Membrane Binding and Pore Formation by a Cytotoxic Fragment of

Subscriber access provided by Gothenburg University Library

Article

Membrane Binding and Pore Formation by a Cytotoxic Fragment of Amyloid # Peptide Nabin Kandel, Tianyu Zheng, Qun Huo, and Suren A. Tatulian J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07002 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 18, 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 26

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

Membrane Binding and Pore Formation by a Cytotoxic Fragment of Amyloid β Peptide †

‡

‡

Nabin Kandel , Tianyu Zheng , Qun Huo , and Suren A. Tatulian*,§ †

Physics Graduate Program, Department of Physics, University of Central Florida, Orlando, Florida, USA ‡

Department of Chemistry and NanoScience Technology Center, University of Central Florida, Orlando, Florida, USA §

Department of Physics, Physical Sciences Bldg., Room 456, University of Central Florida, 4111 Libra Drive, Orlando, Florida 32816, USA AUTHOR INFORMATION Corresponding author * E-mail: [email protected]. Tel.: +1-407-823-1543. Fax: +1-407-823-5112 Notes The authors declare no competing financial interest.

1

ACS Paragon Plus Environment


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

ABSTRACT: Amyloid β (Aβ) peptide contributes to Alzheimer’s disease by a yet unidentified mechanism. In brain tissue, Aβ occurs in various forms, including an undecapeptide Aβ25-35, which exerts neurotoxic effect through mitochondrial dysfunction and/or Ca2+-permeable pore formation in cell membranes. This work was aimed at biophysical characterization of membrane binding and pore formation by Aβ25-35. Interaction of Aβ25-35 with anionic and zwitterionic membranes was analyzed by microelectrophoresis. In pore formation experiments, Aβ25-35 was incubated in aqueous buffer to form oligomers and added to Quin-2-loaded vesicles. Gradual increase in Quin-2 fluorescence was interpreted in terms of membrane pore formation by the peptide, Ca2+ influx and binding to intravesicular Quin-2. The kinetics and magnitude of this process were used to evaluate the rate constant of pore formation, peptide-peptide association constants, and the oligomeric state of the pores. Decrease in membrane anionic charge and high ionic strength conditions significantly suppressed membrane binding and pore formation, indicating the importance of electrostatic interactions in these events. Circular dichroism spectroscopy showed that Aβ25-35 forms the most efficient pores in β-sheet conformation. The data are consistent with an oligo-oligomeric pore model composed of up to 8 peptide units, each containing 6 to 8 monomers.

2


Page 2 of 26

Page 3 of 26

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


1. Introduction The Aβ peptide plays a central role in initiation and progression of Alzheimer’s disease.1 Aβ is derived from the amyloid precursor protein (APP), a bitopic glycoprotein in neuronal membranes, by cleavage in the transmembrane and extracellular juxtamembrane regions by γand β-secretases, respectively.2,3 Poor sequence specificity of γ-secretase produces Aβ molecules of varying lengths, with the 42- and 40-amino acid residue peptides (Aβ1-42 and Aβ1-40) being the dominant species.4 Many shorter forms of the peptide are found in human brain, which result from proteolysis and N- or C-terminal trimming by amino- or carboxy-peptidases. Among these, the undecapeptide Aβ25-35 (GSNKGAIIGLM) has received much attention because of its presence in AD brain tissue and its strong cytotoxicity.5-9 The existing data suggest that Aβ25-35 exerts its cytotoxic action through mitochondrial membrane damage and leakage of proteins such as malate dehydrogenase, citrate synthase, and cytochrome c, culminating in caspase-3 and caspase-9 activation and apoptotic cell death.10-13 While Aβ25-35 has been shown to augment the expression of proteins associated with the mitochondrial permeability transition pore, 14,15 there is strong evidence that the peptide itself binds to membranes and forms ion conducting pores in biological and model lipid membranes.1621 Aβ25-35 was shown to bind to anionic lipid membranes mostly through electrostatic interactions, and suppression of such binding alleviated Aβ25-35-induced cytotoxicity.17,22 The peptide formed voltage-dependent channels in lipid membranes that conducted both cations and anions with a permeability sequence Ca2+ > K+ ≥ Na+ > Cl-.16,18 Among several Aβ fragments tested, Aβ25-35 caused maximum rise in intracellular Ca2+ levels in cultured neurons, close to the effect of the full-length Aβ1-42.21 Interaction with membranes significantly affected the structure of Aβ25-35. In aqueous buffer, the peptide assumed mixed α-helix/β-sheet structure,23,24 whereas in the presence of anionic lipid vesicles the helical structure was replaced by β-sheets and β-turn structure.24 Biophysical studies showed that Aβ25-35 tends to form β-turn conformation at moderate concentrations (~30 µM) and at higher concentrations transitions to β-sheet protofilaments that bind to anionic lipid membranes.25 Membrane binding was mostly peripheral, in contrast to other data showing insertion of Aβ25-35 into the hydrocarbon chain region of membranes.26-29 Neutron diffraction studies suggested two populations of membrane-bound Aβ25-35, peripheral and membrane-inserted,30,31 with a larger fraction of the latter for zwitterionic phosphatidylcholine membrane compared to anionic phosphatidylcholine/phosphatidylserine membranes.30 Solid state NMR showed that a deeper membrane insertion was achieved when vesicles were prepared using pre-mixed lipid-peptide samples compared to addition of the peptide to pre-formed vesicles.32 These data provide solid evidence for membrane binding/insertion of Aβ25-35, but the mode of peptide-membrane interactions, pore formation, and the underlying structural changes remain unclear. The mechanism of membrane pore formation by Aβ25-35 has been studied mostly by computational approaches. A molecular dynamics (MD) analysis proposed a mixed parallelantiparallel 8-stranded β-barrel, with Lys28 side chain turned inside, thus forming a water and ion conducting channel with an inner diameter of 3.5 to 4.0 Å.33 A similar structural model was proposed by MD and mass-spectrometry studies. 34 In contrast, MD and X-ray diffraction studies suggested that membrane-inserted Aβ25-35 was in a mostly α-helical conformation, with the helical axis significantly tilted relative to the membrane normal 31,35,36 Cholesterol-assisted, 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

Ca2+-permeable channels formed by Aβ22-35 and Aβ25-35 were modeled as octameric pores with an inner diameter of 14.6 Å composed of 8 α-helical peptides and 16 cholesterol molecules, with a ~40 degrees tilt with respect to the membrane normal.19,20,37,38 The inconsistency between Aβ25-35 pore structures reported by various groups apparently results from different starting structures used in MD simulations. Chang et al.33 created an a priori, pre-formed octameric β-barrel structure and inserted it into a lipid bilayer. The β-barrel model reported by Do et al.34 was based on best agreement with ion-mobility mass-spectrometry-derived collision cross section. Tsai et al.35,36 and Dies et al.31 used the partially α-helical NMR structures of detergent-solubilized Aβ2539,40 and Fantini and co-workers19,20,37,38 used the 25-35 stretch of the α-helical structure of 35 , Aβ1-40 in sodium dodecylsulfate micelles.41 The final pore conformation of a membrane-inserted peptide in MD simulations is obviously determined by the choice of the initial peptide structure. Here, we present a biophysical analysis of interaction between Aβ25-35 and phospholipid membranes, kinetics of peptide-induced Ca2+ transport across membranes, and the structure of the peptide in aqueous buffer and in membrane-bound state. Microelectrophoresis experiments have allowed evaluation of membrane binding constants and binding site densities. The kinetics and magnitudes of peptide-induced changes of intravesicular Quin-2 fluorescence were used to determine second-order rate constants of pore formation, peptide-peptide association constants, and the oligomeric state of the pore structure. Membrane binding of Aβ25-35 and pore formation are robustly controlled by membrane electrostatics; both processes are promoted by negative surface potential of membranes. The data suggest a supramolecular structure for the functional pore composed of up to 8 hexameric and/or octameric β-barrel-like units.

