Evidence for Inhibition of Lysozyme Amyloid Fibrillization by Peptide

Subscriber access provided by BOSTON UNIV

Article

Evidence for Inhibition of Lysozyme Amyloid Fibrillization by Peptide Fragments from Human Lysozyme: A Combined Spectroscopy, Microscopy and Docking Study Rajiv K Kar, Zuzana Gazova, Zuzana Bednarikova, Kamal H. Mroue, Anirban Ghosh, Ruiyan Zhang, Katarina Ulicna, Hans-Christian Siebert, Nikolay E. Nifantiev, and Anirban Bhunia Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00165 • Publication Date (Web): 26 Apr 2016 Downloaded from http://pubs.acs.org on April 27, 2016

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.

Biomacromolecules 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 39

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

Biomacromolecules

Evidence for Inhibition of Lysozyme Amyloid Fibrillization by Peptide Fragments from Human Lysozyme: A Combined Spectroscopy, Microscopy and Docking Study

Rajiv K. Kar,a Zuzana Gazova,b,c Zuzana Bednarikova,b,d Kamal H. Mroue,e Anirban Ghosh,a Ruiyan Zhang,f Katarina Ulicna,b,g Hans-Christian Siebert,f Nikolay E. Nifantiev,h Anirban Bhuniaa,*

a

Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India b Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Kosice, Slovakia c Department of Medical and Clinical Biochemistry Faculty of Medicine, Safarik University, Trieda SNP 1, 040 11 Kosice, Slovakia d Department of Biochemistry, Institute of Chemistry, Faculty of Science, Safarik University, Srobarova 2, 041 54 Kosice, Slovakia e Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA f RI-B-NT Research Institute of Bioinformatics and Nanotechnology, Franziusallee 177, 24148 Kiel, Germany g Institute of Biology and Ecology, Faculty of Science, Safarik University, Srobarova 2, 041 54 Kosice, Slovakia h N. D. Zellinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation

*To whom correspondence should be addressed: Dr. Anirban Bhunia. E-mail: [email protected] ; [email protected]

1 ACS Paragon Plus Environment

Biomacromolecules

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 Degenerative diseases such as Alzheimer's and prion diseases, as well as type II diabetes, have a pathogenesis associated with protein misfolding, which routes with amyloid formation. Recent strategies for designing small-molecule and polypeptide anti-amyloid inhibitors are mainly based on mature fibril structures containing cross β-sheet structures. In the present study, we have tackled the hypothesis that the rational design of anti-amyloid agents that can target native proteins might offer advantageous prospect to design effective therapeutics. Lysozyme amyloid fibrillization was treated with three different peptide fragments derived from lysozyme protein sequence R107-R115. Using low-resolution spectroscopic, high-resolution NMR, and STD NMR-restrained docking methods such as HADDOCK, we have found that these peptide fragments have the capability to affect lysozyme fibril formation. The present study implicates the prospect that these peptides can also be tested against other amyloid-prone proteins to develop novel therapeutic agents.

Keywords: Amyloid aggregation, Lysozyme, Amyloid inhibitor, Fluorescence, Atomic Force Microscopy (AFM), STDNMR, Simulated Annealing, HADDOCK


Page 2 of 39

Page 3 of 39

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

Biomacromolecules

INTRODUCTION Amyloid fibrillization of proteins is the responsible factor for causing severe diseases that affect the quality of life for millions of people worldwide. Typically, these include complications like type II diabetes, Alzheimer’s and prion diseases.1 The molecular basis of these diseases is routed by the protein misfolding and formation of ordered β-sheet structures.2,3 Despite various efforts, the detailed pathway of amyloidogenesis is still far from complete understanding.4 The structural characteristics of assembled amyloid fibrils include similar ultrastructures, identical biochemical properties, pronounced hydrophobic features, and enhanced fluorescence of the dye Thioflavin T (ThT) upon binding.5 On the contrary, the biochemical properties of native proteins are not homologous or similar to each other by any means.5,6 Human lysozyme is a small globular protein with a 129 amino-acid sequence and four disulphide linkages, which has been extensively used as an in vitro model for the study of protein amyloidogenesis.7 Under conditions such as low pH and high temperature, lysozyme has the ability to undergo structural changes leading to the formation of amyloid fibrils in a two-state transition pathway.8,9 The formed fibrils have typical features of cross β-sheet morphology that are well characterized using deep UV resonance Raman spectroscopy, transmission electron microscopy, and fluorescence spectroscopy using ThT.10,11 In particular, these fibrils have hydrophobic packing-like structures that are oriented either in parallel or antiparallel fashion, similar to that of steric zipper arrangement.12 It is worth mentioning that although valuable atomic-level insights on native lysozyme and fibril structures have been obtained, the exact pathway of protein misfolding is still poorly understood.4 The reason is related to short lifetime of transition state conformations that are difficult to observe by NMR and X-ray techniques.13,14 On the other hand, low-resolution spectroscopic methods have been used to analyse various characteristics such as secondary 3 ACS Paragon Plus Environment

