Conformational Plasticity of the Cell-Penetrating Peptide SAP As

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Conformational Plasticity of the Cell-Penetrating Peptide SAP as Revealed by Solid-State F-NMR and Circular Dichroism Spectroscopies 19

Sergii Afonin, Vladimir Kubyshkin, Pavel K. Mykhailiuk, Igor V. Komarov, and Anne S. Ulrich J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b02852 • Publication Date (Web): 13 Jun 2017 Downloaded from http://pubs.acs.org on June 16, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Conformational Plasticity of the Cell-Penetrating Peptide SAP as Revealed by Solid-State 19F-NMR and Circular Dichroism Spectroscopies Sergii Afonin,† Vladimir S. Kubyshkin,‡,∥ Pavel K. Mykhailiuk,‡,⊥ Igor V. Komarov,§ and Anne S. Ulrich†,‡,* †

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology (KIT), P.O.B. 3640, D-76021 Karlsruhe, Germany; ‡ Institute of Organic Chemistry, KIT, Fritz-Haber-Weg 6, D-76131 Karlsruhe, Germany; § Institute of High Technologies, Taras Shevchenko National University of Kyiv, Prosp. Glushkova 4-g, 02033 Kyiv, Ukraine

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 44

ABSTRACT

The cell-penetrating peptide SAP, which was designed as an amphipathic poly-L-proline helix II (PPII), was suggested to self-assemble into regular fibrils that are relevant for its internalization. Herein we have analyzed the structure of SAP in the membrane-bound state by solid-state

19

F-

NMR, which revealed other structural states, in addition to the expected surface-aligned PPII. (2S)-2-Amino-2-[3-(trifluoromethyl)bicyclo[1.1.1]pent-1-yl]ethanoic acid and two rigid isomers of trifluoromethyl-4,5-methanoprolines (CF3-MePro) were used as labels for 19F-NMR analysis. The equilibria between different conformations of SAP were studied and were found to be shifted by the substituents at Pro-11. Synchrotron-CD results suggested that substituting Pro-11 by CF3-MePro governed the coil-to-PPII equilibrium in solution and in the presence of a lipid bilayer. Using CD and 19F-NMR, we examined the slow kinetics of the association of SAP with membranes and the dependence of the SAP conformational dynamics on the lipid composition. The peptide did not bind to lipids in the solid ordered phase and aggregated only in the liquid ordered “raft”-like bilayers. Self-association could not be detected in solution or in the presence of liquid disordered membranes. Surface-bound amphipathic SAP in a non-aggregated state was structured as a mixture of non-ideal extended conformations reflecting the equilibrium already present in solution, i.e. before binding to the membrane.


2

Page 3 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Cell-penetrating peptides (CPP) occur natively or are rationally designed polypeptide sequences and are generally defined by their function: an ability to enter living cells without compromising cell viability.1,2,3 Primarily due to this property, broad practical interest exists in applying CPPs as biologically inspired carriers for intracellular drug delivery, gene therapy, or for the biotechnological manipulation of cell behavior in cell cultures and multicellular organisms.4,5,6,7 Cargoes of diverse types can be effectively attached to a CPP molecule, among which the most widely demonstrated are various fluorescent molecules; however, proteins, enzymes, nucleic acids, nanoparticles, and even liposomes were also shown to be delivered to cells.8,9,10,11 The entry into a cell of a covalently coupled (cargo-CPP) conjugate, or the CPPpromoted uptake of non-covalently associated cargoes in model cell culture studies, are usually designed to achieve non-toxicity to the cells. In in vivo applications, minimizing systemic stress on the neighboring non-targeted cells and tissues is of great concern. Currently, the best practical examples are the CPP-based technologies for medical applications, including several systems that are in clinical trials.12,13 Molecular mechanisms for cell entry were discussed controversially in the literature: the internalization process per se is generally believed to discriminate between endocytosis-related and endocytosis–independent pathways. This dichotomy dominates the field and is somewhat misleading. In a recent database survey, approximately one-quarter of the CPP systems were assigned to one of the above processes. The remaining greater than 55% of the cases were classified as “unknown” or “other.”14 In any case, should the final destination of a cargo be the cell cytosol, even internalization by endocytosis has to involve direct membrane translocation mechanisms (i.e., endosome escape without disruption of the endosomal membrane). In a


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 44

complex living cell system, CPP or CPP-cargo conjugates undergo dynamic re-distribution of individual molecules by being i) non-interacting (does not reach the cell, is precipitated or aggregated), ii) bound to the cell surface but not internalized, iii) degraded by the action of proteases or bound to environmental scavengers, iv) internalized by endocytosis and located in the inner compartments, or iv) involved in transbilayer diffusion and/or located in the cytosol. It is this complex equilibrium that must be sensitive to the environmental conditions, the type of the cargo and the concentration of the conjugate. It should also be different in different type cells. Typically, only one, and not always the dominating pool of molecules, will be detected in the cell entry monitoring experiment, and a full description of the equilibrium or quantification of the abovementioned states is often impossible. The variability of the known CPP sequences, although highly advantageous for drug discovery, renders the elucidation of the cargo delivery mechanisms even more problematic. Generally, CPPs are defined by their function but are classified by (i) their origin (e.g., sequences derived from native heparin-, DNA- or RNA-binding proteins, from signaling or antimicrobial peptides that are designed de novo or found in screening libraries), (ii) their physical-chemical properties (e.g., polar cationic, hydrophobic, primary or secondary amphipathic); or more rarely (iii) their secondary structures (α-helical, β-stranded, cyclic, nonstructured).12,14,15,16 The obligatory transbilayer movement, non-stereospecificity, the sometimes common origin, and a strong chemical similarity with other non-specific bioactive peptides (membranolytic, hallmarked by antimicrobial peptides (AMPs) or fusogenic peptides) suggests that most CPPs are intrinsically membrane-active peptides (MAP). Indeed, a number of studies demonstrated a strong overlap of activities among the three peptide classes.17,18 Moreover, mechanistic descriptions of the translocation of peptides across lipid bilayers utilize models that