2. Materials and Methods 2.1. Materials Most chemicals, including non-fluorescent Ca2+ ionophore 4-Br-A23187, hexafluoroisopropanol (HFIP), salts, buffers, were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fisher Scientific (Hanover Park, IL, USA). All lipids, i.e., 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and cholesterol were from Avanti Polar Lipids (Alabaster, AL, USA), with ≥ 98% purity. The synthetic Aβ25-35 peptide, acetylated at N-terminus and amidated at C-terminus, was purchased from Peptide 2.0 Inc. (Chantilly, VA), with ~98% purity. Quin-2 tetrapotassium salt was from EMD Chemicals (San Diego, CA, USA). 2.2. Experimental procedures The lyophilized peptide was dissolved in HFIP at 2 mM concentration. To measure the secondary structure of Aβ25-35, 10 µL of the stock solution was transferred into a glass vial, dried with a stream of nitrogen, followed by desiccation for 30 min. For circular dichroism (CD) measurements, 400 µL of aqueous buffer was added to the dry peptide and stirred for several hours, using a magnetic stir bar. CD spectra were measured at different times of incubation, using a 1 mm path-length quartz cuvette, on a J-810 spectropolarimeter with a fluorescence attachment (Jasco, Tokyo, Japan). For Fourier transform infrared (FTIR) measurements, certain amount of HFIP-dissolved peptide was dried on a CaF2 window, and a D2O-based buffer (50 Mm NaCl, 50 mM K,Na-phosphate, pD 7.2, corresponding to pH-meter reading 6.8) was added 4


Page 4 of 26

Page 5 of 26

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


to the dry peptide to achieve 50 µM final concentration. Another CaF2 window was used to seal the sample between two windows separated by a 50 µm Teflon spacer, and spectra were measured at 2 cm-1 resolution on a Vector-22 FTIR spectrometer (Bruker Optics, Billerica, MA, USA), as described earlier.42 Large unilamellar vesicles were prepared using POPC, POPG, and cholesterol, following the procedures described earlier.43 Briefly, chloroform solutions of the lipids were mixed at desired proportions, dried, suspended in an aqueous buffer by vortexing, and extruded through a 100 nm pore-size polycarbonate membrane, using a mini-extruder (Avant Polar Lipids). The total lipid concentration was 5 mM. The buffer contained varying concentrations of KCl, 20 mM Tris-HCl (pH 7.2), and 6 mM Quin-2. The vesicle sample was passed through a Sephadex G-50 desalting column (10 inches long, 0.7 inches inner diameter) to remove external Quin-2. The column was equilibrated with a buffer containing 20 mM Tris-HCl (pH 7.2), NaCl at same concentration as intravesicular KCl, plus 30 mM myo-inositol to iso-osmotically replace 6 mM Quin-2 tetrapotassium salt. Lipid concentration in the elution fractions were estimated based on a calibration curve obtained be measuring right-angle static light scattering at 500 nm, using identical lipid vesicles of known concentrations. The concentration of lipid vesicles was adjusted so that the addition of an iso-osmotic solution of CaCl2 resulted in final lipid and CaCl2 concentrations of 0.2 mM and 6 mM, respectively. Thus, Quin-2-loaded unilamellar lipid vesicles with CaCl2 in the external medium were obtained. To monitor membrane permeabilization by the peptide, Quin-2-loaded vesicle sample was placed in a rectangular quartz cuvette (4 mm × 4 mm, inner cross-section) and Quin-2 fluorescence was measured on the J-810 spectropolarimeter with a fluorescence attachment at excitation wavelength 339 nm. Emission spectra were collected between 450 and 600 nm. Once a stable baseline was established, certain amount of peptide, stirred in a buffer for 2.5 hours, was added, and fluorescence spectra were measured periodically for 20 minutes. Time-dependent increase in Quin-2 fluorescence upon addition of the peptide was interpreted in terms of Ca2+ transport into the vesicle, binding to Quin-2 and fluorescence enhancement. In negative and positive control measurements, the added peptide was replaced by blank buffer or nonfluorescent Ca2+ ionophore 4-Br-A23187, respectively. On completion of fluorescence measurements, CD spectra were recorded on same sample to estimate the peptide structure in its membrane-bound state. All measurements were conducted at 22oC. Membrane pore formation by the peptide was quantitatively described within a theoretical framework described earlier.43 Peptide binding to the vesicles was measured by microelectrophoresis. Certain concentrations of peptide, stirred in an aqueous buffer (20 mM Tris-HCl + varying concentrations of NaCl, pH 7.2) for 2.5 hours, were added to extruded lipid vesicles (200 µM total lipid concentration). All samples were degassed using a vacuum pump before measurements. The sample was transferred into a DTS 1070 folded capillary zeta cell and ζpotentials were measured on a ZetasizerNano ZS90 DLS system equipped with a green laser (532 nm, 4 mW) and an Avalanche photodiode detector (quantum efficiency >50% at 532 nm) (Malvern Instruments Ltd., Malvern, UK). Following ζ-potential measurements, 100 µL of same sample was placed in a 67.758 UV cuvette (Sarstedt AG & Co, Nümbrecht, Germany) and dynamic light scattering size distribution was measured on the same instrument. 2.3. Membrane binding theory

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 26

Membrane binding of the peptide was described using the Gouy-Chapman-Stern theory,44 which allows determination of membrane binding constant and the density of membrane binding sites based on the dependence of vesicle ζ-potential on peptide concentration. The total surface charge density of the vesicle membrane, σ, is the sum of intrinsic charge density, σ0, determined by the presence of anionic lipid, and charge density determined by bound peptide, σb:

σ = σ0 +σb

(1)

In the presence of 1:1 electrolyte, such as NaCl, σ is given by the Gouy-Chapman equation:  Fψ 0    2 RT 

σ = 8εε 0 RTC sh

(2)

where ε is the relative dielectric constant of water (at 22oC, ε = 79.6), ε0 = 8.854×10-12 C2/(N⋅ m2) is the permittivity of free space, R = 8.314 J/(mol⋅ K) is the gas constant, T is the absolute temperature (T = 295 K in our experiments), C is the total molar concentration of the 1:1 electrolyte, F = 96,485.333 C/mol is the Faraday constant, ψ0 is the surface potential. The relationship between ψ0 and the measured ζ-potential is given by:

ζ = ψ 0 e −δ / λ

(3)

where δ is the shear layer thickness, i.e. the distance between the actual membrane surface and the slipping plane, and λ is the Debye length:

λ=

εε 0 RT

(4)

F 2 ∑ z i2 Ci

In (4), zi and Ci are the charge number and the molar concentration of ith ion. Our data are consistent with δ = 3 Å, corresponding to a layer of membrane-bound water molecules. The charge density produced by bound peptide, σb, is:

σb =

ze[ Pb ] γ [ L] ALipid

(5)

where [Pb] is the concentration of membrane-bound peptide particles, in monomeric or oligomeric form, z is the charge number of the peptide particles, e is the elementary charge, γ is the fraction of lipid exposed to externally added peptide (for 100 nm vesicles, γ = 0.52), [L] is the lipid concentration, and ALipid is the cross-sectional area per lipid molecule. Membrane binding of the peptide is described as a bimolecular process45: P f ,0 + γL f ⇒ Lb

(6) 6


Page 7 of 26

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


where P means peptide, L means lipid, the subscripts f and b mean free and bound, and the subscript 0 means peptide at membrane surface. The dissociation constant is: KD =

[ P f ,0 ]γ [ L f ]

(7)

[ Lb ]

where the brackets mean concentrations. Assuming NL lipids correspond to a peptide-binding site, the following relationships hold: [Lb] = NL[Pb], [Pf,0] = [P0] – [Pb], γ [Lf] = γ [L] - NL[Pb]. The concentration of peptide particles with oligomeric number z obeys Boltzmann distribution of charged particles in an electrostatic field: [ P0 ] =

[ P] exp( − zFψ 0 / RT ) z

(8)

where [P] is the total monomer concentration of the peptide. In general, the denominator (the oligomeric number) may be different from the charge number in the exponent, but for a peptide with a single positive charge per monomer, they both have the same value of z. Inserting these relationships into Eq. (7), we obtain a quadratic equation for [Pb], which is solved as follows:

[Pb ] = a ± a 2 − b

(9)

where

a≡

b≡

1 γ [ L]   K D + [ P0 ] +  2 N L 

(9a)

γ [ L][ P0 ]

(9b)

NL

Data analysis shows that only the negative sign in front of the square root in Eq. (9) should be used as the positive sign yields physically meaningless values for [Pb]. The ultimate membrane binding isotherm is:

σ0 +

ze  a − a 2 − b  = 8εε RTC sh Fψ 0  0  γ [ L] ALipid   2 RT 

(10)

Theoretical binding isotherms have been constructed by numerically determining values of ψ0 at each peptide concentration for given values of KD and NL, according to Eq. (10), and then the ζpotentials according to Eq. (3). Values of σ0 were evaluated through the measured ζ-potentials of blank vesicles (in the absence of peptide) using Eqs. (2), (3), (4).