Biomacromolecules

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

structure, fluorescence properties, amino-acid side chain orientation, and hydrodynamic radius.15,16 Similarly, valuable information regarding the association and the conformational transition of native protein to amyloid form has been obtained using computational tools to support the experimental evidence.17 These insights are useful for determination of the molecular basis of amyloidogenesis by providing additional information on various molten globular state and transition state conformations like protofibrils, metastable state, and oligomeric state.18 The motivation behind these efforts lies in the attempt to find suitable candidates (drugs, polypeptides, and naturally occurring biomolecules) that can inhibit or decelerate amyloidogenesis.19,20 Several applications of polypeptide agents with anti-amyloid properties have been reported in the literature, whereby these agents are designed based on their properties like βsheet binders/breaker and hydrophobicity.21,22 Though the selection of random peptide inhibitory sequence is found to be valuable in many cases, the bulk approach is based merely on rational designing.23 Several of these rationalization techniques involve selection of internal segment of fibril-forming peptides,24 insertion of proline residues or substitution of amino acids,25 modification of side chain in fibrillogenic motifs,26 modification of peptide termini,27 cyclization of peptides,28 insertion of D-amino acids,29 and covalent attachment of modified residues which mimic β-sheets.30 In this study, we have investigated the ability of three short lysozyme-derived peptides (Scheme I) (henceforth referred to as Lz-peptides) to affect formation of lysozyme amyloid fibrils. The amino acid sequences of the studied Lz-peptides correspond to either a primary amino acid sequence of the amyloidogenic part of the human lysozyme R107-R115 (Lzpeptide),or to a

modification by the point mutation to change the charge and/or

hydrophobicity of the peptide (LzK and LzKW peptides). The interference of the studied peptides with formation of lysozyme amyloid aggregates has been examined using 4 ACS Paragon Plus Environment

Page 4 of 39

Page 5 of 39

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

Biomacromolecules

experimental and computational methods. The obtained results indicate that lysozyme peptide fragments have the capability to affect lysozyme fibril formation due to their affinity to interact with monomer/multi-mer lysozyme. This inhibitory effect at an atomic resolution can pave the way for further development of therapeutic agents for lysozyme or other amyloidogenic proteins.

Experimental Section Materials. Human lysozyme (E.C. 3.2.1.17), NaCl, NaH2PO4.2H2O and Thioflavin T were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were obtained from Sigma or Fluka with analytical grade. Lz-peptides. Designed peptides were prepared in a solid phase peptide synthesizer using routine Fmoc chemistry (Aapptec Endeavor 90). The peptides were then purified with reverse phase HPLC system (Shimadzu, Japan) using C18 column by linear gradient elution method. Dual solvent system (water and acetonitrile) with 0.1% trifluoroacetic acid (TFA) was used for purification. The molecular weight and purity level of the peptides were confirmed using MALDI-TOF. Reagents such as deuterium oxide (D2O) and 4,4-dimethyl-4-silapentane-1sulfonic acid (DSS) were purchased from Cambridge Isotope Laboratories, Inc (Tewksbury, MA). Human Lysozyme Amyloid Fibrillization. Lysozyme was dissolved in a 0.5 ml microcentrifuge tube (Eppendorf tube) containing citric-phosphate buffered saline (0.1 M citric acid, 0.2 M Na2HPO4, and 100 mM NaCl), pH 2.8 to the final concentration of 10 µM and then incubated for 2h at 65⁰C and 1200 rpm. Formation of amyloid fibrils was confirmed using Thioflavin T assay and atomic force microscopy (AFM).


