Conformational ensembles of neuromedin C reveal a progressive coil-helix transition within a binding-induced folding mechanism

Abstract

Neuromedin C (NMC) is a peptide that regulates various processes in the central nervous system and gastrointestinal tract through its interaction with the bombesin receptor subtype-2 (BB₂R). Hence, BB₂R antagonists hold the potential to treat disorders that occur as a result of NMC dysfunction or misregulation. However, their efficient design requires a detailed understanding of the structural features of NMC, which hitherto are unknown. Herein, we describe the conformational ensembles of NMC in an aqueous solution, at five different TFE concentrations to decode its folding pathway and under its SDS micelle bound state. NMC displays a disordered but well defined backbone architecture that undergoes a progressive coil-helix transition with increasing TFE concentration, first at the C-terminus and then at the N-terminus. NMC also adopts a C-terminal α-helical conformation upon binding to SDS micelles. This micelle binding is directed by hydrophobic interactions that concur with the unfavourable deprotonation of His⁸ and its further insertion into the micelle. Moreover, NMR relaxation data reveal that the acquisition of the micelle bound α-helical conformation constrains the NMC flexibility more than the confinement itself. This comprehensive study of the structural behaviour of NMC provides essential mechanistic information that could be useful for the development of new therapeutics to treat neurological, cancer-related or eating disorders.

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1 Introduction

Neuromedin C (NMC) is an endogenous decapeptide (GNHWAVGHLM-NH₂) that is highly conserved in mammals.¹ It exerts a variety of biological effects both on the central nervous system (CNS) and in the gastrointestinal tract.¹ Together with gastrin-releasing peptide (GRP) and neuromedin B (NMB), NMC belongs to the bombesin-like peptide family. Bombesin is a 14-residue peptide originally isolated from the amphibian Bombina bombina.² In addition its two mammalian analogues (GRP and NMB), NMC functions as a neurotransmitter, paracrine hormone, growth factor and it retains the full hormone activity of GRP.³

NMC exerts its physiological function mainly by its interaction with the subtype-2 bombesin receptor (BB₂R), which is a member of the G-protein coupled receptor superfamily⁴ that is located in the gut and in the CNS.⁵ For instance, NMC mediates neurotransmission and neuromodulation,⁶ and it is able to excite specific neurons by decreasing the resting potassium conductance and increasing the non-specific conductance.⁷ NMC can also reduce the appetite, and therefore can act as a anorexia inducer,⁸ likely through its interaction with bombesin receptors in the central amygdala.⁹ In addition, its intravenous administration increases growth hormone levels in calves,¹⁰ while it can also act as an autocrine growth factor in human small-cell lung cancer.¹¹ Moreover, NMC has been shown to regulate growth and/or differentiation of human tumors in a wide range of tissues, including carcinomas of pancreas, stomach, breast, prostate and colon.¹² Accordingly, a novel protein vaccine comprising six covalently linked repeats of NMC was successful in suppressing the proliferation of breast tumors cells.¹³

As a result of NMC's pharmacological profile, BB₂R antagonists are considered as prospective anticancer therapeutics¹⁴ and for the treatment of other illnesses.¹⁵ RC-3095, a peptidomimetic of NMC, was shown to produce long-lasting tumor regressions in different human models,¹⁶ as well as to show beneficial effects during the treatment of tumor necrosis factor-dependent chronic inflammatory conditions.¹⁷ More recently, a N-terminal modified NMC with acyclic tetraamines for binding of ^99mTc ([^99mTc]-demomedin C) was successfully targeted in BB₂R expressing tumor cells as a potent agonist inducing selective intracellular calcium release and triggering GRP receptor mediated internalization of the radioligand.¹⁸ However, its tolerability, background radioactivity and retention in tumor lesions warrant future studies as these pharmacological aspects have led to the rejection of other ^99mTc-bombesin analogs.¹⁹

The efficient design of more potent antagonists of BB₂R requires a detailed understanding of the structure–activity relationships of NMC. However, very limited structural information on NMC is currently available. In contrast to bombesin or NMB, NMC was predicted not to adopt a α-helical conformation upon binding to non-polar sites, due to reduced hydrophobic interactions that arise from the replacement of Leu³ by His³ in NMB.²⁰ Polverini et al. used CD spectroscopy to suggest that NMC could adopt a helical-like conformation upon binding to lipids.²¹ Furthermore, NMR spectroscopy was applied to solve the solution structure of the NMC–Ni²⁺ complex that consists of two connected turns,²² also likely adopted in the NMC–Cu²⁺ complex that could be physiologically involved in metal transport along the CNS.²³ More recently, we used replica exchange molecular dynamics (REMD) to demonstrate that NMC, in a simulated aqueous environment, adopts different conformations resembling β-turns that are stabilized by different hydrogen bonds formed and broken along the trajectory.²⁴

Although computer simulations can reveal the intrinsic conformational features of a peptide as encrypted in its sequence, caution should be taken about the thoroughness of the sampling. Therefore, we aim here to complement these preliminary computational results with further structural evidences. We combined different biophysical techniques to study the conformational ensemble of NMC in an aqueous solution, and at five different 2,2,2-trifluoroethanol (TFE)/water concentrations (i.e. at 10%, 25%, 40%, 60% and 90% TFE). As TFE can typically induce α-helicity in polypeptides,²⁵ we complemented these data by analyzing the α-helical structure of NMC bound to SDS micelles, characterizing the NMC–SDS micelle complex and evaluating independently the structuring and the binding effects on the peptide flexibility. These data constitute a comprehensive overall picture about the conformational preferences of NMC under different environments and represents a new structural platform for the future development of BB₂R antagonists.

2 Results

2.1 NMC is an intrinsically disordered peptide that undergoes a progressive coil-helix transition with increasing TFE concentration

It is already known that NMC displays a native random coil conformation in an aqueous solution.²¹ However, so far no experimental insight describes how the NMC conformation changes upon varying the dielectric environment, as might occur when it binds to BB₂R. Hence, we approached this folding process by studying the effect of increasing TFE concentrations on the NMC conformation. TFE has been widely used as a kosmotropic agent to study protein folding.²⁵ Moreover, it has a low effect on the pH of acetate buffered solutions when it is added up to 90% (ΔpH ∼ 0.4),²⁶ which makes it highly suitable for our study.

The CD spectrum of NMC in an aqueous solution indicates that the peptide is disordered, as evidenced by the minimum located at 197 nm. A slight increase in the TFE percentage, from 0 to 10%, scarcely modifies the spectrum profile as well as the secondary structure content. However, the addition of TFE at percentages higher than 10% markedly enhances the intensity of the region between 190 and 202 nm and reduces that of the band located between 206 and 240 nm, which results in a notable increase of the α-helical content, while decreasing the percentages of β-strands, turns and random coil regions (Fig. 1A and Table S1†).

Fig. 1 Analysis of the NMC conformation under different experimental conditions. (A) CD spectra of NMC in the presence of different percentages of TFE in 10 mM acetate buffer (pH 4.0) at 15 °C. (B) SSP values of NMC, as calculated from ¹H_N, ¹⁵N, ¹H_α, ¹H_β, ¹³C_α and ¹³C_β chemical shift values at pH 4.0 and 15 °C at 0% (), 10% (), 25% (), 40% (), 60% () and 90% () of TFE, and in the presence of 150 mM SDS (). (C) NMR solution ensembles of the ten lowest energy structures of NMC calculated at different TFE/water ratios in 10 mM acetate buffer (pH 4.0) at 15 °C. Backbone atoms are represented as ribbons, while the side chains are shown as atom coloured sticks. Images were generated using the UCSF Chimera software.