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 26

2.4. Kinetics of membrane pore formation The kinetics of membrane permeabilization by the peptide was analyzed as described earlier.43 Briefly, peptide particles (monomers or oligomers) bind to the membrane from the aqueous phase and form pores, resulting in increase in Quin-2 fluorescence to a limiting value Feq. If pore formation is considered a second order process, then the kinetics of this process can be described as follows:

(

Feq

[ Pb ] Feq − Ft

)

=

1 + kat [ Pb ]

(11)

where Ft is the fluorescence intensity at time t after peptide addition, ka is the second-order rate constant in units M-1s-1, and [Pb] is defined as above. Values of [Pb] at each total peptide concentration were determined though Eq. (9) and values of Feq/{[Pb](Feq – Ft)} were calculated using Ft and Feq from the experimental data. The second-order rate constants of pore formation, ka, were determined as the slope of the time dependence of the term at left-hand side of Eq. (11). It has been shown that pore formation in a lipid membrane by peptides is much slower than a diffusion-controlled process,43 i.e., the experimentally observed rate constant of pore formation, kexp, constitutes the rate-limiting step of the process. In this case: k a = K p k exp

(12)

where Kp is the peptide-peptide affinity constant within the membrane. Values of kexp were found from a single exponential fit of experimental kinetic data and combined with ka (see above) to determine Kp.

2.5. The oligomeric state of the pore Following a theoretical framework described earlier,43 the equilibrium intensity of Quin-2 fluorescence relative to the maximum value produced by Ca2+ ionophore (Frel = Feq/Fmax) is related to the number of peptide particles in the pore, n, through: Frel = q n −1 ( n − nq + q )

(13)

where q=

1

(13a)

α + α −1

α = 1+

2

1 4 K p [ Pb ]

(13b)

8


Page 9 of 26

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


Values of n were evaluated through Eq. (13) using experimental values of Frel along with Kp and [Pb] determined as described above.

3.

RESULTS AND DISCUSSION

3.1. Peptide structure in aqueous media The secondary structure of Aβ25-35 in aqueous media was assessed by FTIR and CD spectroscopy. FTIR data show that initially the amide I band of the peptide comprises a wide component between 1647 cm-1 and 1640 cm-1 and a shoulder around 1666 cm-1 (Figure 1a), indicating mostly unordered and β-turn conformations.46 Dramatic structural change occurs between 3 and 15 minutes of exposure of the peptide to aqueous buffer, evidenced by conversion of the unordered component into a prominent band at 1617-1616 cm-1, indicative of intermolecular β-sheet structure. Further structural changes involve increase in the β-turn structure (1673-1671 cm-1) at the expense of β-sheet structure, as well as appearance of less pronounced structural components such as α-helix, irregular structure, and intramolecular βsheet structure (features around 1660 cm-1, 1642 cm-1, and 1628 cm-1, respectively). The CD spectrum initially showed a relatively weak minimum around 202 nm, which shifted to a deeper minimum at 208 nm upon further incubation of the peptide in buffer (Figure 1b), implying gradual transfer of the dry peptide into the buffer and transition from unordered to type I β-turn structure.47,48 FTIR and CD data thus suggest formation of heterogeneous conformations by Aβ25-35 in aqueous buffer, dominated by intermolecular β-sheet/β-turn structures.

Figure 1. Secondary structure of Aβ25-35 determined by FTIR and CD. (a) FTIR spectra of Aβ25-35 at different times of exposure to D2O-based buffer containing 50 mM NaCl and 50 mM Na,K-phosphate, pD 7.2 (pH meter reading 6.8), as indicated. (b) CD spectra of Aβ25-35 stirred in a buffer containing 145 mM NaCl and 50 mM Tris-HCl, pH 7.2, for different time periods, as indicated.

3.2. Membrane binding 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 26

The peptide used in this work was N-terminally acetylated and C-terminally amidated, so at neutral pH it had a single positive charge due to the side chain amino group of Lys28. The peptide was stirred in an aqueous buffer for 2.5 hours and added to anionic vesicles composed of 60 mol % POPC (zwitterionic phospholipid), 30 mol % POPG (acidic phospholipid), and 10 mol % cholesterol (non-ionic sterol), at total lipid concentration of 200 µM. It has been shown earlier that Aβ25-35 undergoes fibrillogenesis upon incubation in aqueous media for more than 24 hours, while at shorter times it forms soluble oligomers.21 Together with our data indicating stabilization of β-sheet/β-turn structure around 2.5 hours of stirring in buffer (Figure 1), the peptide sample most likely contained oligomers of various aggregation numbers, in intermolecular β-sheet conformation. Increasing peptide concentrations resulted in significant reduction of the negative ζ-potential of vesicles (electrostatic potential at the shear plane, at or a few Å away from membrane surface), indicating membrane binding of Aβ25-35 (Figure 2). Increasing NaCl concentration suppressed both the surface potential of the vesicles and the effect of the peptide, indicating that Aβ25-35-membrane interaction was to a large extent governed by electrostatic forces. In each experiment, the ζ-potential of blank vesicles was used to calculate the intrinsic surface charge density of the membranes, σ0. Data at various ionic strengths were consistent with a shear layer thickness of 3 Å, suggesting the vesicles carried a layer of membrane-bound water molecules. At high peptide concentrations, the ζ-potential levelled off at a negative value, implying a limited surface area per peptide binding site. The extrapolated values of ζ-potentials at high peptide concentrations were used to calculate the limiting surface charge densities, σ∞. Considering that the charge density due to peptide adsorption is σads = σ∞ σ0, and that σads = ze/NLALipid, the number of lipids per bound peptide particle (monomer or oligomer) is: NL =

ze

(14)

ALipid (σ ∞ − σ 0 )

where z is the charge (or, for a peptide with a single charge per monomer, the aggregation number) of the membrane binding species. An average value of ALipid = 55.4 Å2 has been obtained using cross-sectional areas of 59 Å2 for POPC and POPG (in membranes containing 10 mol % cholesterol, at 22oC) and 23 Å2 for cholesterol.49,50 Peptide binding to the membrane was described for z values varying from 1 to 8. Theoretical binding curves for z =1, 4, and 8 are shown in Figure 2 for NaCl concentrations of 10 mM, 30 mM, and 75 mM. Best fit values of NL and the intrinsic dissociation constants, KD, are presented in Table 1. The range of the experimental error of measured ζ-potentials prevents identification of a single oligomeric form of membrane binding species (Figure 2a,b,c). Realistically, peptide aggregates of varying oligomeric forms may bind to the vesicles. However, theoretical curves with z > 8 were off the range of experimental data, implying large particles were either absent or did not bind to membranes. Thus, Aβ25-35 assemblies, ranging from monomers to octamers, form upon incubation in buffer for 2.5 hours and bind to anionic membranes. Estimates if NL by Eq. (14) show that around 25-40 lipid molecules correspond to a monomer binding site, which increases proportionally with increasing aggregation number of the

10


Page 11 of 26

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 2. Binding of Aβ25-35 to unilamellar lipid vesicles measured by microelectrophoresis. Dependence of vesicle ζ-potential on peptide concentration in aqueous buffer containing 20 mM Tris-HCl (pH 7.2) and NaCl at concentrations 10 mM (a), 30 mM (b) and 75 mM (c). Theoretical binding isotherms for peptide aggregation numbers z = 1, 4, and 8 are shown as solid, dashed, and dotted lines, respectively. Total lipid concentration was 0.2 mM, and the lipid composition of vesicles was 60 mol % POPC, 30 mol % POPG, 10 mol % cholesterol. Panels (d), (e), (f) show peptide concentration dependence of apparent binding constants calculated using the intrinsic dissociation constants tabulated in Table 1 and the surface potentials from panels (a), (b), (c), respectively. Solid, dashed, and dotted lines correspond to peptide aggregation numbers z = 1, 4, and 8, respectively.

peptide (Table 1). The intrinsic dissociation constants are in the range (2.5-4.4)×10-5 M for monomers, (1.6-2.0)×10-4 M for tetramers, and (4.2-5.6)×10-3 M for octamers. Higher KD values for species of higher aggregation numbers imply tighter membrane binding of smaller particles. In reality, however, particles with higher z values bind more than those with lower z values because, according to the Boltzmann distribution law, the concentration of charged species at the membrane surface is proportional to exp(-zψ0F/RT) (note that the surface potential, ψ0, is negative). The apparent binding constants, Kb,app = (1/KD) exp(-zψ0F/RT), are indeed much higher for assemblies with higher aggregation numbers, as demonstrated in Figure 2d,e,f. Values of Kb,app decrease with increasing salt concentrations, as expected. Strong electrostatic effects in interaction of Aβ25-35 with anionic phospholipid membranes are consistent with earlier reports indicating suppression of membrane binding of the peptide by increasing NaCl concentration.22 The apparent binding constant of the C-terminally amidated Aβ25-35 was 5×104 M-1, 22 which is in the range of data shown in Figure 2d,e,f corresponding to low values of z at peptide concentrations ≥ 10 µM.