Biomacromolecules

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

Thioflavin T (ThT) Fluorescence Assay. Thioflavin T (ThT) is a dye used to visualize and quantify the amyloid fibrilization of proteins, as the fluorescence intensity of ThT is increased in the presence of fibrillar amyloid aggregates. ThT was added to the lysozyme samples (10 µM) to a final concentration of 20 µM. Measurements of fluorescence were performed in a 96-well plate by a Synergy MX (BioTek) spectrofluorimeter. The excitation was set at 440 nm, and the emission was recorded at 485 nm.31 The excitation and emission slits were adjusted to 9.0/9.0 nm and the top probe vertical offset was 6 mm. All experiments were performed in triplicate, and the final value represents the average of measured values. Effect of Lz-peptides on Human Lysozyme Fibrillization. The ability of Lz-peptides (Lz, LzK, LzKW) (Scheme 1) to inhibit the human lysozyme from forming amyloid aggregates was determined for Lz-peptides in concentration range (10 pM - 1 mM) at fixed 10 µM lysozyme concentration. The samples were then exposed to conditions leading to the formation of amyloid fibrils described above. The extent of lysozyme fibrillization was observed by the ThT assay. The fluorescence intensities were normalized to the fluorescence signal of amyloid fibrils formed in the absence of Lz-peptides. Each experiment was performed in triplicate, and the final fluorescence value is the average of the measured values. Atomic Force Microscopy. Samples of human lysozyme alone and in the presence of Lzpeptides were dropped on a freshly cleaned mica surface. After 5 min of adsorption, the surface was washed multiple times with ultra-pure water, and the samples were left to dry in air. Unfiltered images were collected using a Scanning Probe Microscope (Veeco di Innova, Bruker AXS Inc., Madison) in a tapping mode under ambient conditions, using uncoated silicon cantilevers NCHV (Bruker AFM Probes, Camarillo) with nominal resonance frequency 320 kHz and spring constant 42 N/m.


Page 6 of 39

Page 7 of 39

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

Biomacromolecules

Intrinsic Fluorescence Spectroscopy. The excitation-emission properties of sensitive probes are a useful tool to monitor the interaction of the ligand with the macromolecule.32 The presence of Trp residue in all peptide fragments thus helps in accounting for the biophysical estimation of binding studies by acting as an intrinsic fluorescence probe.33 All fluorescence experiments were performed using Hitachi F-7000 FL spectrometer, using quartz cuvette of 0.1 cm. The experimental condition was set at a temperature of 25oC, with excitation/emission slits fixed at 5 nm. All peptides, lysozyme monomer, and fibrils were prepared in citric-phosphate buffered saline buffer at pH 2.8. The excitation wavelength was set to 280 nm and emission was fixed within a range of 300-400 nm. Increasing concentration of lysozyme monomers was used for titration as per the requirement of experiments, and is explained thereafter in a subsequent section. Estimate of binding constants (KD – equilibrium dissociation constant) was measured with anisotropic values, using a standard single-site binding curve fitting method (Equation 1).34 Here, f is the fractional saturation of peptide with respect to lysozyme monomers, L denotes the concentration of monomer added in µM, and KD represents the equilibrium dissociation constant in µM. f = Bmax× L x (KD + L)-1

Equation 1

Anisotropy was recorded using polarized accessory which is also based on intrinsic fluorescence property of Trp residue.34 Equation 2 was used to obtain fluorescence anisotropy values (r), where IVV and IVH are the vertical and horizontally polarized components with respect to excitation by vertically polarized light at 280 nm. G denotes the sensitivity factor of the instrument. 5 µM concentration of each peptide was titrated with an increasing concentration of lysozyme monomer from 0-25 µM. r= (IVV – G×IVH)/(IVV+ 2 ×G×IVH)

Equation 2


Biomacromolecules

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

Quenching experiments for Lz-peptides were performed in both free as well as bound form to monomers, with addition of increasing concentration of acrylamide from 0.0 to 0.5 M. The obtained spectra were further used for calculating Stern-Volmer’s constant (KSV), using Equation 3, where F0 is the collected fluorescence intensity in the absence of quencher; F is the fluorescence intensity obtained with the successive addition of quencher in increasing concentration, and [Q] is the quencher concentration in molar units. F0/F = 1 + KSV[Q]