4

Page 5 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


are common for CPPs and AMPs. The same static structural modes of lipid-peptide interactions, namely, the “barrel-stave-”, “toroidal-” or other types of distorted pores, are implied.16,19 A stable pore should lead to a sizable loss of membrane integrity and exert a membranolytic function, while the formation of transient and reversible pores should allow peptide translocation without any non-reparable membrane perforation stress. However, direct correlations between the dynamic structures of CPPs and AMPs in their membrane-interacting processes (i.e., comparing the membrane-bound vs. the solution-free conformations), as well as general structure-membrane perturbation activity studies regarding CPPs remain scarce.20 The similarities between various AMPs suggest a potential benefit from the application of the structural methods successfully used on one type of peptide to the other. Solid-state NMR occupies a unique methodological niche, as it allows the structural elucidation of conformationally flexible MAPs in their relevant membrane-associated states with and without a cargo.21,22 Therefore, we have developed a solid-state NMR method based on the use of

19

F-

labels. The main benefit of this method is unprecedented NMR sensitivity, in comparison with alternative approaches that utilize other NMR-active nuclei (2H, 13C, or 15N).21,23,24 The labels are carefully designed to impart a minimal impact on peptide structure and function and can therefore be regarded as non-perturbing.23,25,26,27,28,29,30,31 Currently,

19

F- solid-state NMR labels

for probing aliphatic non-polar residues, proline, serine/threonine and phenylalanine/tyrosine are available, while proper structural substituents for other amino acids, especially charged amino acids, are poorly available.32,33 The cell penetrating peptide SAP (“Sweet Arrow Peptide,” Figure 1.) is a good example of a rational attempt to develop an effective CPP without the major drawbacks of canonical sequences. The SAP sequence obtained from the maize γ-zein storage protein was designed by


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 44

the E. Giralt group.2,34 As with many CPPs, the SAP is short (18 residues), is amphipathic when folded (secondary amphipathicity), contains arginine and is cationic (three arginine residues provide a net charge of +3). It is effective as a carrier in the all-L and all-D forms, works as a CPP when covalently, non-covalently, poly- and mono-topically bound to cargoes, and effectively delivers such diverse cargoes as nanoparticles and fluorescent dyes.35,36,37,38,39 SAP is a proline-rich peptide, approximately 50% of the sequence are proline residues. This compositional feature was postulated by design to impose folding as an amphipathic poly-Lproline type II helix (PPII) and to increase the proteolytic stability. The most intriguing feature of the SAP is its low toxicity compared to other CPPs, thus the name “the sweet arrow”. Structurally, based on circular dichroism (CD) spectroscopy observations, an amphipathic PPII fold was suggested for SAP in aqueous solutions as well as an ability to aggregate in the same conformation.2 Aggregation was proposed from CD and electron microscopy (EM) studies, and its unique cylindrical micellar structure, with the homo-trimeric SAP arrangement as a core, was assumed as a model for the aggregate. The mechanism of SAP-membrane interactions was speculated to be different between the monomer and the fibril. Amphipathic PPII-folded SAP monomers bind to lipid bilayers and may directly translocate to the cytosol, while the polar (type I) aggregates are suggested to associate electrostatically with the juxta-membrane proteins or with the near-membrane polysaccharides. Internalization of this SAP state proceeds passively through the co-attachment to the membrane binding sites, followed by endocytosis. A relationship to “raft”-mediated endocytosis was proposed as a key feature of the CPP activity of SAP.35,36 These latter suggestions remain speculative, as they are based on the observations of fluorescently labeled SAP that was co-incubated with living cells.


6

Page 7 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Postulated structural features of SAP. (A) Three-letter code primary sequence and numbering; (B) Molecular model of SAP folded as an ideal PPII helix. Color code: cationic arginine residues – blue, hydrophobic leucine and valines – yellow, prolines - gray; (C) Secondary amphiphilicity of the PPII-folded SAP; and (D) Key elements of the suggested2,40 SAP oligomer – trimeric core unit (left) and the dorsal-assembled fibril (right). (E) Conformationally constrained amino acids, used in this study (19F-NMR reporting trifluoromethyl groups are highlited). In this study, we decided to investigate the molecular basis of the membrane transduction mechanism of SAP by resolving the structure(s) in the lipid membrane-bound state and by characterizing its interaction with lipids. To achieve this goal, we synthesized

19

F-labeled SAP

analogs using two unnatural amino acids as structural labels that were designed as isosteres of Leu/Val and Pro and studied peptide structural preferences in membrane-mimicking


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 44

environments by circular dichroism and solid-state NMR. Our initial intention was to confirm the hypothetical mechanism described above and to apply our

19

F-NMR approach to a cell-

penetrating peptide that contained a non-helical structure. Finding inconsistencies with the initial hypothesis, we explored the conformational preferences of SAP using unnatural amino acids as conformational perturbants and by employing synchrotron radiation circular dichroism (SRCD) spectroscopy and

19

F-NMR. As a result, we characterized the structural, orientational and self-

assembly behavior of SAP in the membranes, studied the effect of the lipid composition, lipid phase state and peptide concentrations, and report herein on some novel features of the SAP-lipid bilayer interaction as well as demonstrate the involvement of conformations other than the PPII conformation of SAP.