3.3. Membrane permeabilization 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

3.3.1. Anionic membranes. Vesicles loaded with 6 mM Quin-2 were placed in buffers containing 6 mM CaCl2 and the peptide was added, resulting in peptide dose-dependent increase in Quin-2 fluorescence (Figure 3). This effect was interpreted in terms of peptide-induced pore formation in the vesicle membrane, Ca2+ influx and binding to Quin-2, causing enhancement in fluorescence. In positive control experiments, non-fluorescent Ca2+ ionophore 4-Br-A23187 was added, which caused sharp and strong increase in Quin-2 fluorescence (Figure 3). In negative control experiments, blank buffer was added, which did not cause any changes other than the sample dilution effect (not shown). At low ionic strengths, the peptide-induced increase in Quin-2 fluorescence reached more than half of the effect of the ionophore, with a single-exponential time constants within the range 0.01-0.05 s-1, corresponding to time constants of 0.33-1.67 min. Increasing salt concentration strongly suppressed both the rate and the level of fluorescence increase. At NaCl concentrations ≥ 180 mM, only the highest peptide concentration (66.7 µM) induced measurable fluorescence increase, with rate constants exceeding 30 min (Figure 4, Table 2). These data indicate that membrane pore formation by Aβ25-35 is under electrostatic control, consistent with microelectrophoresis data on peptide-membrane interactions. Despite the similar trends of membrane binding of the peptide (Figure 2) and pore formation (Figures 3, 4), i.e., reduction of both effects with increasing salt concentration, the electrostatic effects involved in pore formation appear to be more complex as the experimental single-exponential rate constant, kexp, and the second-order rate constant, ka, involved in this process show a biphasic dependence on the ionic strength (Figure 4b,c). Both kexp and ka initially increase with increasing ionic strength up to 100-150 mM, then sharply drop at higher ionic strengths, indicating a dual effect of salt concentration. This can be interpreted in terms of enhancement of peptide-peptide interactions and suppression of peptide-membrane interactions by high ionic conditions. (Increasing salt concentration screens the particle charge by means of counter-ion accumulation, thus alleviating the electrostatic repulsion between like charges and weakening the attraction between opposite charges). At low salt concentrations, the cationic peptide efficiently binds to the anionic membranes, and peptide-peptide interactions play a major role in pore formation, while higher salt concentrations hinder pore formation by inhibiting membrane binding of the peptide. The number of peptide particles in the pore, n, varies within the range 3-10 (Table 2). Statistical analysis of these data identifies a bimodal distribution, i.e., two populations of pores, those containing 3-5 units, occurring at lower ionic strengths ([NaCl] ≤ 50 mM) and those containing 7-9 units, occurring at higher ionic strengths ([NaCl] ≥ 75 mM). Note that these units are oligomers of aggregates containing 1 to 8 monomers. Thus, the ion-conducting pores constitute a heterogeneous assembly of oligo-oligomers. The correlation between the secondorder rate constants, the affinity constants, and aggregation numbers of peptide units (Table 2) leads to a conclusion that the most efficient pores are formed by oligomers of relatively large aggregates, with z and n values between 6 and 8, such as hexamers of hexamers or octamers of octamers. Data of Figure 3 and Table 2 show that peptide-induced increase in Quin-2 fluorescence never reaches the maximum level caused by the Ca2+ ionophore, which suggests that even though all vesicles had ample amount of bound peptide, even at highest peptide concentrations not all vesicles contained efficient Ca2+ transporting pores. To analyze this conjecture, the number of pores per vesicle has been evaluated. First, the average size of vesicles was 12


Page 12 of 26

Page 13 of 26

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. Kinetics of Quin-2 fluorescence increase, resulting from Ca2+ influx into the vesicles in buffers containing 20 mM Tris-HCl (pH 7.2), 30 mM myo-inositol, 6 mM CaCl2, and NaCl at concentrations 30 mM (a), 75 mM (b), 150 mM (c) and 180 mM (d). Fluorescence of intravesicular Quin-2 was measured for 248 s, then Aβ25-35, pre-incubated in a similar buffer for 2.5 h with stirring, was added at peptide-lipid molar ratios of 1:10 (squares), 1:5 (circles), or 1:3 (triangles) accompanied with consecutive fluorescence measurements. Data of peptide-induced rise in fluorescence are fitted with single exponential lines, using rate constants summarized in Table 2 as kexp. Data presented by rhombs are obtained upon addition of non-fluorescent Ca2+ ionophore 4-Br-A23187. In all cases, the maximum effect induced by 4-Br-A23187 is normalized to 1.0. Membrane lipid composition was 60 mol % POPC, 30 mol % POPG, 10 mol % cholesterol. Total lipid concentration was 0.2 mM.

Figure 4. Dependence of the relative equilibrium intensity of Quin-2 fluorescence (a), single-exponential rate constants describing the increase of Quin-2 fluorescence following addition of the peptide (b), and 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

the second-order rate constant of pore formation (c) on the ionic strength of the buffer. Lipid and peptide concentrations are 0.2 mM and 66.7 µM, respectively. In panel c, solid, dashed, and dotted lines correspond to the number of peptide molecules in the membrane-binding species z = 1, 4, and 8, respectively. All data are presented as mean ± standard deviation from three experiments.

determined. As shown in Figure 5, the vesicle diameter distribution, measured by dynamic light scattering, was presented by a relatively sharp peak centered at 130 nm. At peptide concentrations of 5, 10, 20, 40 and 66.7 µM, the peak upshifted to 137, 145, 162, 190, and 190 nm, respectively. At 40 and 66.7 µM peptide, an additional peak appeared corresponding to larger particles (~2000 nm), at the expense of the initial population of smaller particles. The size of blank vesicles (130 nm), extruded through 100 nm pore-size polycarbonate filters, agrees with earlier measurements.52,52 The elasticity of lipid membranes, which are in liquid-crystalline state, apparently allows passage of vesicles across filters with smaller pores. Addition of increasing concentrations of the cationic peptide to anionic vesicles causes vesicle aggregation, especially at concentrations ≥40 µM. Using the total lipid concentration of 0.2 mM, an average vesicle diameter of 140 nm, and the cross-sectional area per lipid (0.554 nm2, see preceding section), the number of lipids per vesicle is ~2.137×105, corresponding to a “molar” concentration of vesicles of 0.936 nM. At lowest concentrations of membrane-bound peptide, i.e. 2.38 µM, 0.337 µM, and 0.059 µM for monomers, tetramers, and octamers at 300 mM NaCl (Table 2), the numbers of respective assembles per vesicle would be 2540, 360, and 63. At lower NaCl concentrations these numbers would be up to 7-fold greater. If indeed 36 to 64 peptide monomers are required to build a functional pore (hexamers of hexamers or octamers of octamers), then under our experimental conditions each vesicle would contain only a few such structures. Considering the presence of smaller membrane-bound aggregates, which make less effective or ineffective pores, only a fraction of vesicles would contain fully functional pores, which would explain why Frel never reaches 1.0. These data are consistent with earlier studies showing Aβ1-40-indiced Ca2+ influx into anionic vesicles to a level amounting to 0.1 to 0.4 fraction of the maximum effect induced by an antimicrobial peptide .53

14


Page 14 of 26

Page 15 of 26

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. Size distribution of vesicles composed of 60 mol % POPC, 30 mol % POPG, 10 mol % cholesterol in aqueous buffer containing 10 mM NaCl + 20 mM tris-HCl (pH 7.2) at Aβ25-35 peptide concentrations as indicated. Total lipid concentration was 200 µM.