Equation 3

Red Edge Excitation Shift (REES) experiment was used to account for the solvent accessibility of Lz-peptides in both free solution as well as in the presence of lysozyme monomers. The excitation wavelength used in the experiment ranged from 280-304 nm, and the monitoring of the emission profile was accounted in a range from 320-400 nm, along with keeping all the remaining parameters constant. Circular Dichroism (CD) Spectroscopy. Collection of circular dichroism (CD) spectra was carried out using Jasco J-815 spectrometer. The solution was prepared with citrate-phosphate buffer at pH 2.8. All CD spectra were collected at room temperature with accumulation of three scans (data interval of 1 nm) at a speed of 100 nm/min over the wavelength range of 190-260 nm. Quartz cuvette of path length 1 mm was used. The obtained spectral data were base subtracted from buffer, and millidegree values were converted into molar ellipticity (θ) using equation 4, where θ is in units of deg.cm2.dmol-1; mo is in millidegree, M is in g.mol-1; L is in cm; and C is in g.L-1. Molar Ellipticity (θ) = moM/10 ×L×C

Equation 4

NMR Spectroscopy. All NMR experiments were recorded on a Bruker AVANCE III 500 MHz spectrometer, equipped with a SMART 5 mm probe, at either 288 or 298 K temperatures. Data acquisition, processing, and spectral assignment were performed using 8 ACS Paragon Plus Environment

Page 8 of 39

Page 9 of 39

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

Biomacromolecules

Topspin v3.1 software and Sparky (T.D. Goddard and D.G. Kneller, University of California, San Francisco). Samples were prepared using 1 mM solution of peptides in citrate-phosphate buffer, pH 2.8, containing 10% D2O and DSS was used as internal standard (0.0 ppm). Twodimensional total correlation spectroscopy (TOCSY) spectra were recorded with 80 ms mixing time. Nuclear overhauser effect spectroscopy (NOESY) spectra were recorded with 150 ms mixing time and 2K (t2) ×456 (t1) data points.35 Spectral width was set to 12 ppm in both dimensions. The spectrum was recorded with 16 dummy scans, followed by 120 scans in t1 increment.36 After zero filling in t1, data matrices of 4K (t2) × 1K (t1) were obtained. Transferred NOESY (trNOESY) was performed for each peptide (1 mM) against various concentrations of lysozyme monomer, ranging from 5 to 20 µM, with three mixing times of 100, 150 and 200 ms. The experimental set up was almost identical as described above. TOCSY experiments for lysozyme and Lz-peptides were also carried out with a 1:1 concentration ratio to correlate with the docking experiment. Saturation-Transfer Difference NMR (STD NMR) Experiments. All samples containing Lz-peptides were prepared with two times lyophilisation for the buffered solution and then prepared with 99.9% D2O. Final pH of the sample was adjusted to 2.8. All STD NMR experiments were recorded on a Bruker Avance III 500 MHz spectrometer at a temperature of 298 K. The spectra were obtained either for Lz-peptides alone or at a molar ratio of peptides: macromolecule = 1:500, where macromolecule represents lysozyme monomers. A total of 3K scans were recorded with selective saturation of macromolecule at -1.0 ppm (on resonance frequency) and at 40 ppm (off resonance frequency).35 40 Gaussian-shaped pulses with 1% truncation and 1 ms delay between two selective pulses of 49 ms at 30 dB were used in this study. The signals attributed to saturation transfer were obtained by subtracting the offresonance spectra from the on-resonance ones using phase cycling method.35 Data processing was performed in Topspin v3.1. 9 ACS Paragon Plus Environment