8

Page 9 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EXPERIMENTAL SECTION General. Analytical 1H-NMR spectra were routinely recorded on Bruker Avance 400 and Bruker Avance DRX 500 spectrometers and were referenced to tetramethylsilane. MALDI-TOF mass spectra were acquired on a Bruker Autoflex III instrument. Chromatographic (HPLC) analyses were performed using a Jasco HPLC system equipped with a diode-array detector, employing Vydac C18 columns: 4.6 mm x 250 mm for analytical work at a flow rate of 1.5 ml/min, 10 mm x 250 mm columns for semi-preparative work at 6 ml/min, and 22 mm x 250 mm columns for preparative work at 20 ml/min. Separations were achieved using linear gradients of solvent A (90% water, 10% acetonitrile) and solvent B (90% acetonitrile, 10% water). Additionally, 5 mM hydrochloric acid was used ubiquitously as an ion-pairing agent to avoid a fluorine background that was due to trifluoroacetic acid.25 All lipids (DMPC - 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine, DOPC - 1,2-di-(9Zoctadecenoyl)-sn-glycero-3-phosphocholine,

DMPG

-

1,2-ditetradecanoyl-sn-glycero-3-

phospho-(1'-rac-glycerol), BSM - N-(octadecanoyl)-sphing-4-enine-1-phosphocholine, chol cholest-5-en-3ß-ol, POPC - 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine) were obtained from Avanti Polar Lipids and were used without further purification. All other chemicals were of commercial grade (purchased from Merck, Acros, ABCR, Fisher, Iris Biotech, Biosolve or Enamine). Freshly deionized (18Ω) non-degassed ultrapure water was prepared using a Milli-Q system from Millipore and was used for all aqueous preparations. Peptide Synthesis.

19

F-labelled amino acids for solid phase peptide synthesis (SPPS) were

prepared on a semi-preparative scale using Fmoc protection as previously described.28,29,41 All standard SPPS building blocks were obtained from Bachem. Original SAP and SAP analogs


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 44

(Table 1) were synthesized manually starting from proline preloaded chlorotrityl resin according to.29 Arginine was loaded in the Pbf-protected form. The crude peptides were cleaved from the resin using a TFA/H2O/TIS cocktail, precipitated with diethyl ether and then lyophilized from acetonitrile/water (1/1 vol/vol). The dry product was re-dissolved (50 mg/ml) in methanol and purified at room temperature on a semi-preparative or preparative (see above) scale by HPLC as previously described.25,29 Table 1. Peptides Used in This Work and Their Modifications. name

sequencea/

substitution

substituting position (-X-) SAP

VRLPPPVRLPPPVRLPPP

n.a.

SAP-Bpg-3

VR-X-PPPVRLPPPVRLPPP Leucine to CF3-Bpgb

SAP-Bpg-7

VRLPPP-X-RLPPPVRLPPP

SAP-Bpg-9

VRLPPPVR-X-PPPVRLPPP Leucine to CF3-Bpg

Valine to CF3-Bpg

SAP-cMePro-11 VRLPPPVRLP-X-PVRLPPP Proline to CF3-cis-MeProc SAP-tMePro-11

VRLPPPVRLP-X-PVRLPPP Proline to CF3-trans-MeProd

SAP-Bpg-13

VRLPPPVRLPPP-X-RLPPP

SAP-Bpg-15

VRLPPPVRLPPPVR-X-PPP Leucine to CF3-Bpg

Valine to CF3-Bpg

a

amino acids are in one-letter code. b CF3-Bpg:(2S)-2-Amino-2-[3-(trifluoromethyl)bicyclo[1.1.1]pent-1-yl]ethanoic acid. c CF3-cis-MePro:(1R,3S,5R,6R)-6-trifluoromethyl-2-azabicyclo[3.1.0]hexane-3-carboxylic acid. d CF3-transMePro:(1S,3S,5S,6S)-6-trifluoromethyl-2-azabicyclo[3.1.0]hexane-3-carboxylic acid.

Solid-State NMR: Sample Preparation. Mechanically-oriented bilayer samples were prepared as previously described.21,25,26,29 Briefly, the appropriate amounts of dry peptide and lipid were co-dissolved in either methanol or methanol/chloroform (2/1 vol/vol) and spread as solutions over 10-17 cover glass slides (Marienfeld Laboratory Glassware) such that the total amount of material per slide was 0.7-0.8 mg for 18 x 7.5 mm and 0.6-0.7 mg for 15 x 7.5 mm glass plates (typically 40 µl per slide). The solvent was allowed to evaporate under an air flow


10

Page 11 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for 30-60 min, and the open-edge glass slides were further dried in a vacuum for at least 4 hours to remove all traces of solvents. The glass slides with spontaneously formed and oriented multiproteobilayers were then stacked manually and immediately hydrated by placing a stack in a closed plastic container containing an oversaturated aqueous potassium sulfate solution (96% relative humidity). These containers were incubated at 48°C for 12-24 hours, and the hydrated samples were finally wrapped in water-insulating polymer films (using Parafilm or Nescofilm for the first 5-6 wrapping layers and Sarogold film or polyethylene foil for the outermost 4-5 layers). The samples were stored at -20°C between the measurements. The degree of orientation and the level of hydration of the samples were assessed by acquiring 1

H and 31P-NMR spectra before and after the 19F-NMR experiments. If resonances from oriented

lipids were broadened, the material was considered to have partially dried out; hence, it was rehydrated and re-measured. Re-hydration was achieved by completely removing all of the wrapping layers, dipping the stack into pure H2O for 15 seconds and subsequently placing the stack into the hydration chamber for 12 hours, followed by re-wrapping as described above. Solid-State NMR: Measurements. Solid-state 19F- and 31P-NMR spectra of oriented samples were recorded using a Bruker Avance 500 NMR system equipped with a wide bore magnet. The 19

F spectra (470.6 MHz) were collected using a 1H19F double resonance variable angle flat coil

probe (Doty Scientific), typically with a 90 deg pulse of 2.1 µs. To acquire

19

F spectra (2 µs

acquisition time, and 1 s recycling delay), composite anti-ringing (“aring”) pulse sequence was applied along with the TPPM (Two Pulse Phase Modulation) proton decoupling. The sequence is equivalent to a single-pulse excitation. The

31

P spectra (202.5 MHz) were measured using a

standard Bruker 1H/19F-X variable angle flat coil probe, typically using a 90 deg pulse of 7.1 µs. For the 31P-NMR experiments, a Hahn-echo pulse sequence with TPPM proton decoupling was