These results indicate that NMC adopts a predominant α-helix conformation upon addition of TFE, but only when the percentages are higher than 10%.

2.2 The Trp⁴–Gly⁷ region holds the higher α-helicity tendency

Although CD spectroscopy provided general information on the folding behaviour of NMC upon increasing the TFE/water ratio, it does not give any insight at the residue level on how the structuring process occurs. Therefore, we carried out the NMR study of NMC at different TFE/water ratios. The ¹H-, ¹⁵N- and ¹³C-NMR assignments were obtained at 15 °C for different NMC aqueous solutions containing 0%, 10%, 25%, 40%, 60% or 90% TFE. Chemical shifts of the backbone atoms (i.e. N, HN, H_α and C_α) were obtained for all residues, except those corresponding to N and HN of Gly¹ (that could not be achieved at any TFE percentage) and Asn² (which could only be accomplished at 0%, 10% and 25% TFE). The C_α resonance of Asn² at 40% TFE could not be observed either. All the N, H and C atoms at the side chains were assigned. The chemical assignments of NMC have been deposited to BMRB under the accessions codes 25519 (0% TFE), 25520 (10% TFE), 25521 (25% TFE), 25522 (40% TFE), 25523 (60% TFE) and 25524 (90% TFE).

Chemical shifts corresponding to H_N, N, H_α, H_β, C_α and C_β were used to determine the secondary structural propensity (SSP) scores (see Section 4.5) at different TFE/water ratios (Fig. 1B). The SSP plots obtained suggest that the structures of NMC at 0% and 10% TFE are similar, except for the residues located at the C-terminus, which seem to slightly enhance their helicity at 10% TFE. While both the structures seem to be randomly coiled, the increase in the TFE percentage above 10% clearly induces an enlargement of the α-helix content, which is more prominent in the central region of the peptide (Trp⁴–Gly⁷). On the other hand, most of the SSP values obtained at 90% TFE are higher than 0.5, which indicates that the corresponding residues display a well-defined α-helical structure.

These results are in agreement with the CD data and prove that the peptide central region (Trp⁴–Gly⁷) holds the higher α-helical tendency.

2.3 The folding pathway of NMC upon increasing the TFE concentration

The ¹H-, ¹⁵N- and ¹³C-NMR assignments were used to calculate the solution structures of NMC at each TFE/water ratio. The geometrical restrains used during the calculations were taken from the NOEs intensities and automatically assigned using the CYANA software²⁷ (Fig. S1†). These assignments provided NMR ensembles with all dihedral angles located in favoured and allowed regions and with low backbone RMSD, except for the most disordered NMC structures obtained at 0% and 10% TFE. The ensembles obtained have been deposited in the PDB under the accessions codes 2n0b (0% TFE), 2n0c (10% TFE), 2n0d (25% TFE), 2n0e (40% TFE), 2n0f (60% TFE) and 2n0g (90% TFE), and all of them satisfy all convergence criteria for successful structure calculations (Table 1).

Table 1 Structural statistics for the different conformers of NMC obtained at different d₃-TFE/water ratios and in the presence of d₂₅-SDS micelles

	0% TFE	10% TFE	25% TFE	40% TFE	60% TFE	90% TFE	SDS
a Restraint statistics reported for unique, unambiguous assigned NOEs.b Violations are only reported when present in six or more structures.c Coordinate precision is given as the average pair-wise Cartesian coordinate root mean square deviations over the ensemble.d Values obtained from the PROCHECK-NMR analysis^68 by using the Protein Structure Validation Server (PSV).^69
Structural computed conformers	18	17	20	20	19	20	20
Restrains^a
Short-range (|i − j| ≤ 1)	47	71	91	121	80	79	99
Medium-range (1 < |i − j| < 5)	3	2	23	49	24	35	39
Long-range (|i − j| ≥ 5)	0	0	2	7	3	0	0
NOE constrains per restrained residue	7.1	9.0	12.8	19.3	13.1	12.1	13.8
Torsion angles restraints	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Restraints statistics^b
Distance violations > 0.0 Å	5	4	3	4	2	3	2
Torsion angle violations > 0°	0	0	0	0	0	0	0
Target function value (Å^2)
Average/best	0.0	0.0	0.02	0.0	0.02	0.0	0.0
Pairwise RMSD of residues 3–8 in Å^c
Backbone N, CA, C′	0.73 ± 0.26	0.63 ± 0.29	0.08 ± 0.07	0.01 ± 0.00	0.06 ± 0.04	0.25 ± 0.1	0.17 ± 0.7
Heavy atoms	1.87 ± 0.51	1.40 ± 0.38	0.43 ± 0.30	0.05 ± 0.01	0.41 ± 0.20	1.05 ± 0.32	0.57 ± 0.35
Ramachandran plot^d
Most favoured regions (%)	54.8	47.1	47.9	55.0	75.7	82.9	60.7
Additional allowed regions (%)	45.2	52.9	52.1	45.0	23.4	17.1	39.7
Generously allowed regions (%)	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Disallowed regions (%)	0.0	0.0	0.0	0.0	0.0	0.0	0.0

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The NMC ensemble calculated in an aqueous solution displays a native random coil conformation. Nevertheless, the corresponding backbone RMSD is lower than that expected for a fully unstructured ensemble, mostly as a result of the short range NOEs detected between central amino acids, which create a backbone architecture resembling a distorted S (Fig. 1C).

The NMC structure obtained at 10% TFE does not differ significantly to that found in water, which is in perfect agreement with previously reported CD and SSP data (Fig. 1A and B). However, while the depicted S-like conformation between His³–His⁸ is still preserved, the C-terminal region appears to adopt a more extended structure (Fig. 1C). This subtle but remarkable difference was already revealed by the SSP data, but it can also be observed from the overlapping of the ¹⁵N-HSQC spectra of NMC at 0% and 10% TFE, wherein the chemical shift perturbations of Val⁶, Gly⁷ and Leu⁹ are larger than those of Asn², His³, Trp⁴ or Ala⁵ (Fig. 2A).

Fig. 2 Overlapping of the ¹⁵N-HSQC spectra of NMC obtained at different d₃-TFE/water ratios. (A) ¹⁵N-HSQC spectra of NMC in the presence of 0% (red) and 10% (purple) TFE. (B) ¹⁵N-HSQC spectra of NMC in the presence of 10% (purple) and 25% (green) TFE. (C) ¹⁵N-HSQC spectra of NMC in the presence of 25% (green) and 40% (orange) TFE. (D) ¹⁵N-HSQC spectra of NMC in the presence of 40% (orange) and 60% (black) TFE. (E) ¹⁵N-HSQC spectra of NMC in the presence of 60% (black) and 90% (cyan) TFE. The arrows indicate the cross-peak shift from the lower to the higher TFE percentage.

CD and SSP data already suggested that an increase in the TFE content from 10% to 25% implies a larger structural rearrangement than that occurring when the TFE percentage increase from 0% to 10%. Moreover, the overlapping of the ¹⁵N-HSQC spectra obtained at 10% and 25% TFE additionally shows that this rearrangement mainly occurs at the C-terminus, as is evidenced by the chemical shift perturbations displayed by the Val⁶–Met¹⁰ stretch (Fig. 2B). The NMC structure obtained at 25% TFE does not exhibit the S-like conformation observed at 0% and 10% TFE. In contrast, residues at the C-terminus (up to Trp⁴) roll up and adopt a helical-like turn, whereas the N-terminal region bends back towards the central residues (Fig. 1C).