Biomacromolecules

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 39

Lysozyme Peptide Structure Prediction. The lack of NOEs in the trNOESY spectrum reveals that the peptides do not adopt any specific secondary structure in the presence of macromolecule (further details are discussed below). Conformation prediction for peptides was performed using simulated annealing (SA) process.37 Briefly, an initial model of peptides was built using linear sequence in the tleap module of Amber. A short minimization of the peptide sequence was performed using force-field ff99SB-ILDIN to clear all the steric clashes.38 The SA procedure includes temperature scale variation of 0-50K; 50-100K; 100150K; 150-200K; 250-250K; 250-300K; 300-325K; 325K constant; 325-300K; in each step for a time scale of 5 ps each, with integration step of 0.5 fs. A production run of 5 ns (integration time step - 2 fs) was continued, and the trajectory was recorded at an interval of 5ps. The trajectory was processed with Perl scripts of MMTSB tool-set for hierarchical kmeans cluster analysis, based on RMSD values with a cut-off value of 2 Å distances.39,40 Selection criteria for each cluster were made for groups having more than 200 conformations. Representation coordinates of each cluster were selected with conformation having a minimum distance to the centroid of the clusters. Docking Calculation with HADDOCK. Representative peptide conformations, viz. Lz, LzK, and LzKW, were docked with lysozyme monomer using High Ambiguity Driven protein-protein Docking (HADDOCK).41 Active residues in peptide structures were provided as input that are based on STD NMR analysis.42,43 For the lysozyme coordinates, average solvent

accessibility

was

calculated

using

NACCESS

(http://www.bioinf.manchester.ac.uk/naccess/). Based on the average SASA and standard cut-off of 50%, accessibility for the surface exposed residues is fixed as passive residues for lysozyme coordinates. Docking results were further collected as cluster based on the internal standards of HADDOCK server. Analysis and selection of the bestdocked complex were made by comparing the HADDOCK score and i-I-RMSD for the best cluster.43 10 ACS Paragon Plus Environment

Page 11 of 39

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

Biomacromolecules

RESULTS AND DISCUSSION A growing body of evidence suggests that anti-amyloid agents are suitable candidates to frame into therapeutic elements for the alleviation of amyloid-related diseases.44,45 One of the approaches to screen for potential inhibitors of protein amyloid aggregation is based on finding short protein segments from the primary structure of the studied protein that are able to interact with protein as well as affect fibrillization of protein to amyloid aggregates.22,20 Tjernberg et al. found that peptide KLVFF (fragment Aβ16−20) shows significant binding to Aβ peptides, preventing their self-assembly into fibrils.46 Soto et al. have focused on the inhibition of the Aβ peptide fibrillization by designed peptides with comparable hydrophobicity and sequences as central hydrophobic region of Aβ peptide, namely the Aβ17−21 motif LVFFA.47,48 Of note is that the native protein and ordered fibril structures are less toxic compared to the intermediate protofibril, oligomer, pore and oligomeric aggregate structures.49 Thus, suitable candidates that inhibit fibrillization of native proteins are valuable to investigate further for designing novel therapeutic agents. We have explored the potential of three peptide fragments (Lz peptides) derived from human lysozyme sequence to inhibit formation of amyloid fibrils of this protein (Scheme 1). The amino acid sequence in the Lz-peptide corresponds to the R107-R115 primary sequence of the amyloidogenic part of the human lysozyme, whereas the LzK and LzKW peptide sequences represent point mutation modifications that change the charge and/or hydrophobicity of the peptide. The purpose of introducing Lys residue in LzK and replacing Lys and Trp in LzKW was to explore the effect of charge and/or hydrophobicity on antiamyloid activity against the lysozyme. Using various biophysical techniques we have examined the interaction of Lz-peptides with monomers of human lysozyme protein and their effects on lysozyme amyloid fibrillization. 11 ACS Paragon Plus Environment

Biomacromolecules

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

Scheme 1. Representation of structural region of lysozyme (highlighted in yellow), based on which the peptide fragments have been derived in the present study. Primary sequences of respective Lzpeptides (R107-R115) are denoted here as R1-R9 and displayed in the lower panel.

Lysozyme Amyloid Fibrillization. Formation of lysozyme amyloid fibrils is well reported in the literature and can be facilitated by high temperature and low pH conditions.8,31 We formed lysozyme amyloid fibrils by incubation of the protein in acidic pH 2.8, high temperature (65 °C) and constant stirring (1200 rpm). The formation of lysozyme fibrils was confirmed independently by ThT fluorescence assay and atomic force microscopy (AFM). Fluorescence intensity of ThT was enhanced in the presence of fibrils compared to the presence of native lysozyme (Figure S1A, Supporting Information). Formation of the lysozyme amyloid fibrils has typical sigmoidal growth curve shown in Figure S1B (Supporting information). Visualization of aggregates was performed with AFM, where the typical morphology of amyloid fibrils was observed (Figure S2A, Supporting Information). Furthermore, the presence of cross β-sheet structures in the formed fibrils was confirmed by 12 ACS Paragon Plus Environment

