Asymmetric α-helicity loss within a peptide adsorbed onto charged colloidal substrates

doi:10.1016/S0021-9797(03)00092-4

Journal of Colloid and Interface Science

Volume 261, Issue 2, 15 May 2003, Pages 255-263

https://doi.org/10.1016/S0021-9797(03)00092-4 Get rights and content

Abstract

A combination of circular dichroism and solution ¹H NMR spectroscopy provides a localized description of the distribution of $α$ -helical structure within the capped peptide DDDDAAAAARRRR (4DAR5) in aqueous solution and adsorbed onto anionic and cationic colloidal substrates. The adsorption-induced conformational changes are different from those observed upon heating 4DAR5 in solution, in which case the alanine segment remains largely $α$ -helical and the transition to a coil structure propagates from the termini. Adsorption is driven by electrostatic complementarity, which places the charged peptide segment adjacent to the substrate of opposite charge. A similar pattern of $α$ -helicity loss is observed whether the peptide is adsorbed onto anionic or cationic colloidal silica, despite inverse orientations; significant $α$ -helicity loss occurs within the central alanine segment and the terminal arginine segment, whereas $α$ -helicity is retained in the aspartate segment. This pattern of adsorption-induced conformational change illustrates the complex and subtle balance among the intramolecular and intermolecular factors that influence the conformations of adsorbed peptides and proteins.

Introduction

An understanding of the conformational behavior of peptides at solid–solution interfaces is desired for prediction of the functionality of immobilized biologically relevant peptides and for insight into mechanisms of protein adsorption and denaturation in the adsorbed state. The interfacial interactions established upon adsorption to a substrate may induce a structural change within the peptide (relative to its conformation in solution), and this change may affect the entire molecule or may be localized within a particular segment. Circular dichroism (CD) spectroscopy is commonly used to study peptide and protein conformations in solution or adsorbed onto colloidal substrates [1], [2], [3], but a CD spectrum provides only a global measure of the secondary structural features present in a molecule, and it cannot provide detailed information regarding the secondary structure of specific domains within the peptide or protein. A residue-specific technique such as NMR spectroscopy must be used if localized structural information is desired, and localized conformational information has indeed been obtained for some adsorbed peptides using solid state NMR [4], [5], [6]. However, in the absence of a bulk water phase, the peptide may not be in a fully hydrated state, which may affect the conformation and orientation observed in the solid state [7]. Moreover, solid state ¹H NMR spectra of resolution sufficiently high for conformational studies can be difficult to obtain [8], [9], and solid state NMR studies of other nuclei often require the use of isotopically labeled molecules. In this work, we combine solution ¹H NMR and CD spectroscopic data to obtain localized structural information for a peptide adsorbed onto charged colloidal substrates in an aqueous environment.

Our previous CD study of short, neutral triblock (anionic–uncharged–cationic) peptides that are $α$ -helical in aqueous solution showed that adsorption onto anionic or cationic colloidal silica is driven by electrostatic complementary and is associated with a partial loss of $α$ -helicity [3]. The question addressed in the present work is whether the adsorption-induced loss of $α$ -helical structure occurs uniformly throughout the adsorbing molecule, or whether $α$ -helicity loss is localized in particular segments of the peptide molecule depending on their identity, sequence position, and orientation with respect to the substrate. For example, $α$ -helicity may be lost primarily from the segment of the peptide that is least $α$ -helical or most $α$ -helical in solution or from the segment that is positioned nearest to or furthest from the surface. In this work, ¹H NMR is used to investigate the distribution of $α$ -helicity within a peptide in solution and in the presence of anionic and cationic colloidal silica. In conjunction with information from CD regarding the overall $α$ -helicity of these molecules, the resulting pattern of $α$ -helicity loss upon adsorption is used to elucidate the effect of peptide–surface interactions on peptide conformation in this model system.