The NMC solution structure obtained at 40% TFE shows the formation of a short but well defined α-helix at the C-terminus (Ala⁵–His⁸) as a result of an increased compactness of the structure displayed at 25% TFE (Fig. 1C). On the other hand, the N-terminal region remains unstructured, but adopts a newly extended conformation. Therefore, the increase in the TFE percentage from 25% to 40% implies an overall structural rearrangement that is also evident from the comparison of the corresponding ¹⁵N-HSQC spectra, wherein the entire resonances shift (Fig. 2C).

At 60% TFE, the chemical shifts of the residues between Val⁶ and Met¹⁰ do not show remarkable differences in comparison with those obtained at 40% TFE. However, more noticeable are the shifts of the cross peaks corresponding to His³ and Trp⁴ (Fig. 2D). These variations result from the preservation of the α-helical structure between Ala⁵ and His⁸ already formed at 40% TFE, whereas the residues at the N-terminus fold back also adopting a new α-helical conformation (Fig. 1C).

The increase in the TFE percentage mostly resulted in an overall chemical shift variation of the ¹H–¹⁵N cross peaks towards high field, especially in the ¹H dimension (Fig. 2), which can be attributed to the lower capacity of TFE to form hydrogen bonds relative to water.²⁸ This was also the case when the TFE percentage increased from 60% to 90%, except for His³ and Trp⁴, whose cross peaks shifted towards low field, suggesting a further structural rearrangement at the N-terminus (Fig. 2E). The structure of NMC obtained at 90% TFE shows an enlargement of the Ala⁵–His⁸ α-helical stretch depicted at 60% TFE towards Leu⁹, but also towards Trp⁴ and His³ (Fig. 1C). Hence, the more compact conformation adopted by the N-terminus when going from 40% to 60% TFE becomes fully α-helical at 90% TFE.

Thus, NMC is a disordered peptide in an aqueous solution, although its central region exhibits a constrained backbone architecture resembling a distorted S. Upon increasing TFE/water ratios, the C-terminal region first stretches to fold back into an α-helical structure, which also occurs at the N-terminus but only at higher TFE concentrations.

2.4 NMC binds to SDS micelles

TFE is known to induce α-helicity in most polypeptides through a process that mimics their embedding into regions of low dielectric constant and high viscosity.²⁵ Hence, the use of different TFE/water ratios allowed the modelling of a folding route, which could mimic that occurring during its interaction with BB₂R.

In addition, we complemented these results by studying the biophysical characteristics of NMC upon interaction with SDS micelles, which were chosen as a model system.

We ran diffusion-oriented (DOSY) NMR experiments on a solution containing NMC alone or in the presence of SDS micelles. The resulting diffusion coefficients (D) were independent of the NMC concentration, indicating that no self-aggregation occurred in both the samples. Assuming that the slight viscosity change linked to the presence of SDS micelles equally affects the reference (i.e. acetate and DSS signals) and the NMC signals, it is clear that NMC reduces its overall mobility in the presence of SDS micelles (Fig. 3A), which potentially suggests their binding.

Fig. 3 Study of the binding between NMC and SDS. (A) Overlap of the 2D-DOSY spectra of NMC in 10 mM acetate buffer (pH 4.0) alone (blue) and in the presence of 150 mM d₂₅-SDS (red). The spectra were referenced to the acetate and DSS signals. (B) Difference curves arising from the subtraction of the enthalpy curves obtained during the titration of a 35 mM SDS solution into a solution containing NMC at 10 μM (), 20.8 μM (), 42 μM () or 62.5 μM () from that obtained when titration was carried out in free-NMC solution. All the titrations were carried out at 15 °C using acetate buffer solution (10 mM) at pH 4.0. This panel only shows the data collected for the addition ongoing from 0 to 2 mM SDS concentration. (C) Difference enthalpy values for the initial exothermic enthalpy depicted in panel B (black) and for the exothermic enthalpy shown in the following panel D (red) as a function of protein concentration. Points are the experimental data, while lines represent the fitting of this data to a linear correlation. (D) The same plot as depicted in panel B but showing the titration region ongoing from 2 to 9 mM SDS concentration.

Isothermal titration calorimetry (ITC) was then used to thermodynamically characterize the binding process. The titration curve of SDS in acetate buffer resulted in the appearance of initial exothermic peaks accounting for the low-temperature energy-favoured demicellization.²⁹ These peaks became less exothermic as they approached the midpoint of the inflection (critical micelle concentration; cmc),³⁰ to finally become endothermic as a result of the micelle dilution effect²⁹ (Fig. S2†).

A similar trend was observed when titrations were carried out on solutions containing NMC (Fig. S2†). However, several differences that could be ascribed to the SDS–NMC interaction were observed. Difference enthalpograms reveal an initial exothermic heat flow that rapidly levels off as the SDS concentration increases (Fig. 3B). This effect likely corresponds to specific and cooperative electrostatic interactions occurring between the SDS sulfate group and cationic His.³¹ The amplitude of this variation scales linearly with NMC concentration (Fig. 3C) and the slope obtained (α_ΔH ∼ −79 ± 5 kJ mol⁻¹ mM⁻¹) indicates that there is a large change in the ΔH of the system upon a small change in both protein and surfactant [α_ΔH is proportional to (d²H)/(dn_proteindn_SDS) and measures the enthalpy of NMC–SDS interaction³²].

Next, the enthalpy difference became slightly endothermic, which could be attributed to a conformational rearrangement of NMC.³³ Then, the curves began to deviate exothermically from the control curve at a SDS concentration of ∼3.3 mM, which corresponds to the onset for binding of NMC to SDS (critical aggregation concentration; cac).³⁴ The subsequent exothermic variation is attributed to the association of SDS and NMC,³³ whose saturation is at the inflection point and corresponds to cmc, which slightly increases with the NMC concentration (3.9–4.2 mM). SDS injected beyond this point remains in the micellar form, having fewer NMC molecules to interact and leading to the final asymptotic curve (Fig. 3D). The amplitude of this curve linearly scales with the NMC concentration and the obtained α_ΔH ∼ −0.92 ± 0.09 kJ mol⁻¹ mM⁻¹ (Fig. 3C) proves that the nature of the interaction between SDS micelles and NMC is much weaker than an electrostatic one.

The cac and cmc values were additionally used to calculate the Gibbs free energy change of aggregation (ΔG_mic) and the Gibbs free energy change of aggregation in the presence of NMC (ΔG_ag) through the application of the charged phase separation and mass-action model.³⁵ The obtained ΔG_mic and ΔG_ag values were −24 ± 2 and −25 ± 1 kJ mol⁻¹, respectively, which proves that the micellar behaviour of SDS and the formation of NMC/SDS mixed micellar junctions are both similarly thermodynamically favoured. Moreover, ΔH_mic and ΔH_ag, both <−0.1 kJ mol⁻¹ (Fig. 3D), are much smaller that the terms TΔS_mic or TΔS_ag (∼24 kJ mol⁻¹), revealing that both the aggregation of SDS in the absence and in the presence of NMC is entropy driven.

According to Lindman and Thalberg,³⁵ the free energy to drive 1 mol of monomeric SDS into NMC-bound micelle (ΔG_ps = ΔG_ag − ΔG_mic) is indicative of the binding strength of SDS onto NMC. The obtained ΔG_ps ≈ −1 kJ mol⁻¹ reveals that the binding between NMC–SDS is only slightly thermodynamically favoured in comparison to that occurring between SDS–SDS molecules.