3.3.2. Zwitterionic membranes. To further assess the effect of membrane electrostatics on membrane binding and pore formation by Aβ25-35, microelectrophoresis and Ca2+ flux experiments have been conducted using vesicles composed of 90 mol % POPC and 10 mol % cholesterol. In the absence of the peptide, the vesicles exhibited negative ζ-potential of around -6 mV, corresponding to σ0 = -5.926 mC/m2 (Figure 6a). A relatively small negative surface potential of phosphatidylcholine membranes is routinely observed and ascribed to binding of anions, such as Cl-.44,54,55 The ζ-potential showed little dependence on peptide concentration, suggesting weak binding of Aβ25-35 to zwitterionic membranes. At peptide concentrations exceeding 40 µM, positive ζ-potentials have been recorded, arguably resulting from peptide aggregates in the buffer. Therefore, only data at [P] ≤ 40 µM have been considered. The dependence of ζ-potential on peptide concentration was described by a theoretical binding isotherm (Eq. 10) using same intrinsic KD and NL values corresponding to vesicles with 30 mol % POPG in a buffer containing 75 mM NaCl (Table 1, last row). Theoretical curves generated for z = 1, 4, and 8 were in the range of experimental error of measured ζ-potentials (Figure 6a).

Figure 6. (a): Dependence of ζ-potential of vesicles composed of 90 mol % POPC and 10 mol % cholesterol on Aβ25-35 concentration in aqueous buffer containing 75 mM NaCl and 20 mM Tris-HCl (pH 7.2). Theoretical binding isotherms for peptide aggregation numbers z = 1, 4, and 8, simulated using KD and NL values shown in Table 1 for 75 mM NaCl (last row), are shown as solid, dashed, and dotted lines, respectively. (b): Kinetics of peptide-induced Ca2+ transport across lipid vesicle membranes composed of 90 mol % POPC and 10 mol % cholesterol in 75 mM NaCl, 30 mM myo-inositol, 6 mM CaCl2, 20 mM Tris-HCl (pH 7.2). Procedures and the meanings of the symbols are the same as in Figure 3. Data of peptide-induced rise in fluorescence are fitted with single exponential lines, using rate constants summarized in Table 3 as kexp. Total lipid concentration was 0.2 mM in both panels.

As expected, removal of the anionic lipid from the membranes strongly inhibited the peptide’s ability to induce Ca2+ flux across the membrane; the increase of Quin-2 fluorescence saturated at low values of Frel = 0.0169, 0.0419, and 0.1018 at peptide concentrations of 20 µM, 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

40 µM, and 66.7 µM, respectively (Figure 6b). Parameters describing the kinetics of pore formation and Ca2+ flux are summarized in Table 3. The observed single-exponential rate constant of Ca2+ flux, kexp, was 2-3-fold lower compared to the vesicles with 30 % POPG in 75 mM NaCl (cf. Tables 2 and 3). Membrane-bound peptide concentration significantly decreased in case of POPC/cholesterol membranes, especially for oligomers with z = 4 to 8 (cf. [Pb] values in Tables 2 and 3). This is explained by Eq. 8: the concentration of oligomers is inversely proportional to the aggregation number, z. The Boltzmann factor exp(-zFψ0/RT), which overcompensates this effect by means of electrostatic accumulation of the cationic peptide at the anionic membrane surface (large negative ψ0), becomes negligible in case of zwitterionic membranes (small ψ0). Smaller values of [Pb] yield larger second-order rate constants, ka, and association constants between oligomers, Kp. Peptide oligomers bound to membranes containing POPG might be restrained, in terms of lateral mobility, through ionic and/or H-bonding interactions between Lys28 side chain and POPG head-group, whereas in zwitterionic membranes this constraint would be eliminated, resulting in more efficient peptide-peptide interaction and larger Kp values. Thus, membrane surface electrostatics modulates binding of Aβ25-35, pore formation, and Ca2+ transport. The negative surface potential facilitates membrane binding of the cationic peptide, especially the multiply charged oligomers, which then form the pore assembly. Similar effects of membrane anionic charge on membrane binding, followed by hydrophobic membrane insertion and pore formation, have been reported for Aβ1-40 and Aβ1-42.53,56 In case of zwitterionic lipid membranes, the peptide-membrane Coulombic attraction is diminutive. This limits membrane binding and subsequent pore formation, while lending more lateral mobility to membrane-bound peptides. In addition, Ca2+ concentration is enhanced by the Boltzmann factor at the surface of anionic, but not zwitterionic, membranes, which facilitates Ca2+ transport and thus contributes to larger overall effects in the former case.

3.4. Peptide structure in the presence of lipid vesicles CD spectra of Aβ25-35 were recorded following measurements of Ca2+ flux on same J-810 spectropolarimeter to estimate the in situ peptide structure in its functional, membrane-bound state. These data are presented in Figure 7 along with the second derivatives, which amplify the spectral features and enhance the resolution. The spectra were different from those measured in aqueous buffer in the absence of vesicles (gray lines in Figure 7). In addition to the minimum at

16


Page 16 of 26

Page 17 of 26

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 7. CD spectra (black solid lines) and second derivatives (black dotted lines) of Aβ25-35 in the presence of lipid vesicles composed of POPC, POPG, and cholesterol at 6:3:1 molar ratio, measured at the end of Ca2+ flux measurements, shown in Figure 3, in buffers containing 20 mM Tris-HCl (pH 7.2), 30 mM myo-inositol, 6 mM CaCl2, and NaCl at concentrations 10 mM (a), 30 mM (b), 50 mM (c), 75 mM (d), 150 mM (e), 180 mM (f), 225 mM (g), and 300 mM (h). In each panel, the CD spectra and the second derivatives of the peptide incubated in aqueous buffer for 2.5 hours in the absence of lipid vesicles are shown in gray solid and dotted lines, respectively, as reference. Note that positive features of the second derivatives reflect negative features of the measured spectra. The second derivatives have been amplified 15-fold for better perception.

208 nm, CD spectra of membrane-bound Aβ25-35 displayed additional features at higher wavelengths. In low ionic strength buffers, a shoulder around 221-222 nm was present (Figure 7a,b), implying partial α-helical structure. This is reminiscent of the effect of membrane charge and lipid composition on Aβ structure reported earlier (reviewed in Ref. 56). At intermediate ionic strengths (50 to 150 mM NaCl), the spectra showed a minimum around 211-215 nm (Figure 7c,d,e), indicating predominant β-sheet structure. At higher salt concentrations, the minimum at 208 nm, signifying β-turn structure, again became dominant (Figure 7f,g,h). In high ionic strength buffer, the counterions screen the membrane anionic surface charge and thereby inhibit membrane binding of the cationic peptide and increase the fraction of free peptide. Binding of divalent cations (Ca2+, Mg2+) to anionic membranes has been shown to exert a similar effect on membrane binding and permeabilization by islet amyloid polypeptide.57 Considering that only a fraction of the peptide binds to membranes at all salt concentrations, the CD signal around 208 nm is attributable to the free peptide in the buffer. Furthermore, in view of the fact that the second-order rate constants of pore formation and Ca2+ transport reach highest values at NaCl concentrations of 50-150 mM (Figure 4c), where the peptide assumes β-sheet structure, it is reasonable to conclude that Aβ25-35 in β-sheet conformation forms efficient membrane pores.

4. CONCLUSIONS This work provides a quantitative analysis of membrane binding and pore formation by the Aβ2535 peptide. Both effects are robustly controlled by membrane electrostatics; the anionic membrane surface charge facilitates binding of the cationic peptide and subsequent formation of Ca2+-permeable pores. The peptide assumes β-turn structure in buffer, and membrane binding promotes β-sheet conformation. The data suggest that the membrane binding species are small oligomers, mostly ranging from tetramers to octamers, and that the pore structure comprises up to eight such oligomers. Thus, the pores are oligo-oligomers, such as hexamers of hexamers or octamers of octamers, with relatively large numbers of monomers in the pore. The pore formation is consistent with a model where small β-barrel-like structures bind to the membrane, then cluster by means of lateral diffusion within the membrane. The interior of each unit is likely occluded by peptide side chains, and the pores may be formed between six to eight units. The inner pore diameter is presumably 7 Å or larger to allow for transport of hydrated Ca2+ ions. How do these relatively large oligomeric states of membrane-embedded Aβ25-35 compare with earlier Aβ pore models? Octameric Aβ25-35 channels, in β-barrel or α-helical conformations, have been modeled.19,20,33,37,38 A detailed analysis of Aβ25-35 structure suggested the presence of 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

larger oligomeric structures, e.g., multimers of tetramers that could form membrane pores.34 Octadecameric and eicosameric β-barrel membrane pore models for Aβ1-42, Aβ9-42, Aβ17-42 , and an N-terminally truncated and pyroglutamylated Aβ peptide have been modeled by MD methods.58,59 Hexamers of 6-stranded β-barrels of Aβ1-42, with 36 monomers in the assembly, have been modeled to form ion conducting pores in cell membranes.60 Aβ Arctic mutant has been reported to form pore-like structures composed of 40-60 molecules.61 Further MD studies suggested formation of tetramers or pentamers of 16-stranded β-barrels of Aβ9-42 and Aβ17-42, with 64-80 peptide monomers in the pore.62 These results are in line with our findings suggesting pore formation by oligomerization of oligomeric peptide assemblies. Detailed biophysical characterization of membrane pores formed by cytotoxic peptides, such as full-length Aβ or its fragments, may help elucidate the mechanism of toxicity and develop therapeutic means to combat Alzheimer’s and other diseases.

