT-IR spectra in the conformational context of 310 helices. Oligomers of Aib are known to adopt predominantly 310 helical structures, even at short peptide lengths. The Amide I band is sensitive to the details of peptide secondary structure, but the competency of this band to distinguish between alpha- and 310 helical secondary structure remains an open question. The 310 helix is shorter than an alpha-helix of the same number of residues and exhibits an i to i+3 hydrogen bonding pattern, instead of an i to i +4 pattern of the alpha-helix. These differences bring amide oscillators of a 310 helix slightly closer in space and in shorter periodicity of hydrogen-bonding partnership as compared to an alpha-helix. We have collected infrared spectra of isotopomers of hexamers of Aib (e.g., Z-Aib-Aib-Aib-Aib-Aib-Aib-OtBu and Z-Aib∗-Aib-Aib∗-Aib-Aib-Aib-OtBu, Aib∗ = 13C enrichment at ∗C=O) in dichloromethane (a nonpolar aprotic solvent) and methanol (a polar, protic solvent) to examine the effects of carbon-13 enrichment on the spectra. Differences between the spectral lineshapes and isotopically-induced shifts (∼40 cm−1) of the Amide I bands of these peptide isotopomers in these two solvents will be discussed.

Pyrophosphate Activation in Hypoxanthine−Guanine Phosphoribosyltransferase with Transition State Analogue

Isotope-edited difference Raman and FTIR studies complemented by ab initio calculations have been applied to the transition state analogue complex of HGPRT.ImmHP.MgPP(i) to determine the ionic states of the 5'-phosphate moiety of ImmHP and of PP(i). These measurements characterize electrostatic interactions within the enzyme active site as deduced from frequency shifts of the phosphate groups. The bound 5'-phosphate moiety of ImmHP is dianionic, and this phosphate group exists in two different conformations within the protein complex. In one conformation, a hydrogen bond between the 5'-phosphate of ImmHP and the OH group of Tyr104 in the catalytic loop appears to be stronger. With the stronger H-bond, the OH of Tyr104 approaches one of the P..O bonds from the bridging oxygen side to cause distortion of the PO(3) moiety, as indicated by a lowered symmetric P..O stretch frequency. The asymmetric stretch frequencies are similar in both phosphate conformations. Bound PP(i) in this complex is fully ionized to P(2)O(7)(4-). Bond frequency changes for bound PP(i) indicate coordination to Mg(2+) ions but show no indication of significant P..O bond polarization. Extrapolation of these results to reaction coordinate motion for HGPRT suggests that bond formation between C1' of the nucleotide ribose and the oxygen of PP(i) is accomplished by migration of the ribocation toward immobilized pyrophosphate.

The organization and assembly of a beta-sheet formed by a prion peptide in solution: an isotope-edited FTIR study.

Insight into the details of protein misfolding diseases requires a detailed understanding of the conformation and dynamics of multistrand beta-sheet aggregates. Here, we report an isotope-edited FTIR study of a model peptide directed at the elucidation of residue-level details of the structure and mechanism of a beta-sheet aggregate. A series of specifically isotope-labeled derivatives of a short peptide (H1) derived from residues 109 through 122 of the prion protein PrPC have been synthesized and characterized by FTIR. On the basis of the analysis of variable temperature FTIR spectra of these peptides in solution, the organization of strands within the beta-sheets has been determined; at equilibrium, the strands form a beta-sheet in which the hydrophobic core (112-122) participates in the sheet structure, resulting in the alignment of residue 117 in all of the strands. The peptides initially form a kinetically trapped intermediate beta-sheet, with a distribution of strand alignments, which can be rearranged into the stable equilibrium conformation by an annealing cycle. These observations are discussed in terms of the biological significance of residue 117 of the prion protein and the mechanism of beta-aggregate nucleation in prion proteins.

Conformational changes in G-CSF/Receptor complex as investigated by isotope-edited FTIR spectroscopy.

Conformations of G-CSF and the extracellular domain of its receptor as well as their complex have been investigated by employing isotope-edited FTIR spectroscopy. To determine unambiguously the protein conformations of G-CSF and the receptor in the complex, we have prepared uniformly 13C/15N isotope labeled G-CSF to resolve its amide I' band from that of the receptor in the IR spectrum of the complex. By comparing the IR spectra of the isotope-labeled G-CSF and the receptor with that of the complex, we have provided spectral evidence that the AB loop region involving the unique 310 helix segment of G-CSF likely undergoes a conformational change to a regular alpha-helix upon binding to the receptor. The IR data also indicate a possible minor increase in alpha-helical conformation for the receptor in the complex. Furthermore, FTIR spectra of G-CSF, the receptor, and their complex demonstrate clearly that protein conformations of both G-CSF and the receptor have been dramatically stabilized by complex formation. Specifically, the melting transition (Tm value) of the alpha-helix in G-CSF is increased by nearly 30 degrees C and that of the beta-strand in the receptor by nearly 15 degrees C in the G-CSF/receptor complex. We estimate from the current FTIR data that the native conformations of approximately 15% of all receptor residues are stabilized by G-CSF binding. On the other hand, the entire alpha-helical content of G-CSF appears to be stabilized in the complex. Together, these results indicate that formation of the ligand/receptor complex results in not only conformational changes in the receptor but also significant structural changes in the ligand. This adds insight to the general consensus that binding of ligand to cytokine receptors induces mostly structural changes in the receptor which lead to receptor oligomerization and signal transduction. The current data also suggest a possible physiological role of the 310 helix present in G-CSF for its receptor binding activity.