2.5 Solution structure of NMC bound to SDS micelles

Upon showing that NMC binds to SDS micelles, we wanted to assess this binding effect on the peptide structure. In contrast to bombesin or NMB, NMC was predicted not to adopt a membrane-inserted α-helical conformation due to the reduction of hydrophobic interactions upon replacement of Leu³ by His³, which would be needed to effectively build the α-helix.²⁰ However, the CD spectrum of NMC bound to SDS micelles indicated the contrary: the presence of micelles induced a redshift in the minimum, while the intensity of the regions between 191 and 193 nm and 200 and 240 nm increased and decreased, respectively (Fig. 4A). This indicates that NMC increases its α-helicity upon binding to SDS micelles at a level similar to that shown at 60% TFE content (Table S1†).

Fig. 4 Structural study of NMC bound to SDS micelles. (A) CD spectra profiles of NMC collected at 15 °C in the presence and in the absence of SDS. (B) Overlapping of the ¹⁵N-HSQC spectra of NMC recorded at 15 °C in 10 mM acetate buffer (pH 4.0) alone (red) and in the presence of 150 mM d₂₅-SDS (purple). (C) NMR ensemble of the ten lowest energy structures of NMC calculated in the presence of SDS micelles. (D) Structural alignment between His³ and Met¹⁰ of the mean NMC structure (averaged over the entire unfolded ensemble by using MOLMOL software) obtained in the presence of 60% TFE (blue) and when it is bound to SDS micelles (orange). (E) Fluorescence emission spectra of NMC collected at 15 °C in 10 mM acetate buffer (pH 4.0) alone and in the presence of SDS micelles.

NMR was then used to obtain residue level insights on this helical rearrangement. The comparison of the ¹⁵N-HSQC spectra of NMC obtained in the absence and in the presence of d₂₅-SDS show notable chemical shift perturbations as a result of the binding. This occurs for all residues except for the cross peaks of Asn² (i.e. HN–N and HN^δ–N^δ), which indicates that the chemical environment of Asn² is only slightly modified upon micelle interaction (Fig. 4B). All ¹H, ¹⁵N, and ¹³C resonances (except those for N and HN of Gly¹) were unambiguously assigned, deposited to BMRB (25525), and used to calculate the SSP values, which were also comparable to those obtained at 60% TFE (Fig. 1B). Hence, CD and SSP data strongly suggest that the structure of NMC bound to SDS micelles must be similar to that obtained at 60% TFE.

The geometrical restrains automatically obtained from the NMR assignment and the ¹H–¹H-NOE intensities (Fig. S1†) were used to calculate the solution structure of NMC bound to d₂₅-SDS micelles. The ensemble obtained, deposited to the PDB under the accession code 2n0h, has an excellent Procheck-NMR score satisfying all convergence criteria for structure calculations (Table 1). The structure reveals that the binding process induces the formation of an α-helical stretch between Ala⁵ and Leu⁹, whereas the N-terminus retains the native random coil conformation (Fig. 4C). In fact, this structure is very similar to that obtained in 60% TFE, which is ascertained by the low RMSD value arising from the alignment of the averaged structures of both ensembles (0.52 Å for the backbone atoms) (Fig. 4D).

Our results show that NMC binds to SDS micelles through a process that implies the formation of an α-helical stretch at the C-terminus, whereas the N-terminal segment remains disordered.

2.6 Trp⁴ is embedded into the SDS micelle

We then wanted to understand the molecular architecture of the complex formed between NMC and SDS micelles. Initially, we used fluorescence spectroscopy to determine whether Trp⁴ is inserted into the micelles. The fluorescence spectrum of NMC shows an emission maximum at 356 nm, typical of a solvent exposed indole group. However, when NMC binds to SDS micelles, the fluorescence maximum undergoes to a hypsochromic shift, from 356 to 345 nm, suggesting the incorporation of the Trp⁴ side chain into a less polar environment (Fig. 4E). In contrast, the quantum yield of Trp⁴ also decreases in agreement with what was found during other peptide–SDS micelle interactions.³⁶

The extent to which Trp⁴ was buried into the micelles was determined using the acrylamide quenching experiments. Equal amounts of acrylamide were added to solutions containing either free NMC or NMC–SDS micelles complex. The presence of micelles decreased the Stern–Volmer constant (K_sv) of NMC threefold (∼13 M⁻¹ for the free form vs. ∼4 M⁻¹ for the complex) (Fig. S3†), indicating a high degree of protection of the Trp⁴ side chain against the solvent. Hence, the Trp⁴ side chain inserts into the SDS micelles during the binding process.

2.7 Mapping the interactions between NMC and SDS micelles

The atomic contact map between NMC and SDS micelles was obtained from overlapping of the ¹H–¹H-NOESY spectra obtained in the presence of 150 mM d₂₅-SDS and that obtained in the presence of 50 mM SDS. The change in the SDS concentration did not alter the NMC structure, as evidenced by the comparison of both ¹⁵N-HSQC spectra (Fig. S4†).

The use of non-deuterated SDS resulted in the appearance of new NOEs that were unambiguously assigned to specific intermolecular NMC–SDS contacts. The side chain of Trp⁴ is fully embedded into the SDS micelles since its indole group exhibits strong NOEs with different SDS methylene groups. In addition, the HN of Val⁶, Leu⁹ and Met¹⁰, as well as the H_α of Trp⁴, Ala⁵, Val⁶ and Leu⁹ display different NOEs with the aliphatic tail of SDS, proving that the backbone at the C-terminus is also inserted into the SDS micelles. NOEs signals connecting the Val⁶ and Met¹⁰ side chains with protons of C1 in SDS were also found, indicating that these regions protrude from the hydrophobic core of the micelle (Fig. 5A and S5†) (Table 2).

Fig. 5 The mapping of the interactions observed between NMC and SDS micelles. (A) Overlapping of the ¹H–¹H NOESY spectra of NMC obtained at 15 °C when it was bound to SDS micelles (red) and when it was bound to d₂₅-SDS micelles (blue). The chemical structure of SDS is shown above the NMR spectra. Carbon atoms are numbered arbitrarily. (B) Model representation of the interaction between NMC and SDS micelles. Aliphatic chains of SDS are coloured in grey, while the corresponding sulfate group is coloured in yellow (sulphur atoms) and red (oxygen atoms). NMC residues are coloured based on the previously determined free energy transfer of each amino acid from water to lipid bilayers (ΔG = −1.8 kcal mol⁻¹, red; ΔG = 1.8 kcal mol⁻¹, blue).⁴⁰ His³ is coloured taking the ΔG value determined for protonated His, while His⁸ is coloured based on the ΔG value determined for neutral His.

Table 2 NOE connectivities found between NMC and SDS micelles^a

NMC
	CH2 (1)^b	CH2 (2)^b	CH2 (3–11)^b
a The intensities of the NOE signals are divined as: “+++” high intensity; “++” medium intensity; “+” low intensity.b Atoms are numbered arbitrarily according the chemical structure of SDS.
HN-V6			++
HN-L9			+
HN-M10			+
Hα-W4			++
Hα-A5			++
Hα-V6			++
Hα-L9			+
Hβ2-W4			+
Hβ3-W4	++		++
Hβ3-H8			+
Hβ3-M10	+
Hγ2-M10	+
Hε1-W4	+	+	++
Hε3-W4		+	++
Hζ2-W4	+		++
Hδ1-W4	+	+	++
Hζ3-W4		+	+++
Hδ2-H8			++
Hγ1-V6	++
Hγ2-V6	++

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When analyzing the structure of NMC upon binding to SDS micelles (Fig. 4C), it is difficult to understand how this interaction can occur since an amphipathic-like architecture is lacking. For instance, His⁸ points towards the same face of the helix than Trp⁴, while His³, Val⁶ and Met¹⁰ are in the opposite one.