Page 12 of 39

Page 13 of 39

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

Biomacromolecules

CD spectroscopy (Figure S3, Supporting Information), where a characteristic negative peak was observed at 218 nm.50 Interestingly, Lz-peptides alone have no ability to form amyloid fibrils under the given conditions. AFM images for Lz-peptides after exposing to the conditions for lysozyme fibrillization are shown in Figure S2B-D (Supporting Information). Interference of Lz-peptides with Amyloid Fibrillization of Lysozyme. We have studied the effect of Lz-peptides on lysozyme fibrillization using ThT fluorescence assay. The relative fluorescence intensities obtained for the concentration range of Lz-peptides (from 10 pM to 1 mM) and 10 µM of lysozyme are presented in Figure 1. The fluorescence intensities were normalized to the fluorescence signal detected in the lysozyme amyloid aggregates alone (taken as 100%). The obtained results suggest that Lz and LzK peptides are able to influence the formation of lysozyme amyloid aggregates. Specifically, they are able to inhibit the formation of amyloid fibrils to a certain extent, as the fluorescence signal is lower than that observed for amyloid aggregates alone. It should be noted that the inhibiting activity against fibril system is inversely proportional to the fluorescence intensity obtained with ThT assay. The largest decrease in fluorescence intensities was observed for the LzK-peptide (Figure 1B). Starting at 100 nM concentration, the fluorescence intensities were decreasing with increasing concentration and reached ~ 50% of ThT fluorescence detected for lysozyme amyloid fibrils alone. This corresponds to about 50% inhibition of lysozyme fibrillization. The lowest concentration with the 50% inhibitory activity was 100 nM. A similar trend was observed for Lz-peptide (Figure 1A), where the fluorescence values were slightly higher (~ 60% of fluorescence measured for lysozyme fibrils), indicating about 40% of inhibiting activity. On the other hand, the intensities for LzKW-peptide ranged from 90 to 100% (Figure 1C), which indicates none or minimal inhibitory activity.


Biomacromolecules

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. The fluorescence intensities detected after human lysozyme fibrillization using ThT assay in presence of increasing concentration of Lz (A), LzK (B) and LzKW (C) peptides. The fluorescence intensities were normalized to the fluorescence intensity of the lysozyme fibrils alone (taken as 100%).

For further support, we also have measured the growth curves of lysozyme fibril formation using ThT assay in the absence and presence of Lz-peptides (Figure S4, Supporting information). In the case of Lz and LzK peptides, the fluorescence signal is decreased in plateau phase, and achieves about 60% and 50% of fluorescence detected for lysozyme. Similar fluorescence values were observed for lysozyme and its fibrillization in presence of LzKW peptide. Atomic force microscopy (AFM) was used for visualization of the effect of Lz-peptides on human lysozyme fibrillization. Lysozyme fibrils have typical amyloid morphology – long, unbranched fibrillar aggregates (Figure 2A). The presence of Lz or LzK peptides during lysozyme amyloid fibrillization leads to a reduction in the amount of fibrillar aggregates; the corresponding fibrils are shorter, and their amount is significantly lower (Figures 2B and 2C). In case of LzKW peptide, the AFM image is similar to the image of the untreated amyloid fibrils (Figure 2D), confirming the very weak ability of this peptide to affect lysozyme fibrillization. On the contrary, the individual Lz-peptides fragments do not have any amyloid formation property as discussed above (Figure S2, C-D, Supporting Information).


Page 14 of 39

Page 15 of 39

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

Biomacromolecules

Figure 2. Representative AFM images obtained after amyloid fibrillization of human lysozyme alone (A) and in presence of 100 nM concentrations of short peptides Lz (B), LzK (C) and LzKW (D). Bars in the images represent 1 µm.

Changes in human lysozyme secondary structure after fibrillization alone and in the presence of either 10 µM or 60 µM Lz-peptides were estimated using Far-UV CD spectroscopy (Figure 3) and quantified using deconvolution of the CD spectra using CDNN software (Table S1, Supporting Information). Human lysozyme fibrils formed in acidic conditions showed a negative band at 222 nm, corresponding to the high content of β-sheet in secondary structure (Figure 3, red curves) (Table S1, Supporting Information). The spectra for fibrillization of human lysozyme in presence of Lz peptide (Figure 3A) showed a shift of local minimum and changes in shape of the spectra indicating lower content of β-sheets and higher content of α-helices compared to lysozyme fibrils. The effect of


Biomacromolecules

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. Far - UV CD spectra of human lysozyme after 2 h fibrillization in acidic conditions alone (red curves) and in presence of 10 µM (blue curves) and 60 µM (pink curves) concentrations of Lzpeptides: Lz (A), LzK (B) and LzKW (C).