ACKNOWLEDGMENTS Financial support from Florida Department of Health, Ed and Ethel Moore Alzheimer’s Disease Research Program (grant 7AZ27), is gratefully acknowledged. The authors are thankful to Greta Apostoli for her assistance with some of membrane permeabilization experiments and to Greg Goldblatt for his help with FTIR measurements.

REFERENCES 1. Selkoe, D. J.; Hardy, J. The Amyloid Hypothesis of Alzheimer's Disease at 25 Years. EMBO Mol. Med. 2016, 8, 595–608. 2. Chow, V. W.; Mattson, M. P.; Wong, P. C.; Gleichmann, M. An Overview of APP Processing Enzymes and Products. Neuromolecular Med. 2010, 12, 1–12. 3. Agostinho, P.; Pliássova, A.; Oliveira, C. R.; Cunha, R. A. Localization and Trafficking of Amyloid-β Protein Precursor and Secretases: Impact on Alzheimer's Disease. J. Alzheimers Dis. 2015, 45, 329–347. 4. Benilova, I.; Karran, E.; De Strooper, B. The Toxic Aβ Oligomer and Alzheimer's Disease: An Emperor in Need of Clothes. Nat. Neurosci. 2012, 15, 349–57. 5. Kubo, T.; Nishimura, S.; Kumagae, Y.; Kaneko, I. In-Vivo Conversion of Racemized BetaAmyloid ([D-Ser 26]A Beta 1-40) to Truncated and Toxic Fragments ([D-Ser 26]A Beta 2535/40) and Fragment Presence in the Brains of Alzheimer's Patients. J. Neurosci. Res. 2002, 70, 474–483. 6. Cuevas, E.; Lantz, S. M.; Tobón-Velasco, J. C.; Newport, G. D.; Wu, Q.; Virmani, A.; Ali, S. F.; Santamaría, A. On the In-Vivo Early Toxic Properties of A-Beta 25-35 Peptide in the Rat Hippocampus: Involvement of the Receptor-For-Advanced Glycation-End-Products and Changes in Gene Expression. Neurotoxicol. Teratol. 2011, 33, 288–296. 7. Behensky, A. A.; Yasny, I. E.; Shuster, A.M.; Seredenin, S. B.; Petrov, A. V.; Cuevas, J. Stimulation of Sigma Receptors with Afobazole Blocks Activation of Microglia and Reduces Toxicity Caused by Amyloid-β 25-35. J. Pharmacol. Exp. Ther. 2013, 347, 458–467.

18


Page 18 of 26

Page 19 of 26

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


8. Behensky, A. A.; Yasny, I. E.; Shuster, A. M.; Seredenin, S. B.; Petrov, A. V.; Cuevas, J. Afobazole Activation of σ-1 Receptors Modulates Neuronal Responses to Amyloid-β 25-35. J. Pharmacol, Exp, Ther. 2013, 347, 468–477. 9. da Luz Eltchechem, C.; Salgado, A. S.; Zângaro, R. A.; da Silva Pereira, M. C.; Kerppers, II.; da Silva, L. A.; Parreira, R. B. Transcranial LED Therapy on Amyloid-β Toxin 25-35 in the Hippocampal Region of Rats. Lasers Med. Sci. 2017, 32, 749–756. 10. Li, J.; Ji, X.; Zhang, J.; Shi, G.; Zhu, X.; Wang, K. Paeoniflorin Attenuates Aβ 25-35Induced Neurotoxicity in PC12 Cells by Preventing Mitochondrial Dysfunction. Folia Neuropathol. 2014, 52, 285–290. 11. Li, L.; Du, J.; Zou, L.; Xia, H.; Wu, T.; Kim, Y.; Lee, Y. The Neuroprotective Effects of Decursin Isolated from Angelica gigas Nakai against Amyloid β-Protein-Induced Apoptosis in PC 12 Cells via a Mitochondria-Related Caspase Pathway. Neurochem. Res. 2015, 40, 1555–1562. 12. Lahmy, V.; Long, R.; Morin, D.; Villard, V.; Maurice, T. Mitochondrial Protection by the Mixed Muscarinic/σ1 Ligand ANAVEX2-73, a Tetrahydrofuran Derivative, in Aβ25–35 Peptide-Injected Mice, a Nontransgenic Alzheimer’s Disease Model. Front. Cell. Neurosci. 2014, 8, 463, 1–11. 13. Chang, C. P.; Liu, Y. F.; Lin, H. J.; Hsu, C. C.; Cheng, B. C.; Liu, W. P.; Lin, M. T.; Hsu, S. F.; Chang, L. S.; Lin, K. C. Beneficial Effect of Astragaloside on Alzheimer's Disease Condition Using Cultured Primary Cortical Cells under β-Amyloid Exposure. Mol. Neurobiol. 2016, 53, 7329–7340. 14. Moreira, P. I.; Santos, M. S.; Moreno, A.; Oliveira, C. Amyloid Beta-Peptide Promotes Permeability Transition Pore in Brain Mitochondria. Biosci. Rep. 2001, 21, 789–800. 15. Ren, R.; Zhang, Y.; Li, B.; Wu, Y.; Li, B. Effect of β-Amyloid (25-35) on Mitochondrial Function and Expression of Mitochondrial Permeability Transition Pore Proteins in Rat Hippocampal Neurons. J. Cell. Biochem. 2011, 112, 1450–1457. 16. Mirzabekov, T.; Lin, M. C.; Yuan, W. L.; Marshall, P. J.; Carman, M.; Tomaselli, K.; Lieberburg, I.; Kagan, B. L. Channel Formation in Planar Lipid Bilayers by a Neurotoxic Fragment of the Beta-Amyloid Peptide. Biochem. Biophys. Res. Commun. 1994, 202, 1142– 1148. 17. Hertel, C.; Terzi, E.; Hauser, N.; Jakob-Rotne, R.; Seelig, J.; Kemp, J. A. Inhibition of the Electrostatic Interaction Between Beta-Amyloid Peptide and Membranes Prevents BetaAmyloid-Induced Toxicity. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 9412–9416. 18. Lin, M. C.; Kagan, B. L. Electrophysiologic Properties of Channels Induced by A-Beta 25-35 in Planar Lipid Bilayers. Peptides 2002, 23, 1215–1228. 19. Di Scala, C.; Troadec, J. D.; Lelièvre, C.; Garmy, N.; Fantini, J.; Chahinian, H. Mechanism of Cholesterol-Assisted Oligomeric Channel Formation by a Short Alzheimer β-Amyloid Peptide. J. Neurochem. 2014, 128, 186–195. 20. Di Scala, C.; Chahinian, H.; Yahi, N.; Garmy, N.; Fantini, J. Interaction of Alzheimer's βAmyloid Peptides with Cholesterol: Mechanistic Insights into Amyloid Pore Formation. Biochemistry 2014, 53, 4489–4502. 21. Peters, C.; Bascuñán, D.; Opazo, C.; Aguayo, L. G. Differential Membrane Toxicity of Amyloid-β Fragments by Pore Forming Mechanisms. J. Alzheimers Dis. 2016, 51, 689–699. 22. Terzi, E.; Hölzemann, G.; Seelig, J. Alzheimer Beta-Amyloid Peptide 25-35: Electrostatic Interactions with Phospholipid Membranes. Biochemistry 1994, 33, 7434–7441.