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 44

applied (30 µs echo delay, 10-20 ms acquisition time and 2 s recycling delay). The proton decoupling power in each case corresponded to the actual 15-20 kHz decoupling strength. The temperature in the different probe heads was calibrated using a methanol chemical shift thermometer as suggested in.42

19

F spectra were referenced by setting the fluoride resonance of

freshly prepared 1 M NaF aqueous solution (observed at 35°C) to -119.5 ppm. Solid-State NMR: Structure Analysis. The membrane alignment of the major fraction of SAP molecules was characterized by two angles τ and ρ, as previously described in detail.21,23,24 The tilt angle τ is the angle between the membrane normal and the long axis of inertia of the folded peptide (Z axis). The azimuthal angle ρ reflects the rotation of the peptide around its Z axis. Here, we define ρ=0° such that the radial vector passing through the Cα atom of 12th residue, in a plane perpendicular to Z, is rotated to lie in the plane of membrane (i.e. orthogonal to the membrane normal). For any particular orientation given by τ and ρ, the

19

F-19F dipolar coupling of each NMR label is predicted by

converting the corresponding dipolar interaction tensor from its principal axes system into the laboratory frame. The principal axis values of the axially symmetric 19F-19F dipolar tensor were taken as -8 kHz, 16 kHz, and 16 kHz. Averaging by molecular motions that are fast on the NMRtimescale was taken into account by scaling with an order parameter 1 ≥ Smol ≥ 0. In a systematic grid search, for every combination of τ, ρ, Smol the deviation between the calculated NMR parameters from the experimental values was quantified in terms of a root mean square deviation (r.m.s.d.). To identify the best-fit peptide orientation and order parameter, the r.m.s.d. was determined for all combinations of τ and ρ within the range of 0° to 180° in steps of 1°, and for all Smol within the range of 0 to 1 in steps of 0.1. The solution with the lowest overall r.m.s.d.


12

Page 13 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


was considered to be the best-fit, especially when comparing the outcome for different kinds of secondary structures probed. This way, the most plausible secondary structure, together with its best-fit combination of τ, ρ, Smol was obtained from the experimental set of

19

F-19F dipolar

couplings.21,23,24 Initially, the homonuclear 19F-19F dipolar couplings from CF3-Bpg labels in five different SAP positions were collected and used to calculate the orientation of the peptide in the lipid bilayer. The coupling from SAP-cMePro-11 (6th NMR constraint) was later used to verify the results for the PPII conformation. The backbone was modeled as ideal secondary structures: PPII-helix, αhelix, 310-helix, π-helix and β-strand using Accelrys Discovery Studio software, and the geometry of the CF3-group that was rigidly connected to the backbone was described in each case by two angles: i) the angle between C(i-1)-C(i)F3 and the secondary structure long axis (Z axis of the helix/strand); and ii) the angle between the projection of the C(i-1)-C(i)F3 bond onto the X-Y plane, orthogonal to the Z-axis, and the radial axis, which crosses Cα of the 12th amino acid), as previously described.26,43 To estimate the trifluoromethyl-group geometry in SAPcMePro-11, the crystal structure of the CF3-substituted amino acid core29 was superimposed onto the pyrrolidine ring of the L-proline, using ring atoms 1, 2 and 3. Circular

Dichroism

Spectroscopy:

Sample

Preparation.

Lyophilised

material

(gravimetrically determined weights) of dry peptides was dissolved in methanol at a concentration of either 10 or 20 mg/ml and the corresponding aliquot was transferred to a 2 ml Eppendorf tube using a gas-tight Hamilton glass syringe. The solvent was removed first with a gentle stream of N2 followed by an overnight exposure to vacuum. For the lipid-containing samples, in order to ensure complementarity to the solid-state NMR samples, appropriate amounts of dry lipids and peptides were first co-dissolved in excess methanol, dried in a similar


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 44

manner and then processed further as described below. To the vacuum-dried peptide (or peptide/lipid) film, a corresponding volume of solvent (water, 2H2O, methanol, trifluoroethanol or 95% n-propanol) or buffer (10 mM NaH2PO4/Na2HPO4, pH 7.0) was added at room temperature in order to obtain a solution (suspension) at a peptide concentration of 5-50 µM. The solutions (suspensions) were rigorously vortexed/bath-sonified until clear, and the material obtained was immediately transferred to the measurement cuvettes. Circular Dichroism Spectroscopy: Conventional CD Measurements. Circular dichroism spectra were recorded on either a Jasco J-800 or a Jasco J-815 spectropolarimeter. The spectra were recorded between 260 and 185 nm at 0.1 nm intervals. Rectangular quartz glass (Suprasil) cells of 1 mm pathlength from Hellma were used. Three consecutive scans that were obtained at a scan rate of 10 nm/min were averaged for each spectrum, including the backgrounds. The background spectra were subtracted from the sample spectra after the measurements. All spectra were measured at a controlled temperature using the standard Jasco setup of a rectangular cuvette holder that was connected to an external water thermostat. For each spectrum of samples containing peptides, which were collected at a steady-state temperature, a 5 min equilibration period was implemented and identically measured corresponding peptide-free background spectra were acquired. Temperature-dependent spectra were collected in the successive heating (or heating-cooling cycles) series in the range 5-55°C. The heating rate was set to 1°/min, and at each selected temperature at least a 10 min incubation delay was programmed to ensure thermal equilibrium in the cell. No background subtraction was found necessary in the temperature-dependent measurements when relative spectral changes were assessed. Spectral acquisition, including