At pH 4.0 His side chains must be protonated, the pK_a of the imidazole protons ranges from 4.9 to 6.6 in micellar media,³⁷ and therefore highly unlikely to penetrate into the micelles. This has been the case for His containing peptides, where micelle insertion was only observed when the pH increased above the pK_a of His.³⁸ However, we unexpectedly detected unambiguous NOEs between H_β/H_δ2 of His⁸ and the aliphatic methylene groups of SDS (Fig. 5A and S5†) (Table 2), which proves that His⁸ inserts into the micelles even at pH 4.0. This could only occur if the insertion occurs together with His⁸ deprotonation. In fact, the H_δ2 and H_ε2 chemical shift values (highly sensitive to imidazole protonation state) in His⁸ are shifted upfield upon micelle binding in a range comparable to that observed during the pH-induced deprotonation (i.e., ∼0.2 ppm for H_δ2 and ∼0.4 ppm for H_ε2).^38,39 This was not the case for His³, since its H_δ2 and H_ε2 values only underwent a slight downfield shift upon micelle binding (Table 3).

Table 3 Chemical shift values of imidazolinic protons of His³ and His⁸ and of the C-terminal and Asn² amide group atoms when NMC is either free or bound to SDS micelles

	Hδ2^a	Hε1^a	N^a	H1^a	H2^a
a Values are given in ppm.
His^3 in free NMC	7.08	8.42	—	—	—
His^3 in micelle bound NMC	7.28	8.61	—	—	—
His^8 in free NMC	7.14	8.43	—	—	—
His^8 in micelle-bound NMC	7.02	8.03	—	—	—
Ans^2 side chain in free NMC	—	—	113.3	7.53	6.93
Asn^2 side chain in micelle-bound NMC	—	—	112.8	7.52	6.88
C-terminal amide in free NMC	—	—	108.2	7.54	7.15
C-terminal amide in micelle-bound NMC	—	—	105.6	7.20	7.03

---

Our data indicate that NMC binds to SDS micelles only through the insertion of its C-terminal region, while the N-terminal tail remains out of the micelle (Fig. 5B). This is additionally supported by the upfield chemical shifts of the C-terminal amide, whereas the chemical shifts corresponding to the amide side chain of Asn² remain unaltered (Table 3) (Fig. 4B). The binding process occurs through the energetically favoured insertion of Trp⁴, Val⁶, Gly⁷, Leu⁹ and Met¹⁰ have a negative ΔG for transfer form water to lipid bilayers,⁴⁰ which must energetically compensate the deprotonation and the further insertion of His⁸ into the micelle (Fig. 5B).

2.8 The α-helical folding and the micelle binding effects on the NMC flexibility

NMC folds upon SDS micelle interaction adopting a structure similar to that displayed at 60% TFE. This scenario gives the unique opportunity to study separately the influence of the folding and the binding effect on the NMC dynamics. Hence, we acquired the ¹⁵N R₁, R₂, and HET-NOE relaxation data for NMC in an aqueous solution, in the presence of 60% TFE, and under its SDS micelle bound state.

In an aqueous solution, R₁ and R₂ constants are lower than 1.3 s⁻¹, whereas all HET-NOEs are negative, which indicates that both the features are typical of intrinsically disordered peptides (IDP).⁴¹ The addition of 60% TFE increases R₁ and R₂ constants by ∼2–3 times, while most of the HET-NOEs become positive as a result of the rigidity linked to the α-helix formation. These variations are even more pronounced when NMC binds to SDS micelles. In this case, R₂ increases by ∼9 times and most of the HET-NOEs display values close to 0.5, thus proving that the binding additionally constrains the α-helical NMC structure (Fig. 6A–C).

Fig. 6 NMR dynamics of NMC in acetate buffer, in 60% TFE and under its micelle bound state at 14.1 T and 15 °C. (A–C) ¹⁵N relaxation dynamics parameters (A) R₁, (B) R₂ and (C) heteronuclear NOE plotted as a function of the residue number. Error bars in plots indicate the curve-fit root mean square deviation of each point. Mean values are plotted as columns. (D) Order parameters (S²) obtained from Liparizi–Szabo model-free analysis as a function of the residue number.

The R₁ and R₂ constants obtained in an aqueous solution for the Trp⁴-indole group are similar to the backbone ones. However, HET-NOE is ∼0.9 units higher, likely a result of the rigidity linked to the contacts of Trp⁴-H_δ1, H_ε3 with Ala⁵, which must reduce the side chain dynamics. The presence of 60% TFE slightly increases the R₁ and R₂ constants, revealing that the mobility of the Trp⁴ side chain is slightly reduced upon folding. Moreover, the confinement of the indole group into the SDS micelles enlarges by ∼4 times the R₂, thus proving that the binding also additionally constrains the mobility of the Trp⁴ side chain (Fig. 6A–C).

The R₁/R₂ ratios within one standard deviation of the mean were then used to determine the correlation time (τ_c) of each structural ensemble. Calculations carried out with the r2r1_tm and TENSOR 2.0 software gave similar values, being 0.8 ± 0.1 ns in buffer, 2.1 ± 0.1 in 60% TFE, and 6.9 ± 0.1 ns under its micelle bound state. Although the NMC molecular size scarcely changed in the presence of 60% TFE (Fig. 1C), τ_c increased by ∼3 times as a result of the increased viscosity of the TFE/water mixture.⁴² Furthermore, τ_c of the NMC–SDS micelle complex is ∼8 times bigger than that of free NMC, which can be ascribed to the resulting high molecular weight complex. In addition, this τ_c value is also ∼1 ns bigger than that of free SDS micelles,⁴³ which points to the formation of 1 : 1 NMC–SDS micelle complex.

The backbone R₁ and R₂ values and the energy-minimized representative conformers of each NMR-derived solution structure were used to estimate the diffusion tensor (D_∥/D_⊥) using the isotropic, axially symmetric and fully anisotropic diffusion models in the software Quadratic-Diffusion.⁴⁴ The D_∥/D_⊥ values obtained for NMC were 1.2 in buffer, 0.98 in 60% TFE, and 0.8 under its micelle bound state. Hence, the diffusion model that best describes the NMC rotational behaviour under these experimental conditions is the isotropic one (D_∥/D_⊥ < 1.3).

The ¹⁵N-relaxation parameters were then analyzed assuming an isotropic rotational diffusion model and according to the Lipari–Szabo model-free formalism⁴⁵ (Tables S2–S4†). The order parameters (S²; indicative of the amplitude of internal ps–ns timescale motions) of NMC in water have an average value of 0.45 ± 0.12, being within the typical range found in IDPs (S_av² ∼ 0.3–0.6).^46,47 However, these values are not homogeneous along the NMC sequence. The S_av² of the residues between Trp⁴ and His⁸ is notably higher than that arising from the three N-terminal and the two C-terminal residues (ΔS_av² ∼ 0.18) (Fig. 6D). This striking variation clearly proves that central amino acids, although integrated within the fully disordered NMC structure, display much slower motions than the terminal ones, likely as result of their inter-residual interactions.

The acquisition of the α-helical structure in the presence of 60% TFE reduced by ∼46% the flexibility of the central residues (His³–His⁸), as evidenced by ΔS_av² of ∼0.23. This was not the case for the still unstructured two C-terminal residues, whose mobility was nearly unaltered upon folding of the Ala⁵–His⁸ stretch (ΔS_av² ∼ 0.03). The insertion of the folded NMC into SDS micelles additionally reduced the mobility of the central amino acids (His³–His⁸) by ∼20% (ΔS_av² ∼ 0.1), whereas that corresponding to the C-terminal Leu⁹ and Met¹⁰ was reduced by ∼90% (ΔS_av² ∼ 0.36) as a result of their confinement in the SDS micelle (Fig. 6D).