¹H NMR chemical shift values of amino acid $α$ -protons ( $α$ H), side chain $β$ -protons ( $β$ H), and peptide backbone amide protons (NH) are perturbed from their random coil (“intrinsic”) values by sequence, backbone torsional angles, hydrogen bonding, and local electrostatic effects [10], [11], [12]. The reliability of these conformationally sensitive proton chemical shift values as indicators of secondary structure depends on the particular proton type, as discussed in turn below.

The chemical shift values for the backbone $α$ -protons ( $δ_{αH})$ are more sensitive to the amide backbone dihedral angles $φ$ and $ψ$ and are less sensitive to other factors than the chemical shift values for other nuclei. Consequently, $δ_{αH}$ is considered to be a good indicator of secondary structure [11], [13]. The $α$ H resonances for residues in an $α$ -helical conformation are observed to be upfield of the coil value, whereas $α$ H resonances for residues in a $β$ -strand configuration are downfield; for example, for alanine within proteins, the average $δ_{αH}$ for an $α$ -helical conformation is 4.05 ppm, the average $δ_{αH}$ for a coil conformation is 4.25 ppm, and the average $δ_{αH}$ for a $β$ -strand conformation is 4.79 ppm [14]. The reliability of this relation has led to the Chemical Shift Index algorithm, which is commonly used to identify regions of secondary structure within proteins [15].

By contrast, $β$ H chemical shift values ( $δ_{βH})$ are less strongly correlated with secondary structure, although within a fixed sequence context the $β$ H resonances for a residue in an $α$ -helical conformation are downfield of the coil value, whereas the $β$ H resonances for a residue in a $β$ -strand configuration are upfield [10]. However, because the range of observed $δ_{βH}$ values for a given residue (approximately 0.6 ppm [10]) is considerably larger than the calculated contribution from the backbone dihedral angles $φ$ and $ψ$ (approximately 0.05 ppm [13]), these resonances do not provide reliable quantitative estimates of the secondary structural content of peptides. Interpretation of $δ_{βH}$ values is also complicated by the diastereotopic nature of the protons in residues such as aspartate and arginine, which gives rise to two different $β$ H resonances [10]. Nonetheless, limited qualitative conformational information is available from the $δ_{βH}$ values for the peptide described here.

NH chemical shift values ( $δ_{NH}$ ) are sensitive to secondary structure, but their dependence on sequence context [10] makes them difficult to study. In addition, the amide protons are susceptible to H/D exchange in the presence of deuterium oxide as the solvent, which is required in our studies to produce good quality NMR spectra of the adsorbed peptide. These considerations preclude the use of NH resonances as useful conformational indicators in our work.

The scalar coupling between the $α$ -protons and the amide protons (³ $J_{HNα}$ ) is largely dependent on the backbone conformation, specifically on the dihedral angle $φ$ [16], and ³ $J_{HNα}$ values provide a marker for $α$ -helicity. Residues in an $α$ -helical conformation are generally characterized by ³ $J_{HNα}$ values less than approximately 6 Hz, whereas larger values indicate residues in a coil or $β$ -strand configuration [16], [17]. Measurement of ³ $J_{HNα}$ requires the use of water as the solvent. Although we were not able to obtain the necessary high-resolution spectra for adsorbed peptide under these conditions, some conformational information based on ³ $J_{HNα}$ is presented for the peptide in solution.

The peptide studied here has the sequence DDDDAAAAARRRR (abbreviated as 4DAR5), with an N-terminal acetyl cap and a C-terminal amide group. The central alanine segment (A) has a strong propensity for $α$ -helix formation. Placement of the anionic aspartate (D) residues at the N-terminus and cationic arginine (R) residues at the C-terminus leads to a dipolar charge distribution at neutral pH, which, through favorable interaction with the amide backbone helix dipole, stabilizes an $α$ -helical structure. As shown previously [3], 4DAR5 adsorbs reversibly onto charged substrates despite the absence of a net charge on the peptide at neutral pH. Solution ¹H NMR reveals that peptide adsorption is orientation-specific, with the complementary charged end of the peptide adjacent to the substrate surface; the resonances for the residues apposed to the colloid surface (i.e., arginine for an anionic colloidal silica substrate or aspartate for cationic colloidal silica) are absent, while the resonances for the other residues are detected. Loss of signal intensity results from residue-specific immobilization within the Stern layer of the colloid on the time scale of the NMR experiment: residues located within this layer of tightly bound counterions (within approximately 6 Å of the surface [18]) move with the surface of the slowly rotating colloid, which leads to such extreme NMR signal broadening that these signals are absent from the solution NMR spectrum; by contrast, residues that reside outside the Stern layer,¹ such as those repelled electrostatically from the surface, retain sufficient local mobility for detection by solution NMR. Although this selective signal loss provides information regarding peptide orientation, conformational information is consequently not available directly for all residues of the adsorbed peptide.