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

23. Ghobeh, M.; Ahmadian, S.; Meratan, A. A.; Ebrahim-Habibi, A.; Ghasemi, A.; Shafizadeh, M.; Nemat-Gorgani, M. Interaction of Aβ(25-35) Fibrillation Products with Mitochondria: Effect of Small-Molecule Natural Products. Biopolymers 2014, 102, 473–486. 24. Accardo, A.; Shalabaeva, V.; Cotte, M.; Burghammer, M.; Krahne, R.; Riekel, C.; Dante, S. Amyloid β Peptide Conformational Changes in the Presence of a Lipid Membrane System. Langmuir 2014, 30, 3191–3198. 25. Labbé, J. F.; Lefèvre, T.; Guay-Bégin, A. A.; Auger, M. Structure and Membrane Interactions of the β-Amyloid Fragment 25-35 as Viewed Using Spectroscopic Approaches. Phys. Chem. Chem. Phys. 2013, 15, 7228–7239. 26. Mason, R. P.; Estermyer, J. D.; Kelly, J. F.; Mason, P. E. Alzheimer's Disease Amyloid Beta Peptide 25-35 is Localized in the Membrane Hydrocarbon Core: X-ray Diffraction Analysis. Biochem Biophys. Res. Commun. 1996, 222, 78–82. 27. Dante, S.; Hauss, T.; Dencher, N. A. Insertion of Externally Administered Amyloid Beta Peptide 25-35 and Perturbation of Lipid Bilayers. Biochemistry 2003, 42, 13667–13672. 28. Dante, S.; Hauss, T.; Dencher, N. A. Cholesterol Inhibits the Insertion of the Alzheimer's Peptide Abeta(25-35) in Lipid Bilayers. Eur. Biophys. J. 2006, 35, 523–531. 29. Vitiello, G.; Di Marino, S.; D'Ursi, A. M.; D'Errico, G. Omega-3 Fatty Acids Regulate the Interaction of the Alzheimer's Aβ(25-35) Peptide with Lipid Membranes. Langmuir 2013, 29, 14239–14245. 30. Dante, S.; Hauss, T.; Dencher, N. A. β-Amyloid 25-35 Is Intercalated in Anionic and Zwitterionic Lipid Membranes to Different Extents. Biophys. J. 2002, 83, 2610–2616. 31. Dies, H.; Toppozini, L.; Rheinstädter, M. C. The Interaction Between Amyloid-β Peptides and Anionic Lipid Membranes Containing Cholesterol and Melatonin. PLoS One. 2014, 9, e99124, 1-17. 32. Lau, T. L.; Gehman, J. D.; Wade, J. D.; Perez, K.; Masters, C. L.; Barnham, K. J.; Separovic, F. Membrane Interactions and the Effect of Metal Ions of the Amyloidogenic Fragment Abeta(25-35) in Comparison to Abeta(1-42). Biochim. Biophys. Acta 2007, 1768, 2400– 2408. 33. Chang, Z.; Luo, Y.; Zhang, Y.; Wei, G. Interactions of Aβ 25-35 β-Barrel-Like Oligomers with Anionic Lipid Bilayer and Resulting Membrane Leakage: An All-Atom Molecular Dynamics Study. J. Phys. Chem. B 2011, 115, 1165–1174. 34. Do, T. D.; LaPointe, N. E.; Nelson, R.; Krotee, P.; Hayden, E. Y.; Ulrich, B.; Quan, S.; Feinstein, S. C.; Teplow, D. B.; Eisenberg, D. et al. Amyloid β-Protein C-Terminal Fragments: Formation of Cylindrins and β-Barrels. J. Am. Chem. Soc. 2016, 138, 549–557. 35. Tsai, H. H.; Lee, J. B.; Tseng, S. S.; Pan, X. A.; Shih, Y. C. Folding and Membrane Insertion of Amyloid-Beta (25-35) Peptide and its Mutants: Implications for Aggregation and Neurotoxicity. Proteins 2010, 78, 1909–1925. 36. Tsai, H. H.; Lee, J. B.; Shih, Y. C.; Wan, L.; Shieh, F. K.; Chen, C. Y. Location and Conformation of Amyloid β(25-35) Peptide and its Sequence-Shuffled Peptides within Membranes: Implications for Aggregation and Toxicity in PC12 Cells. ChemMedChem. 2014, 9, 1002–1011. 37. Di Scala, C.; Yahi, N.; Lelièvre, C.; Garmy, N.; Chahinian, H.; Fantini, J. Biochemical Identification of a Linear Cholesterol-Binding Domain within Alzheimer's β Amyloid Peptide. ACS Chem. Neurosci. 2013, 4, 509–517.

20


Page 20 of 26

Page 21 of 26

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


38. Fantini, J.; Di Scala, C.; Yahi, N.; Troadec, J. D.; Sadelli, K.; Chahinian, H.; Garmy, N. Bexarotene Blocks Calcium-Permeable Ion Channels Formed by Neurotoxic Alzheimer's βAmyloid Peptides. ACS Chem. Neurosci. 2014, 5, 216–224. 39. Kohno, T.; Kobayashi, K.; Maeda, T.; Sato, K.; Takashima, A. Three-Dimensional Structures of the Amyloid Beta Peptide (25-35) in Membrane-Mimicking Environment. Biochemistry 1996, 35, 16094–16104. 40. D'Ursi, A. M.; Armenante, M. R.; Guerrini, R.; Salvadori, S.; Sorrentino, G.; Picone, D. Solution Structure of Amyloid Beta-Peptide (25-35) in Different Media. J. Med. Chem. 2004, 47, 4231–4238. 41. Coles, M.; Bicknell, W.; Watson, A. A.; Fairlie, D. P.; Craik, D. J. Solution Structure of Amyloid Beta-Peptide(1-40) in a Water-Micelle Environment. Is the Membrane-Spanning Domain Where We Think It Is? Biochemistry 1998, 37, 11064–11077. 42. Goldblatt, G.; Matos, J. O.; Gornto, J.; Tatulian, S. A. Isotope-Edited FTIR Reveals Distinct Aggregation and Structural Behaviors of Unmodified and Pyroglutamylated Amyloid β Peptides. Phys. Chem. Chem. Phys. 2015, 17, 32149–32160. 43. Garg, P.; Nemec, K. N.; Khaled, A. R.; Tatulian, S. A. Transmembrane Pore Formation by the Carboxyl Terminus of Bax Protein. Biochim. Biophys. Acta. 2013, 1828, 732–742. 44. Tatulian, S. A. Surface Electrostatics of Biological Membranes and Ion Binding. In Surface Chemistry and Electrochemistry of Membranes; Sørensen, T. S., Ed.; Marcel Dekker: New York, NY, 1999; pp. 871–922. 45. Qin, S.; Pande, A. H.; Nemec, K. N.; Tatulian, S. A. The N-terminal α-Helix of Pancreatic Phospholipase A2 Determines Productive-Mode Orientation of the Enzyme at the Membrane Surface. J. Mol. Biol. 2004, 344, 71–89. 46. Tatulian, S. A. Structural Characterization of Membrane Proteins and Peptides by FTIR and ATR-FTIR Spectroscopy. Methods Mol. Biol. 2013, 974, 177–218. 47. Venyaminov, S. Yu.; Yang, J. T. Determination of Protein Secondary Structure. In Circular Dichroism and the Conformational Analysis of Biomolecules; Fasman, G. D., Ed.; Plemun Press: New York and London, 1996, pp. 69–107. 48. Perczel, A.; Hollósi, M. Turns. In Circular Dichroism and the Conformational Analysis of Biomolecules. Fasman, G. D., Ed.; Plemun Press: New York and London, 1996, pp. 285– 380. 49. Alwarawrah, M.; Dai, J.; Huang, J. A Molecular View of the Cholesterol Condensing Effect in DOPC Lipid Bilayers. J. Phys. Chem. B. 2010, 114, 7516–7523. 50. Leftin, A.; Molugu, T. R.; Job, C.; Beyer, K.; Brown, M. F. Area Per Lipid and Cholesterol Interactions in Membranes from Separated Local-Field 13C NMR Spectroscopy. Biophys. J. 2014, 107, 2274–2286. 51. Shabbits, J. A.; Chiu, G. N. C.; Mayer, L. D. Development of an In Vitro Drug Release Assay that Accurately Predicts in vivo Drug Retention for Liposome-Based Delivery Systems. J. Control. Release 2002, 84, 161–170. 52. Lunelli, L.; Pasquardini, L.; Pederzolli, C.; Vanzetti, L.; Anderle, M. Covalently Anchored Lipid Structures on Amine-Enriched Polystyrene. Langmuir 2005, 21, 8338–8343. 53. Sciacca, M. F.; Kotler, S. A.; Brender, J. R.; Chen, J.; Lee, D. K.; Ramamoorthy, A. TwoStep Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation. Biophys. J. 2012, 103, 702–710. 54. Manzini, M. C.; Perez, K. R.; Riske, K. A.; Bozelli, J. C. Jr.; Santos, T. L.; da Silva, M. A.; Saraiva, G. K.; Politi, M. J.; Valente, A. P.; Almeida, F. C. et al. Peptide:Lipid Ratio and 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