Our results reveal that NMC in an aqueous solution displays highly different conformational motions along its sequence. Moreover, the NMC α-helical folding markedly reduces the mobility of the central residues, affecting the peptide flexibility to a larger extent than the subsequent structural confinement into SDS micelles.

3 Discussion

NMC modulates different physiological processes such as feeding or tumor growth mostly through its interaction with BB₂R. Although its physiological implications have been known for four decades,^3,6,11 its structural features, either in its free form or when bound to BB₂R, have not yet been reported. Herein, we combined different biophysical techniques to study the structural and dynamical preferences of NMC in an aqueous solution and under its SDS micelle bound state. In addition, we also analyzed the structure of NMC at different TFE/water ratios to decode the NMC folding pathway. TFE is known to induce polypeptide folding in the sense that it deprives peptides of establishing hydrogen bonds with water thereby favouring intra-peptide hydrogen bonding.⁴⁸

Most of the small peptides do not behave as pure random coils because their residues usually do not sample all sterically accessible regions, but rather exhibit local structural preferences.⁴⁹ Hence, we used NMR to gain residue insights on the structural preferences of NMC in an aqueous solution. The ¹⁵N-HSQC spectrum only displays nine signals proving that either there is a main conformational state or that the dynamic equilibrium between different conformers is a fast exchange regime (Fig. 2A). This agrees with our REMD prediction of a low energy structure among the different NMC conformers.²⁴ The NMR ensemble possesses a well-defined backbone architecture resembling a distorted S (Fig. 1C), which is built through short range contacts between the central residues. We already predicted these turns using REMD, since ∼20% of the sampling displayed the segment Trp⁴–Gly⁷ stabilized by a hydrogen bond. In addition, others turns, such as His³–Trp⁴, Trp⁴–Ala⁵ or Gly⁷–His⁸, were only ∼10% sampled.²⁴ This S-like architecture is also adopted in the NMC–Ni²⁺ complex since two turns are formed, one involving the three first residues coordinating the metal and the other linking Ala⁵ to His⁸.²²

NMC folds into a helical structure upon increasing the TFE percentage as result of the reduction of the dielectric constant, which favours the formation of intramolecular hydrogen bonds. However, the ease of adopting this α-helical structure is not the same along the entire sequence. The presence of only 10% TFE induces the stretching of the C-terminal region that folds back into a helical-like structure only when the TFE percentage increases to 25%. The C-terminal helical structuring is finally strengthened at 40% TFE and it does not further change at 60 or 90% TFE. The N-terminal segment is more resistant to undergo the coil-helix transition, since it only acquires a helical-like structure when the TFE content is 60%, becoming fully α-helical at 90% TFE.

In addition, we studied the molecular complex formed between NMC and SDS micelles. ITC was used to characterize the binding process. Initially, it appeared as a highly exothermic event proportional to the NMC concentration and was attributed to electrostatic interactions.^31,33 This showed that NMC is the limiting reactant in this region of the plot and that the binding is notably weak. NMC started to bind SDS at a cac of ∼3.3 mM, consistent with what was observed when using hydrophobically alkali-soluble emulsion polymers (HASE) (cac ∼ 4 mM).⁵⁰ The cac value was independent of the NMC or HASE concentration, although it is concentration-dependent in folded proteins.³³ The binding saturation of NMC occurred at [SDS] ∼ 4 mM, which agrees with the cmc of SDS at 15 °C,²⁹ and it did not change with the NMC concentration, thus differing from what occurs in folded proteins.³³ SDS added beyond cmc remains in micellar form and leads to an asymptotic curve that is related to the NMC hydrophobicity.^34,50 Its α_ΔH value is ∼ 86 kJ mol⁻¹ mM⁻¹ lower than that determined for the electrostatic interactions between NMC and SDS monomers, which proves that the diving force leading to the NMC/SDS micelle binding is weaker than an electrostatic one and likely to be hydrophobic. This α_ΔH is also ∼15 times lower than that determined for folded proteins,³³ which must account for the lower hydrophobicity of NMC in comparison to larger polypeptides. The formation of micelles alone or in the presence of NMC was always thermodynamically favoured through an entropy-driven process. Moreover, the calculation of ΔG_ps (which compares the stability of the interactions between NMC–SDS and SDS–SDS) demonstrated that NMC–SDS was only ∼−1 kJ mol⁻¹ more favoured than the SDS–SDS interaction, being weaker than that determined for HASE–SDS interactions (∼−4 kJ mol⁻¹).⁵⁰

Next, we calculated the solution structure of NMC under its micelle bound state and characterized the architecture of the complex. Although it was predicted that the replacement of Leu³ in NMB by His³ in NMC would hinder the acquisition of a helical membrane-bound structure,²⁰ we have shown that the C-terminal region of NMC folds into an α-helix upon micelle insertion, whereas the N-terminal segment remains unstructured. This folding process must be directly related to the energetically favoured insertion of Trp⁴, Val⁶, Gly⁷, Leu⁹ and Met¹⁰ (residues with a ΔG < 0 of transfer form water to lipid bilayers⁴⁰) into the non-polar micelle, which was experimentally observed through intermolecular NOEs (Fig. 5B). However, this hydrophobic insertion cannot occur without the enclosure of His⁸ into the micelle, which is expected to be fully protonated at pH 4.0, and therefore highly unfavourable. Nevertheless, NMR data reveal that His⁸, but not His³, deprotonates during the NMC insertion. These observations enabled us to hypothesize that the favourable hydrophobic interactions of the residues near His⁸ would energetically compensate for its unfavourable deprotonation and insertion. This idea is also supported by the small ΔG_ps value of the NMC–SDS complex in comparison to other peptide–SDS complexes.⁵⁰

The micelle-bound NMC structure displays Trp⁴, His⁸ and Leu⁹ oriented toward the same face. These residues correlate with Trp⁸, His¹² and Leu¹³ in bombesin, which are essential for the binding to the bombesin family of receptors.⁵¹ Hence, it is likely that Trp⁴, His⁸ and Leu⁹ also reorient as NMC approaches its receptor, which further validates the micelle-bound NMC structure, also within a biologically relevant context.

The ability of NMC to form a short α-helix during its micelle insertion may result in conformation and/or orientation selective interactions. Hence, the wide spectrum of similar but not identical biological activities of bombesin-related peptides raises the possibility that fluctuations of secondary structure can modulate their accessibility to different receptors, a mechanism already proven for neurokins and opioid peptides.⁵² Hence, we completed our structural data by analyzing the dynamics of NMC in water, in the presence of 60% TFE and under its micelle bound state. The fact that NMC at 60% TFE displays a structure similar to that adopted when it is embedded into micelles has allowed us to discriminate the folding and the binding effects on the molecular tumbling and dynamics.