In conjunction with the global measure of $α$ -helicity afforded by CD measurements, the conformational information that is available from $δ_{αH}$ provides a site-specific description of the structure of the 4DAR5 peptide. Using a combination of ¹H NMR and CD data, which has been applied previously only to the conformations of other peptides and proteins in solution and in membrane environments [19], [20], [21], the structures of 4DAR5 in solution and adsorbed onto charged colloidal substrates are elucidated here.

Section snippets

Materials

Crude 4DAR5 peptide (Research Genetics) was purified by reverse-phase HPLC on a C-18 column, followed by lyophilization. Ludox HS-40 aqueous colloidal silica (DuPont) and Ludox CL-30 alumina-capped colloidal silica (DuPont) were obtained from Aldrich; according to the manufacturer's specifications, they have surface areas of 220 and 230 m² g $^{−1}$ , respectively, and particle diameters of 12 nm. Solutions of colloidal silica in D₂O were prepared by repeated dilution of 1 g of colloidal silica with

Conformation of 4DAR5 peptide in solution

The CD spectrum of 4DAR5 in aqueous solution (pH 4.5–11) displays two strong negative bands at 208 nm (amide $π$ – $π^{∗}$ transition) and 222 nm (amide $n$ – $π^{∗}$ ), which are characteristic of an $α$ -helical structure [22]. The negative band at 222 nm $([θ]_{222})$ is taken as a measure of $α$ -helicity [23]. The intensity of $[θ]_{222}$ decreases with increasing temperature as the peptide undergoes a transition to a coil structure (no evidence of $β$ -strand structure is observed under any of the conditions used in this

Summary

Through a combination of CD spectroscopy, which provides an overall indication of secondary structure, and ¹H NMR, which provides amino acid-specific structural information, we have elucidated the detailed conformation of 4DAR5 peptide in solution and in the presence of anionic and cationic colloidal silica. Although 4DAR5 has no net charge, the strong dipolar charge distribution drives adsorption of the peptide onto a charged substrate in a specific orientation that places the complementary

Acknowledgements

The authors thank Professor Anne M. Mayes and Dr. Shuguang Zhang at MIT for helpful discussions and for use of experimental facilities and Professor David S. Wishart at the University of Alberta for generously providing a preprint of Ref. [14]. This material is based on work supported by the National Science Foundation under Grant No. DMR-9996338 to S.L.B. The NMR spectrometers in the Chemistry Department Instrumentation Facility at MIT are supported by the National Science Foundation under

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Asymmetric α-helicity loss within a peptide adsorbed onto charged colloidal substrates

Abstract

Introduction

Section snippets

Materials

Conformation of 4DAR5 peptide in solution

Summary

Acknowledgements

J. Colloid Interface Sci.

J. Colloid Interface Sci.

J. Mol. Struct.

Ann. Rep. NMR Spectrosc.

J. Mol. Biol.

J. Mol. Biol.

Methods Enzymol.

Prog. Biophys. Mol. Biol.

Langmuir

J. Am. Chem. Soc.

J. Am. Chem. Soc.

Biochemistry

Langmuir

Methods Enzymol.

J. Am. Chem. Soc.

J. Biomol. NMR

Asymmetric $α$ -helicity loss within a peptide adsorbed onto charged colloidal substrates