LzK-peptide (Figure 3B) on lysozyme fibrillization was concentration dependent. Lower concentration of LzK-peptide (blue curve) caused minimal changes in the shape of the spectrum, implying a similar content of β-sheet as observed for lysozyme fibrils formed alone. However, the spectrum for lysozyme fibrils after fibrillization with higher concentration of LzK-peptide (60µM, pink curve) has negative bands at 213 nm and 225 nm. This spectrum is similar to the CD spectrum of native lysozyme and indicates the presence of high content of α-helix. The negligible inhibiting activity was observed for peptide LzKW (Figure 3C and Table S1, Supporting Information), as the obtained spectra for both concentrations were similar to the one detected for lysozyme fibrillization alone. These results infer that the largest inhibiting activity was observed for the LzK-peptide and the smallest for the LzKW-peptide, which is in agreement with data obtained using ThT assay. Consequently, these promising results have motivated us to identify the interaction pattern with the help of various high and low-resolution spectroscopic techniques that can provide more valuable insights for robust anti-amyloid polypeptide design at an atomic resolution. Biophysical Study of Lz-peptide Interaction with Lysozyme Monomer Using Fluorescence Spectroscopy. Fluorescence spectroscopy was used in various forms to determine the binding parameters of Lz-peptides to lysozyme monomeric state. The primary goal behind the biophysical characterization is to seek physico-chemical properties of the 16 ACS Paragon Plus Environment

Page 16 of 39

Page 17 of 39

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

Biomacromolecules

interaction between Lz-peptides and lysozyme that mediates the inhibition of structural transition for lysozyme amyloidogenesis. It is well known that Trp residue reflects an emission maximum with λmax ~ 350nm,51 and the same has been used to assess the intrinsic fluorescence property. The change in fluorescence emission maxima for Trp residues in peptides Lz, LzK, and LzKW is useful for analyzing the biophysical interaction pattern. Characteristic emission maxima for the Lz-peptides in free solution were found at approximately 350 nm, which indicates that Trp residues are exposed to the solvent. We observed a pattern of blue shift in emission maxima for fluorescence spectra in the presence of native lysozyme. In particular, a shift in wavelength of 18.8 nm, 18.0 nm, and 19.6 nm was observed for Lz, LzK, and LzKW peptides, respectively, with increasing concentration of native lysozyme monomer (Figure 4). These spectra indicate that all Lzpeptides have stronger affinity towards the native lysozyme structure. It is also noteworthy to mention that the indicative hypsochromic or blue shift is attributed to the deeper insight of Trp residue of Lz-peptides upon binding with the lysozyme protein.32 In contrast, selfassembly of lysozyme did not show any blue-shift effect (Figure 4D), as it is shown for binding of lysozyme to the Lz-peptides. In addition, the fluorescence intensity of 25 µM of lysozyme alone is almost half of the intensity observed for the concomitant titration of lysozyme to the Lz-peptide solutions. The rationale behind such observation can be attributed to the combined effect of Trp fluorescence of lysozyme and Lz-peptides (Figure 4A, B, C), which leads to a two-fold increase in the fluorescence intensity compared to fluorescence spectrum of lysozyme alone (Figure 4D).


Biomacromolecules

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. Fluorescence spectra showing the intrinsic Trp emission maxima for free peptides in solution (red) and those in presence of native lysozyme (blue) at 1:5 molar ratio. The fluorescence emission spectra display the intrinsic emission of Trp residues for Lz (A); LzK (B); and LzKW (C). 5 µM concentration of each peptide was titrated with maximum concentration of lysozyme 25 µM (A, B, C). The fluorescence emission spectra of native lysozyme with incremental concentrations are shown in (D).