The R₁/R₂ ratios were used to determine the τ_c values, which represent the time of the molecule to tumble as a function of size, shape and viscosity. The τ_c of NMC in water is similar to that found for other peptides of similar size.⁵³ The addition of 60% TFE raised τ_c ∼ 3 times, which is attributed to an increased solvent viscosity typical of the TFE/water mixtures⁴² and not to changes in the peptide size.^53,54 The τ_c of the NMC–SDS complex was ∼1 ns bigger than that of free SDS micelles proving that NMC slightly reduces the micellar tumbling rate as a result of the formation of a 1 : 1 complex; the stoichiometry mainly observed in peptide–micelle complexes.^46,55

¹⁵N relaxation data were used to determine the diffusion tensor of NMC in water, at 60% TFE and under its micelle-bound state. All the D_∥/D_⊥ values were <1.3, suggesting an isotropic rotational behaviour. This model has already been adopted to study the dynamics of other small peptides, either in their free form⁵⁶ or under their micelle-bound states.⁴⁶ NMC dynamics were studied applying the model-free approach,⁴⁵ which fits the relaxation data to one of the five models characteristic of the complexity of the residue level dynamics. The nine residues of NMC in water were described by model 2, indicating internal motions (τ_e) on ps–ns timescales. This was also the case for most of the residues of NMC at 60% TFE, except that His³ was fitted to model 4, and Leu⁹/Met¹⁰ that were fitted to model 5, hence suggesting complex internal motions. Five out of the nine residues in the NMC–SDS complex were fitted to model 1, proving their lack of flexibility (Tables S2–S4†).

Residue level mobility was qualitatively compared within the same NMC structure and across the three different NMC structures through the analysis of S². Folded proteins exhibit S_av² of ∼0.8, whereas mobile terminal residues display a S_av² of ∼0.6 (ref. 57) similar to IDPs (S_av² ∼ 0.3–0.6).^46,47 The S_av² of NMC in water was within the typical range of IDPs. However, the S² values notably changed between the central and the terminal residues, pointing towards a constrained mobility of the central residues, a trend also observed at 60% TFE. S² values also revealed that the formation of intramolecular hydrogen bonds linked to the α-helical folding reduces much more the backbone flexibility than the intermolecular hydrophobic interactions associated to the insertion of NMC into the SDS micelles. Hence, the coil-helix transition has a higher impact on the dynamics than the NMC confinement.

The ¹⁵N-relaxation data acquired at 60% TFE could be affected by the increase in the viscosity (Δη ∼ 1 cp at 25 °C (ref. 42)). However, data comparing the NMR dynamics of the Escherichia coli orthologue of frataxin (CyaY) in water (η ∼ 0.89 cp (ref. 42)) and in hen egg white (η ∼ 4 cp (ref. 58)) prove that viscosity does not affect the CyaY fold nor the HET-NOE values, but notably decreased and increased the R₁ and R₂ values, respectively.⁵⁹ The viscosity change scarcely affected the S² values of CyaY, calculated assuming an axially symmetric diffusion model (ΔS_av² ∼ 0.06) (Fig. S6†). In contrast, R₁, R₂, HET-NOE and S² values of NMC were notably enhanced when going from pure water to 60% TFE, thus proving that these changes are associated with structural alterations rather than viscosity modifications.

4 Experimental

4.1 Materials

NMC was purchased from Hölzel Diagnostika Handels GmbH. Sodium dodecyl sulfate (SDS, 99%), deuterated sodium dodecyl sulfate (d₂₅-SDS, 98%), 3-(trimethylsilyl)-1-propanesulphonic acid (DSS), deuterated (d₃-TFE, 99.5%) and non-deuterated 2,2,2-trifluoroethanol (TFE) were acquired from Sigma-Aldrich. All reagents were used without any further purification.

4.2 Circular dichroism (CD) studies

Circular dichroism (CD) spectra of NMC were obtained on a Jasco-715 spectropolarimeter equipped with a thermostated cell holder controlled by a Jasco Peltier element. Far-UV CD spectra were acquired from 260 to 190 nm at 15 °C in a 0.1 cm path length quartz cuvette at a NMC concentration of 30 μM in 10 mM acetate buffer (pH 4.0) containing different percentages of TFE (0%, 10%, 25%, 40%, 60% and 90%). The CD spectrum of NMC was also obtained in a buffered aqueous solution in the presence of 150 mM SDS. The scan speed was 50 nm min⁻¹ with a response time of 1 s and a step resolution of 0.2 nm, whereas 15 scans were accumulated. Base-line spectra were subtracted for all spectra. The secondary structure content was derived from the far-UV CD spectra using the BeStSel on-line platform (http://bestsel.elte.hu/).

4.3 Sample preparation for NMR studies

A 5 mM NMC solution was prepared in 10 mM acetate buffer at pH 4.0 containing 10% of D₂O and 1.6 mM of DSS (added as internal reference). This solution was additionally prepared in the same acetate buffer but in the presence of 10%, 25%, 40%, 60% or 90% d₃-TFE (to study the NMC folding pathway upon addition of a structuring solvent), in the presence of 150 mM d₂₅-SDS (to determine the NMC structure bound to SDS micelles), and in the presence of 50 mM SDS (to map the intermolecular interactions occurring between NMC and SDS micelles).

4.4 NMR spectroscopy

NMR experiments were carried out at 15 °C on a Bruker Avance III spectrometer operating at 14.1 T (600 MHz) and equipped with a 5 mm ¹³C, ¹⁵N, ¹H triple resonance cryoprobe. For sequence-specific assignments, ¹H–¹H-TOCSY experiments⁶⁰ were performed with the MLEV-17 spin-mixing pulse using a mixing time of 80 ms. The ¹H–¹H-NOESY experiments⁶¹ were acquired with a mixing time of 500 ms for the samples containing different d₃-TFE/water ratios, whereas a mixing time of 200 ms was used for the samples containing SDS micelles. 2D ¹⁵N-HSQC and ¹³C-HSQC spectra were also acquired at natural abundance. Spectra were obtained with 2048 data points × 512 increments and with a spectral width of 7184 Hz in both dimensions. Water suppression was achieved by the field-gradient method with the WATERGATE sequence.^{62 1}H and ¹³C chemical shifts were measured relative to the methyl resonance of internal DSS at 0 ppm. ¹⁵N chemical shifts were referenced indirectly using the ¹H, X frequency ratios of the zero-point. The relative diffusion coefficients (D) of NMC were measured by the pulse field gradient spin echo (PGSE) using a standard ledbpgp2s experiment.⁶³ D is a good indicator of the molecular mobility that is relative to the viscosity and to the molecular size. All the spectra were processed using NMRPipe/NMRDraw,⁶⁴ analyzed by Xeasy/Cara⁶⁵ and plotted using the Sparky software.⁶⁶

4.5 NMR structure calculations

The NMR experiments permitted assignment of the resonance frequencies of ¹³C, ¹H and ¹⁵N for NMC. The assignments were then used to calculate the secondary structure propensity (SSP) scores⁶⁷ (http://pound.med.utoronto.ca/software.html). The SSP score is the weighted average of the chemical shifts from different nuclei in a given residue with the relative weighting reflecting the sensitivity of different secondary shifts to structure. An SSP score of 1.0 suggests a fully-formed α-helix, a −1.0 value indicates a β-strand, whereas a 0 score specifies a random coil conformation. The chemical shift assignments were also used to obtain the geometrical restrains resulting from the NOE intensities, which were then used to calculate the NMC solution structures. NOE cross-peak assignment was done using the automated NOE assignment of CYANA,²⁷ a software that was further used to calculate the NMC ensembles. The standard protocol was used with seven cycles of combined automated NOE assignment and structure calculation for 200 conformers in each cycle, of which the 20 structures with lowest target function value were selected for further minimization and analysis. PROCHECK-NMR⁶⁸ was used to analyze the quality of the structures through the Protein Structure Validation Server (PSV) (http://psvs-1_4-dev.nesg.org/).⁶⁹ The MOLMOL software⁷⁰ was used for visualization and UCSF Chimera⁷¹ was used for structural representations.