14

Page 15 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


temperature series, and all processing steps were performed using the preinstalled software package of the spectropolarimeter (Jasco). Circular Dichroism Spectroscopy: SRCD Measurements. SRCD spectra of SAP analogs were measured on the UV-CD12 beamline at the synchrotron facility of the Karlsruhe Institute of Technology (ANKA). The spectra were collected between 270 and 175 nm at 0.1 nm intervals. Measurements were performed at a concentration of 10 mg/ml for the lipid-containing samples in a circular demountable 13 µm CaF2 cell, while for lower concentrations (e.g., 0.5 mg/ml) circular Suprasil cuvettes with a 0.1 mm path length were used. Each spectrum was acquired in three successive scans at a scan rate of 10 nm/min and was averaged prior to baseline corrections. Background spectra were measured in a similar manner, and the material content of the cuvettes differed only in the absence of peptide. Hardware and software that were developed by S. Roth at the UV-CD12 beamline were used for the temperature-dependent measurements. Parameters of the temperature control were taken as close as possible to the conventional CD measurements described above. Data processing and analysis were conducted using CDtool software from the B. A. Wallace group.44


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 44

RESULTS AND DISCUSSION Conformational Variability of SAP and Analogs. All

19

F-labelled peptides and non-

modified SAP (Table 1) were produced by manual SPPS using standard Fmoc-chemistry, identified by MALDI-TOF and purified by preparative HPLC to 100% purity. The artificial amino acids CF3-Bpg and CF3-cis-MePro were introduced into the SAP sequence as previously reported.29 SPPS using the new CF3-trans-MePro was equally successful. Use of enantiomerically pure fluorine-substituted amino acids was essential for the labeling as, contrary to many other amphiphilic peptides, individual SAP diastereomers were not separable by conventional HPLC techniques,23 possibly due to their non-amphipathic (random coil) structuring under HPLC conditions. At this stage, 6 peptides were generated for structural studies (SAP-Bpg-3, SAP-Bpg-7, SAPBpg-9, SAP-Bpg-13, SAP-Bpg-17 and SAP-cMePro-11), while the last peptide together with the SAP-tMePro-11 constituted a pair for the conformational studies. A positive conclusion of the usability of the structural analogs, which is essential for the subsequent solid-state

19

F-NMR

structure analysis, was based on the criterion of structural non-perturbance, as suggested in.28 Each mutation was confirmed to be non-perturbing to the SAP structure by CD measurements in an aqueous environment, under non-aggregating conditions as reported by Giralt et al,2 i.e., employing peptide concentrations below 50 µM at ambient temperatures (e.g., in deionized water at 20°C). Based on qualitative determinations from the overall similarity in the spectra (with the dominating negative CD intensity at approximately 203 nm and close to 0 ellipticity values at approximately 225 nm), SAP and its structural analogs appeared to be present as random coil/PPII mixtures (Figure 2A). Quantitative analysis of the conventional CD spectra was not possible because the only existing data sets for quantification of PPII content (reference sets45 2


16

Page 17 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and 5) in the DICHROWEB46 require data to be acquired down to 178 nm, which is inconsistent with the experimental conditions used here.47 With the notable exception of the two MePro mutants (see below), all of the peptides that were used for structure analysis were indistinguishable in their CD behavior from the original SAP.

Figure 2. (A) Evaluation of the conformation of 19F-labeled SAP analogues by CD-spectroscopy. Peptides are solubilized in aqueous buffer at monomeric concentration and measured at 20°C. All CF3-Bpg labeled analogues assume the same conformation as the parent SAP. (B) Concentration dependence and (C) temperature dependence of the CD signal, comparing aqueous solutions of SAP (top row) with the labeled SAP-Bpg-9 (bottom row). The spectra of the 19

F-labeled analogue are equivalent to the ones of SAP To determine if

19

F-NMR labels modify the conformational equilibrium of SAP, we

additionally have monitored the temperature dependence of the CD spectra. Temperaturedependent spectra were acquired in successive heating series in the range 5-55°C. Here again, for all 6 peptides with the structural labels, increasing the temperature resulted in the non-abrupt lowering of the intensity at approximately 203 nm, which is indicative of the expected48,49 reduction of the PPII-structured fraction in the RC/PPII mixtures. Furthermore, throughout all


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 44

peptide – structural analogs, including the Pro mutant,29 an identical isodichroic point (at approximately 208 nm) was observed, which is compatible with an equilibrium between only two conformations (RC and PPII, for instance) as well as with the fact that the introduction of 19

F-labels does not influence this equilibrium. These measurements confirmed that SAP possesses a significant percentage of PPII in aqueous

environments and allowed us to conclude that all

19

F-labelled analogs structurally resemble the

parent peptide, thus permitting the solid-state 19F-NMR structure evaluation. SAP Structure in Lipid Membranes. For solid-state NMR studies, the peptides were reconstituted, one at a time, in DMPC, a bilayer which compositionally resembles zwitterionic eukaryotic membranes. Solid-state NMR spectra were obtained at a high temperature (55°C), where lipid bilayers are in a fluid liquid disordered (l.d.) phase state. No distortion of the mechanically aligned oriented bilayers was observed, as determined from the lipid-observing solid-state 31P NMR spectra that were obtained from the same samples (data not shown). All 19Flabelled peptides were found to be oriented into rotationally averaged states, and well-resolved 19

F-19F dipolar splittings (Figure 3A) were collected for structure analysis.


18

Page 19 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. SAP structured as PPII in a fluid DMPC bilayer. (A) Experimental solid-state

19

F-

NMR spectra of the 19F-labeled analogs (indicated) reconstituted in the oriented bilayer samples from DMPC at a peptide/lipid ratio of 1/50. The dominant signals represent the major conformation of SAP, and the corresponding dipolar splittings are shown together with the values used in the analysis. Minor signals (marked with dashed lines), originating from less prominent conformations, non-oriented peptides and racemized peptide material, were omitted in the analysis below (see text for detailed discussion). The isotropic position is indicated by a dotted line. (B) Best-fit result for the alignment determination of PPII-folded SAP. (C) Molecular model showing a possible position of the PPII helix on the surface of a fluid DMPC bilayer as determined from solid-state

19

F-NMR analysis. The depth of insertion was not determined and

was chosen arbitrarily.