Membrane Surface Charge Determine the Mechanism of Action of the Antimicrobial Peptide BP100. Conformational and functional studies. Biochim. Biophys. Acta 2014, 1838, 1985– 1999. 55. Petrache, H. I.; Zemb, T.; Belloni, L.; Parsegian, V. A. Salt Screening and Specific Ion Adsorption Determine Neutral-Lipid Membrane Interactions. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7982–7987. 56. Kotler, S. A.; Walsh, P.; Brender, J. R.; Ramamoorthy, A. Differences between Amyloid-β Aggregation in Solution and on the Membrane: Insights into Elucidation of the Mechanistic Details of Alzheimer's Disease. Chem. Soc. Rev. 2014, 43, 6692–6700. 57. Sciacca, M. F.; Milardi, D.; Messina, G. M.; Marletta, G.; Brender, J. R.; Ramamoorthy, A.; La Rosa, C. Cations as Switches of Amyloid-Mediated Membrane Disruption Mechanisms: Calcium and IAPP. Biophys. J. 2013, 104, 173–184. 58. Jang, H.; Arce, F. T.; Ramachandran, S.; Capone, R.; Lal, R.; Nussinov, R. β-Barrel Topology of Alzheimer's β-Amyloid Ion Channels. J. Mol. Biol. 2010, 404, 917–934. 59. Gillman, A. L.; Jang, H.; Lee, J.; Ramachandran, S.; Kagan, B. L.; Nussinov, R.; Teran Arce, F. Activity and Architecture of Pyroglutamate-Modified Amyloid-β (AβpE3-42) Pores. J. Phys. Chem. B 2014, 118, 7335–7344. 60. Shafrir, Y.; Durell, S.; Arispe, N.; Guy, H. R. Models of Membrane-Bound Alzheimer's Abeta Peptide Assemblies. Proteins 2010, 78, 3473–3487. 61. Lashuel, H. A.; Hartley, D.; Petre, B. M.; Walz, T.; Lansbury, P. T. Jr. Neurodegenerative Disease: Amyloid Pores from Pathogenic Mutations. Nature. 2002, 418, 291. 62. Jang, H.; Connelly, L.; Arce, F. T.; Ramachandran, S.; Kagan, B. L.; Lal, R.; Nussinov, R. Mechanisms for the Insertion of Toxic, Fibril-like β-Amyloid Oligomers into the Membrane. J. Chem. Theory Comput. 2013, 9, 822–833.

22


Page 22 of 26

Page 23 of 26

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


Table 1 Parameters characterizing membrane binding of Aβ25-35 to unilamellar lipid vesicles composed of 60 mol % POPC, 30 mol % POPG, 10 mol % cholesterol in aqueous buffer containing 20 mM Tris-HCl (pH 7.2), 30 mM myo-inositol, and NaCl at concentrations 10 mM , 30 mM, or 75 mM, as indicated. Standard deviations of all parameters, based on three independent experiments, ranged from 6 % to 17 % of mean values.

NaCl (mM) 10 30 75

σ0 (mC/m2) -21.19 -22.70 -25.61

z=1 KD (M) NL -5 26.3 2.5×10 -5 40.5 2.5×10 -5 33.5 4.4×10

z=4 KD (M) 1.6×10-4 1.6×10-4 2.0×10-4

NL 105.1 161.8 134.0

23


z=8 KD (M) 5.6×10-3 5.6×10-3 4.2×10-3

NL 210.2 323.6 268.0


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

Page 24 of 26

Table 2 Parameters characterizing membrane pore formation by A25-35 in unilamellar lipid vesicles composed of 60 mol % POPC, 30 mol % POPG, 10 mol % cholesterol in aqueous buffer containing 20 mM Tris-HCl (pH 7.2), 30 mM myo-inositol, and NaCl at varying concentrations. Standard deviations of all parameters, based on three independent experiments, ranged from 3 % to 21 % of mean values.

[NaCl] (mM) 10 10 10 30 30 30 50 50 50 75 75 75 150 150 150 180 225 300

[P] (M) 20 40 66.7 20 40 66.7 20 40 66.7 20 40 66.7 20 40 66.7 66.7 66.7 66.7

Frel

kexp (s-1)

0.1662 0.5094 0.6388 0.1691 0.3564 0.5263 0.1249 0.2779 0.4129 0.1123 0.2137 0.3530 0.0898 0.1244 0.1955 0.1089 0.1758 0.163

5.1810-2 2.6910-2 2.3010-2 1.1410-2 1.8510-2 2.2810-2 4.3310-2 2.5010-2 3.1710-2 2.0410-2 3.6610-2 3.0610-2 1.1910-2 1.6310-2 1.2610-2 3.0010-3 6.1810-4 4.4110-4

z=1 2.86 3.27 3.50 1.88 2.16 2.30 2.01 2.41 2.64 1.76 2.21 2.49 1.75 2.22 2.50 2.47 2.41 2.38

[Pb] (M) z=4 0.766 0.834 0.875 0.515 0.563 0.589 0.523 0.600 0.648 0.493 0.577 0.630 0.315 0.416 0.489 0.459 0.381 0.337

z=8 0.400 0.423 0.439 0.276 0.292 0.301 0.269 0.299 0.319 0.271 0.303 0.323 0.107 0.145 0.174 0.147 0.088 0.059

z=1 5.907103 5.925103 1.255104 1.693104 2.942104 2.923104 6.527104 1.393104 2.452104 2.631104 4.563104 3.297104 1.776104 1.313104 1.296104 2.561103 4.103102 2.385102

ka (M-1s-1) z=4 2.206104 2.323104 5.020104 6.178104 1.129105 1.142105 2.508104 5.596104 9.990104 9.391104 1.748105 1.303105 9.871104 7.010104 6.626104 1.381104 2.595103 1.684103

1


z=8 4.224104 4.581104 1.001105 1.153105 2.176105 2.234105 4.877104 1.123105 2.026105 1.708105 3.328105 2.542105 2.906105 2.011105 1.862105 4.304104 1.121104 9.538103

z=1 1.141105 2.199105 5.458105 1.489106 1.593106 1.282106 1.508105 5.577105 7.739105 1.287106 1.247106 1.076106 1.491106 8.041105 1.028106 8.533105 6.641105 5.407105

Kp (M-1) z=4 4.258105 8.623105 2.183106 5.437106 6.114106 5.004106 5.797105 2.240106 3.153106 4.595106 4.777106 4.255106 8.285106 4.291106 5.256106 4.603106 4.201106 3.819106

n z=8 8.155105 1.700106 4.351106 1.014107 1.179107 9.792106 1.127106 4.495106 6.406106 8.359106 9.096106 8.300106 2.439107 1.231107 1.477107 1.434107 1.185107 2.163107

3.4 2.6 3.1 8.2 6.3 4.4 3.6 4.8 4.6 8.6 7.4 5.7 9.8 7.4 7.5 8.4 6.3 5.9

Page 25 of 26

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


Table 3 Parameters characterizing membrane pore formation by A25-35 in unilamellar vesicles composed of 90 mol % POPC and 10 mol % cholesterol in aqueous buffer containing 75 mM NaCl, 30 mM myo-inositol, 6 mM CaCl2, 20 mM Tris-HCl (pH 7.2). [P] (M) 20 40 66.7

Frel

kexp (s-1)

1.69710-2 4.19710-2 1.01810-1

1.05810-2 1.31410-2 1.05110-2

z=1 1.05100 1.52100 1.88100

[Pb] (M) z=4 5.7310-2 9.8010-2 1.3910-1

z=8 3.0110-3 5.8210-3 9.2910-3

z=1 7.575103 7.684103 7.082103

ka (M-1s-1) z=4 1.388105 1.192105 9.580104

1


z=8 2.642106 2.007106 1.434106

Kp (M-1) z=1 z=4 7.159105 1.312107 5.863105 9.053106 6.737105 9.114106

n z=8 2.497108 1.525108 1.364108

8.1 7.3 6.8


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

TOC Graphic


Page 26 of 26

Membrane Binding and Pore Formation by a Cytotoxic Fragment of

Recommend Documents