4.6 NMR relaxation measurements

¹⁵N longitudinal (R₁) and transverse (R₂) relaxation data, as well as steady-state ¹⁵N HET-NOE data were acquired at 15 °C in an aqueous solution in the presence of 60% TFE and in the presence of 150 mM d₂₅-SDS. In all cases, R₁ values were determined using a series of 11 experiments with relaxation delays ranging from 10 to 2000 ms, while ¹⁵N HET-NOE measurements were performed by 3 s high power pulse train saturation within a 5 s recycle delay. R₁ and ¹⁵N HET-NOE data were acquired using standard pulse sequences,⁷² as well as R₂ data of the NMC solution containing d₂₅-SDS, recorded using 11 different relaxation delays ranging from 8 to 128 ms. R₂ measurements of the solutions prepared in water and in 60% TFE were carried out using the pulse program recently developed by Yuwen and Skrynnikov with modifications, which permits the increase of the relaxation delays thus avoiding cryoprobe heating.⁷³ In these cases, R₂ values were measured using 9 relaxation delays ranging from 58 to 691 ms. Recycle delays were 3 s in both R₁ and R₂ experiments. Thirty two scans in R₁ and R₂, and 200 scans in ¹⁵N HET-NOE spectra per t₁ experiment were acquired. 2048 × 128 complex points were obtained during R₁ and R₂ experiments, whereas 2048 × 164 complex points were acquired in the ¹⁵N HET-NOE experiments.

4.7 NMR relaxation analysis

R₁ and R₂ relaxation data were fitted to a mono-exponential decay function, whereas ¹⁵N HET-NOE data were obtained as the ratio of the peaks intensities from the saturated and unsaturated spectra. Relaxation constants and experimental errors were calculated using the Protein Dynamics Center software (Bruker, Germany). R₁ and R₂ relaxation constants were then used to determine the NMC correlation times (τ_c) at each experimental condition using both the r2r1_tm (Palmer's group, http://www.hhmi.umbc.edu/toolkit/analysis/palmer/r2r1_tm.html) and the TENSOR 2.0 software.⁷⁴ The magnitude and orientation of the rotational diffusion tensor was determined from the R₁ and R₂ relaxation constants and the energy-minimized representative conformers of the NMR-derived solution structures using the software Quadratic-Diffusion.⁴⁴

TENSOR 2.0 was also used to calculate the generalized order parameters describing the amplitudes of internal motions (S²). The ¹⁵N relaxation constants and the energy-minimized solution structures were analyzed according to the molecular diffusion derived by Woessner in combination with the Lipari–Szabo model-free analysis of local flexibility.⁴⁵ The amide bond length was fixed at 1.02 Å. Five different models were tested to characterize the internal dynamics of the NH groups:⁷⁵ model 1 (S²), model 2 (S², τ_e), model 3 (S², R_ex), model 4 (S², τ_e, R_ex) and model 5 (S_f², S_s², τ_e). τ_e is the effective internal correlation time (describes motions on a timescale > 20 ps), R_ex is a chemical exchange term (describes slow timescale motions on the order of μs–ms), and S_f² and S_s² are terms that result from splitting the generalized order parameter into two order parameters reflecting slower and faster motions, respectively. The confidence levels were estimated using 100 Monte Carlo simulations per run in combination with c² and F-test criteria.

4.8 Fluorescence measurements

The intrinsic fluorescence spectra of NMC were acquired at 15 °C on a Varian Cary Eclipse fluorimeter equipped with a Peltier-controlled cell holder. Emission spectra were obtained between 300 and 400 nm using an excitation wavelength of 280 nm. The fluorescence spectra were collected using a 0.1 mM NMC solution in 10 mM acetate buffer at pH 4.0, either alone or in the presence of 50 mM of SDS. The change in the solvent accessible surface area of Trp⁴ was measured by the acrylamide quenching experiments, which permitted the calculation of the corresponding Stern–Volmer constants (K_sv). Aliquots of a 1 M acrylamide aqueous solution were added to the cuvette containing either the peptide alone or the peptide/SDS mixture. Emission spectra were obtained as previously described after each addition of the quencher. The K_sv values were calculated from the following equation,

F₀/F = 1 + K_sv[Q] (1)

where F₀ is the initial fluorescence of the peptide and F is the fluorescence intensity following the addition of soluble quencher, Q.

4.9 Isothermal titration calorimetry (ITC) measurements

ITC experiments were used to measure the thermodynamic parameters of the NMC–SDS interaction. ITC measurements were carried out on a Nano-ITC (TA Instruments©) at 15 °C in duplicate. In a typical experiment, the sample cell (190 μl) was filled with 10 mM acetate buffer (pH 4.0) in the absence of or in the presence of different NMC concentrations (i.e. 10 μM, 20.8 μM, 42 μM and 62.5 μM) and stirred at 250 rpm, while a 35 mM SDS buffered solution was loaded in the injection syringe. Both solutions were degassed before use. SDS solution was titrated into the sample cell as a sequence of 32 injections (the first four injections of 2 μl and the other injections of 1.5 μl). The time between successive injections was 400 s. Raw data corresponding to the heating rate (μJ s⁻¹) was integrated to obtain the observed molar enthalpy change (ΔH) at each SDS concentration. The data corresponding to the titration of SDS into acetate buffer was subtracted from the data corresponding to the titration of SDS into a NMC buffered solution before the analysis of the ΔH variation. Titration of acetate buffer into a NMC buffered solution resulted in small ΔH variations that were not further considered in the data analysis. All data acquisition and analysis were performed using the NanoAnalyze software.

The Gibbs free energy changes of aggregation (ΔG_mic) and aggregation in the presence of NMC (ΔG_ag) were calculated from the ITC data through the application of the charged phase separation and mass-action models (eqn (2)).³⁵

ΔG_mic/ag = (1 + K)RT ln[cac or cmc] (2)

A factor of (1 + K) is needed to calculate the free energy of ionic SDS, where K is the micellar charge fraction with a value of 0.85.⁷⁶ The enthalpy change and free energy changes were also used to calculate the entropy changes of micellization (ΔS_mic) and aggregation in the presence of NMC (ΔS_ag).

5 Conclusions

The results reported in this study allow a rationalization of the structural determinants of NMC in an aqueous solution and describe the different folding steps that potentially occur during its interaction with its natural partners. The description of the NMC–SDS micelle complex revealed that the hydrophobic binding must energetically compensate for the deprotonation and the micelle insertion of His⁸, which results in a low ΔG of interaction. The fact that the solution structure of NMC at 60% TFE mimics the micelle-bound structure provides an excellent opportunity to discriminate the folding and the binding effects on the NMC mobility. We observed that the folding constrains the NMC mobility twice as much as its micelle confinement. Our data contribute to the general understanding of the mechanisms involving peptide–lipid interactions and provide fundamental to insights into the structure–flexibility relationship of bombesin-like peptides. These novel insights into the binding-induced folding mechanism of NMC also constitute a new molecular platform for the future design of antagonists of the bombesin family of receptors.

Acknowledgements

The authors are grateful for the excellent technical assistance from the Serveis Cientificotècnics at the UIB, especially to Dr Joan Cifre for his generous assistance with ITC measurements. K. P. is supported by a FWO Pegasus long-term post-doctoral fellowship (1218713). P. S. is supported by a post-doctoral fellowship (PD/009/2013) from the Conselleria d'Educació, Cultura i Universitats of the Balearic Government and the European Social Fund through the ESF Operational Program of the Balearic Islands 2013–2017. This study is supported by the project AAEE26/2014 from the Conselleria d'Educació, Cultura i Universitats of the Balearic Government and FEDER.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra12753j

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