In the

19

F-NMR spectra, all peptides that were labeled with CF3-Bpg exhibited one

predominant splitting among others (Figure 3A). In particular, CF3-Bpg in place of Leu (SAP positions 3, 9 and 15), where the steric differences introduced by replacement labeling are


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 44

minimal, revealed one large positive splitting of approximately +10 kHz. With CF3-Bpg in place of Val (SAP positions 7 and 13), an additional splitting of approximately +7 kHz was present. Additional splittings may possibly indicate a slight perturbance of the structure by introducing the larger CF3-Bpg amino acid, or a presence of molecules with different orientations, in amounts proportional to the intensities of the minor signals. It is worth noting that in the solution CD, neither of the peptides demonstrated a significant perturbation of the SAP conformational equilibrium, thus suggesting environmental effects. The most complex pattern was observed for the SAP analog with CF3-cis-MePro at position 11. Apart from the ubiquitous +9.5 kHz splitting, two more were detected at +3.5 and -4.5 kHz, with the latter being responsible for approximately 30% of the spectral intensity. Next, from the collected sets of orientational constraints, the compatibility of the SAP structure with various helical conformations (PPII, 310-helix, π-helix, α-helix) was evaluated using only established CF3-Bpg labels (Table 2). Table 2 Numerical Best-Fit Solutions for the Alignment of SAP in Fluid DMPC. conformation r.m.s.d [kHz] Smol

τ [°] ρ [°]

PP II

0.77

1.00 83

139

310-helix

1.94

1.00 91

35

π-helix

> 4.8

-

-

-

α-helix

> 7.3

-

-

-

β-strand

0.63

0.99 60

61

A minimal r.m.s.d. value was observed for the PPII conformation, with τ = ~139°, ρ = ~83° and Smol = ~1.0 (Figure 3B). To clarify whether the peptide with the unexplored Pro-derived 19Flabel (CF3-cis-MePro) also meets this solution, all three splittings that were present in the solid-


20

Page 21 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


state

19

F-NMR spectrum (Figure 3A) were included, one by one, in this calculation. The

calculation confirmed that one constant (-4.5 Hz) among the three possibilities does indeed fit the obtained solution. Interestingly, when the β-strand conformation was assumed, improved formal solutions, in terms of r.m.s.d. value, could be obtained. With an r.m.s.d. below 1.0, orientations of a β-strand folded SAP that cover a range of τ from 90 to 60° (ρ = ~60° in all cases) are compatible with the observed splittings (in absolute terms the best fit was with an r.m.s.d. of 0.63: τ = 60°; ρ = ~61°, Smol = 0.99). However, due to the known amino acid composition of SAP, an ideal β-strand conformation is in conflict with the presence of the equally distributed β-breaking50 Pro-Pro-Pro segments. We concluded therefore that one state of the SAP (among others) in the model membranes corresponds to the PPII conformation under the experimental conditions tested (55°C; DMPC). The peptide, when structured in this manner, is aligned parallel to the lipid bilayer surface; the lipophilic residues point towards the membrane core and the hydrophilic residues of Arg point out of the lipid bilayer (Figure 3C). Condition-Dependent Membrane-Associated States of SAP. In the experiments above, DMPC multi-bilayers were used, as this system is known to orient well and is among the most well-studied and popular biomembrane models. The main lipid phase transition (Tm) occurs in DMPC at a temperature of approximately 24°C and allows convenient experimental access to both gel and fluid bilayer states by changing the temperature. To investigate the lipid phasedependent structural or orientational preferences of SAP, all seven

19

F-labelled analogs were

studied over a temperature range of 15-55°C. Surprisingly, at low temperatures (gel) the solidstate

19

F-NMR spectra predominantly contained quasi-isotropic singlets. Figure 4A shows a

representative temperature series from the sample containing SAP-cMePro-11 at a P/L of 1/50.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 44

All other peptides exhibited virtually the same temperature dependent spectral changes at both P/L = 1/50 and 1/200 (and are therefore not shown). At higher temperatures (above the Tm of DMPC, l.d.-phase of lipids) additional signals, as described above, appeared reversibly at the cost of the quasi-isotropic signals. Therefore, SAP appears to have different modes of interactions (orientation and/or structure) with the zwitterionic DMPC bilayers below (“isotropic” state of SAP) and above their Tm (“ordered” state of SAP). Interestingly, in the DMPC/DMPG (3/1, mol/mol) bilayers, the same phase-dependent “isotropic”-to-“ordered” transition remained, suggesting that the presence of negatively charged lipids does not affect this general behavior of the cationic peptide.


22

Page 23 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Temperature-dependent and time-dependent solid-state

19

F-NMR spectra. (A)In the

gel-phase bilayers of DMPC, only a quasi-isotropic 19F signal is seen with the SAP analog (SAPcMePro-11). Above the main phase transition temperature (indicated as ~Tm), the peptide is aligned in the membranes with multiple orientations simultaneously present (multiple splittings, see text). (B) Immobilization of SAP is a reversible time-dependent process.

In the course of the above measurements, we have noted that the kinetics of the transition between the two SAP states appear to be rather slow (Figure 4B), as different equilibration times (10 min, 0.5 h, 3 h, 6 h, 12 h and 24 h) produced different proportions of the molecules in the two states. At a high temperature (55°C) using a prolonged equilibration time (24 h), a pure “ordered” state was observed and was used in the analysis above (Figure 3A). An “isotropic” state was observed with all SAP analogs, independent of the

19

F-label or label position. The

resonance line was narrow (ca. 2 kHz) and at an isotropic chemical shift frequency; molecularly, this type of NMR signal should represent a situation where the peptide is highly mobile on the NMR-experiment time scale. This can occur when the peptides are “dissolved” and tumble freely, either in the inter-bilayer water bulk, or within the hydrocarbon lipid chains. The latter is rather implausible, due to the charged (net charge +3) nature of the peptide.51 Moreover, prolonged incubation times at the elevated temperatures required for the “ordered” states to occur correlate with the expected partial dehydration of the samples under such conditions. Taken together, these data suggest a generally low affinity of the SAP for the lipid membranes, especially for the gel-state bilayers, and the “ordered” states stemming from the peptide molecules that are forcefully immobilized by the conditions of the experiment.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 44

Lipid “raft”-mediated cell uptake was indirectly observed for the SAP,35,36 therefore we further questioned the effect of the lipid composition by addressing qualitatively the alignment behavior of the peptide in the “raft”-like model bilayers. A ternary bilayer system of the type: short-chain lipid/cholesterol/long-chain lipid, should provide a bilayer where non-“raft” and “raft” domains, i.e., laterally separated l.o., s.o. and l.d. lamellar lipid phases, co-exist.52 We have chosen to study the most extensively studied mixture of DOPC/cholesterol/BSM (2/1/2 molar ratio).53 However, for this system, no full temperature-composition phase diagram exists in the literature, and the “rafts” co-exist with not-“rafts” in a delicate equilibrium.54 Furthermore, even in the binary system PC/cholesterol, there are several phase diagrams reported that significantly differ among themselves (e.g., 55,56,57 for DPPC/cholesterol). Given these background uncertainties and the complex time and temperature-dependent binding of SAP to lipid bilayers (Figure 4), we decided to sample a broad temperature range for each bilayer and to additionally study the less complex single and two-component lipid mixtures. These included DOPC; BSM, DOPC/cholesterol (2/1 mol/mol) and BSM/cholesterol (2/1 mol/mol) bilayers. Such a spectrum of compositions and conditions allowed us to systematically sample pure lamellar lipid phases: i.e. s.o.-, l.o.-, l.d.-phases, and provide mixtures thereof. In these 19F-NMR experiments, a single 19

F-labeled SAP analog was used as a monitor. Because a CF3-cis-MePro-containing peptide

provided the largest spectrum of possible

19

F-NMR signals (i.e., different alignment states or

conformations), this peptide was selected as a probe.


24

Page 25 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. SAP in the “raft”-model membranes. Temperature dependence of the solid-state 19FNMR spectra from SAP-cMePro-11 being reconstituted at a P/L of 1/50 in mechanically oriented bilayers of: (A) BSM, (B) BSM/chol (2/1 mol/mol), (C) DOPC, (D) DOPC/chol (2/1 mol/mol), and (D) classical “raft” ternary mixture of DOPC/BSM/chol (2/2/1).

In pure BSM bilayers (Figure 5A) SAP was found to re-align in the same manner as in DMPC (Figure 4A): below the Tm (i.e., in the s.o.-phase) an “isotropic” signal was observed, which coexisted with the newly appearing “ordered” states when the lipid phase changed to become l.d. upon heating. In DOPC (Figure 5C), where the lipid bilayers are in the l.d.-phase at all temperatures, multiple orientations of SAP were present at all times. The addition of cholesterol to DOPC should have converted the uniform l.d.-phase to an l.d.+l.o. mixture, but it had no influence on the appearance of the

19

F-NMR spectra (Figure 5C, 5D). In contrast, in the


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 44

BSM/chol mixture, (Figure 5B), which exists in the “pure” l.o.-phase at elevated temperatures, “powder”-like spectra are observed, indicating that the SAP has no preferred orientation and is immobilized. The very same lipid system at lower temperatures should be present as in the l.o.+s.o. phase mixture, where the SAP was converted into a mixture of “isotropic” and “powder” states. Finally, in the DOPC/BSM/chol bilayers (Figure 5E), the

19

F-NMR spectra

appeared to reflect a superposition of the respective BSM/chol and DOPC/chol situations with all their lipid phases being present simultaneously. SAP Perturbed With Conformational Probes (4,5-Methanoprolines). In another study, using short model peptides, we discovered that PPII can be stabilized by the conformationally rigid cis-4,5-methanoprolines and destabilized by their trans-isomers at the very same peptide positions (unpublished data). We employed this effect here by probing SAP conformational equilibria with a mild conformational perturbant, which can simultaneously serve as a 19F-NMR reporter – the trans-isomer of the previously employed

19

F-NMR label (Figure 2E), CF3-cis-

MePro.29 As outlined above, using CF3-cis-MePro and CF3-trans-MePro, a pair of SAP analogs with increased (SAP-cMePro-11) and reduced (SAP-tMePro-11) PPII content was therefore produced and cross-compared. In the solid-state 19F-NMR (Figure 6), under conditions that were previously used for the SAP orientational analysis, i.e., in the fluid DMPC bilayers at a 1/50 P/L, SAP-tMePro-11 indeed produced only one dipolar splitting of approximately +9 kHz. The other signals, including the 4.5 kHz coupling of the PPII-structured oriented SAP, completely disappeared, thus confirming our assignment of this splitting having a PPII origin. The remaining rotationally averaged signal


26

Page 27 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(large positive splitting), however, does not appear to originate from a fibril or other type of aggregate. Fully aggregated immobile peptides should resemble themselves in the form of a “powder”-like spectra, similar to (Figure 5B). Thus, another stable conformation should exist for the PPII-deprived SAP in l.d. bilayers.

Figure 6. Solid-state

19F

-NMR spectra of conformationally perturbed SAP analogs in DMPC.

The peptide with destabilized PPII, SAP-tMePro (A) and with stabilized PPII, SAP-cMePro measured in DMPC bilayers in the liquid disordered state (P/L 1/50, 50°C). The oriented samples are placed such that the membrane normal is parallel (upper traces) or perpendicular (lower traces) to the external B0 magnetic field. Sample orientations are drawn schematically. The isotropic frequency is marked as a dotted line, and the dipolar splittings that are averaged due to rotation about the membrane normal are shown.

We previously claimed that replacing the proline at position 11 in SAP with CF3-cis-MePro led to the stabilization of PPII.29 This statement was based entirely on the prominent increase in the CD intensity for this analog of the characteristic PPII band at approximately 225 nm. The


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 44

self-aggregation or fibrilization of SAP (Figure 1) was suggested by Giralt et al2 based on the fact that by increasing the peptide concentration (over the range 5-100 µM) a concentrationproportional increase of the CD signal ceased after reaching approximately 50 µM. Subsequently, in the samples with higher than this threshold concentration, fibrils were observed by transmission EM analysis only after freeze-fixing and freeze-drying of the polypeptide solution.2 The authors argued that due to the amphipathic nature of the SAP, typical procedures for drying the precipitate for the EM specimen would be erroneous because of the exposure to an air-liquid interface in the drying process.58 It should be noted, however, that all published EM photographs obtained in this manner for SAP and its derivatives (as well as for the parent γ-zein proteins) appear to show very similar fibril morphologies (approximately 20 nm diameter), and furthermore, fibrils are never observed in large abundance, as they are distributed sporadically over the field of view with a 50-100 nm spacing.2,37,58,59,60,61,62 This low abundance of fibrils along with the clear absence of massive precipitation in CD samples, suggests that the SAP monomer ↔ oligomer ↔ fibril equilibrium is considerably shifted towards small size species. After discovering a non-PPII conformation for the SAP, and having observed no detectable aggregation in the DMPC bilayers, we challenged the assumption of spontaneous SAP fibrilization by using higher concentrations of SAP and its conformationally perturbed derivatives in the SRCD experiments. SRCD, in addition to having much better signal-to-noise ratios in concentrated or crowded samples, should allow for the collection of UVCD data down to 178 nm for quantitative spectral analysis. This method, therefore, allowed us to measure CD spectra under similar conditions to those used for the solid-state NMR studies, in the 10-100 mM range.


28

Page 29 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


As seen in Figure 7, in our hands, at a concentration as high as 50 mM, SAP and its analogs were soluble in water, and the CD signal shape, which reflects a mixture of PPII and random coil, did not change compared to those obtained at lower concentrations. We did not observe precipitation or an increase in scattering as is usually seen when the peptides aggregate in solution. It should be noted, that in Giralt et al2 and in our previous SAP measurements, the ellipticity at 225 nm in the parent peptide was approximately zero; therefore, all of the spectral changes were reflected only in the 204 nm band and were highly crowded with the contributions from non-helical conformations. Nevertheless, a conformational assessment of SAP and its 19Flabeled analogs that were used in the solid-state NMR analysis described above was performed under nominally non-aggregating low-concentration conditions and are therefore valid.

Figure 7. SRCD of SAP and MePro analogs in water. The three peptides were dissolved in water at 250 µM (A) and 50 mM (B), and the spectra were collected at 25°C. Characteristic wavelengths for the RC and PPII spectral contributions are marked for clarity.

The conformationally perturbed Pro-mutants of SAP, on the contrary, had non-zero values and allowed a ratiometric two-band evaluation. The CD data (Figure 7), based on the intensity ratio


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 44

at 225 nm to 203 nm, suggests that the PPII content in SAP-tMePro-11 deviated from that in the SAP much greater than in the corresponding analog SAP-cMePro-11. This is the only peptide, where the CD features that are associated with PPII are visually reduced with an increase in its concentration. The general temperature behavior of the Pro-substituted samples in solution followed the common trend, which is normally interpreted in systems with an RC/PPII equilibrium as an increase in the RC content at the cost of a reduction of PPII. Upon heating (550°C) the aqueous samples, all three peptides display a coherent lowering of the intensities at 225 and 204 nm. To obtain further insights into the SAP-lipid interactions from the perspective of peptide conformation and to address if these results are due to the presence of lipids, we performed SRCD measurements with the lipid-reconstituted SAP analogs as a function of temperature. Characteristic SRCD spectra are shown in Figure 8. Although the general appearance of the CD spectra for all three peptides continued to resemble that of the RC/PPII mixtures, the equilibrium was shifted towards a reduction of the PPII content. Furthermore, in all cases, heating to 50°C did reduce the spectral features that are associated with PPII and the initial equilibrium was restored when the samples were returned to the initial temperature.


30

Page 31 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. SRCD of SAP and MePro analogs in DMPC multilamellar vesicles. Selected spectra from the temperature series (30°C, 35°C, 40°C, 45°C and 50°C; the series are collected in three consecutive heating/cooling cycles) of the SAP peptides that were reconstituted in DMPC at a P/L of 1/100 at a total material concentration of 15 mg/ml. Characteristic wavelengths are marked for clarity. (A) SAP, (B) PPII-stabilized SAP-cMePro-11, (D) PPII-destabilized SAPtMePro-11.

Conformational Plasticity of SAP. To quantify the peptides´ conformations after having collected CD signals in the 178-260 nm range, all SRCD spectra were subjected to analysis using DICHROWEB.46 We knew from the qualitative analyses (see above) that at least PPII and RC conformations have to be present in the SAP peptides, therefore, we applied the two recommended data sets which contain these conformations (data set 2 and 5) and employed three spectra decomposition algorithms: SELCON3,63 CONTIN/LL,64 and CDSSTR.65 For the parent peptide in solution, seemingly the simplest situation, this approach failed. First, there was no consensus in the results using three algorithms, which is required for confidence in the quantitative results. Moreover, the former two algorithms produced best-fit results with unacceptable r.m.s.d. values (r.m.s.d. >0.1).46 Only CDSSTR analysis was formally acceptable according to an r.m.s.d. judgement. Interestingly, in this case, it was possible to suggest for all spectra a significant amount of other than RC and PPII conformations. For instance, from the spectra in Figure 7A, using the reference set 5 under CDSSTR analysis produced the best-fit results (r.m.s.d. in all cases

Conformational Plasticity of the Cell-Penetrating Peptide SAP As

Recommend Documents