Further solvent exposure of these peptides was confirmed using static quenching mechanism. Acrylamide was selected since it is considered as a ‘neutral’ quencher.32,37 The free forms of all peptides show relatively higher Stern-Volmer quenching compared to the bound state, with KSV values 15.5, 14.5 and 14.4 M-1 for Lz, LzK, and LzkW, respectively. This indicates that the Trp residues of all peptides are well buried in the hydrophobic region of native lysozyme, where the solvent accessibility to the quencher is well protected (Figure 5A).37 The quenching results in presence of native lysozyme showed a lower Ksv value in case of Lz (KSV=4.1 M-1) and LzK peptides (KSV=5.7 M-1) (Figure 5A). Furthermore, using 18 ACS Paragon Plus Environment

Page 18 of 39

Page 19 of 39

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

Biomacromolecules

fluorescence anisotropy experiment, we have calculated the equilibrium dissociation constants of the peptides bound to native lysozyme. The resulting KD values of binding of Lz, LzK, and LzKW to native lysozyme appeared to be 1.85, 2.35 and 1.77 µM, respectively (Figure 5B). The corresponding KD values were further used to derive the binding free energy that yielded a comparative value for all Lz-peptides (~ -8 Kcal.mol-1), as indicated in Figure 5C.

Figure 5. (A) Analysis of solvent exposure of Trp residues of Lz-peptides in free state (blue) and bound to native lysozyme (orange), accounting for Stern-Volmer constant for Lz-peptides. (B) The dissociation constant (KD) calculated on the basis of fluorescence anisotropy experiment, which infers values in the µM range. (C) Calculated free energy of binding for Lz-peptides with native lysozyme protein. Fluorescence anisotropy was calculated with successive increment of macromolecule concentration (native lysozyme) to Lz (D), LzK (E), and LzKW (F).

Fluorescence anisotropy has also been used to gain in-depth information regarding the binding pattern.34 In particular, the rotational dynamics of ligand (Lz-peptides in this case) is decreased with increased interaction with the protein macromolecule (native lysozyme).52 The same inference of Lz-peptide binding to native lysozyme is further reflected with an increase in anisotropy. We have noticed that with increasing concentration of macromolecule, the anisotropy value was also increased for all Lz-peptides (Figure 5D, 5E, and 5F). This 19 ACS Paragon Plus Environment

Biomacromolecules

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

rapid rise in anisotropy value indicates the immediate binding of peptides with lysozyme in the in-vitro system. However, it reaches a plateau with successive addition of native lysozyme, reflecting the saturation of macromolecule binding site (Figure 5D, 5E, and 5F). The dissociation constant (of ca. 1.8 µM) obtained from this experiment was found to be comparable for all Lz-peptides studied here, which correlates well with Figure 5B. Secondary Structural Characteristics in the Binding Phenomenon. After establishing that Lz-peptides have a strong potential to bind with lysozyme and inhibit the progression of amyloid fibril formation, it is important to probe possible changes in secondary structure that might occur upon binding. Circular dichroism (CD) spectroscopy is one of the valuable tools to study secondary structures of biological macromolecules.53 Preliminary analysis of secondary structure characteristics of peptides in solution form was estimated using this technique (Figure S5, Supporting Information). The CD profile for native lysozyme showed typical characteristics peak for negative maxima at 208 nm and 222 nm, which is clearly evident in Figure 6. No structural perturbation was obtained upon addition of peptide fragments into the sample containing native lysozyme, even at higher concentrations of Lzpeptides (up to 1:6 lysozyme:Lz-peptides) (Figure 6), which indicates no changes in lysozyme secondary structure.

Figure 6. Secondary structure changes recorded using CD spectroscopy. Concentration of native lysozyme was 10 µM, to which Lz-peptides were added with increasing concentrations. CD spectra are shown for Lz (A), LzK (B) and LzKW (C).


Page 20 of 39

Page 21 of 39

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

Biomacromolecules

We have also performed reverse titration, where the successive incremental concentrations of native lysozyme were added to the peptide solutions (Figure S5, Supporting Information). The free Lz-peptides do not adopt any particular secondary structure with typical peaks at 200 nm that correspond to random coil motif. Surprisingly, upon addition of lysozyme monomer to the Lz-peptide fragments, there was no change in secondary structure for Lz-peptides, indicating that these short peptides, consisting of 9 amino acid residues each, do not adopt any conformation even upon addition of a macromolecule. Notably, the characteristics α-helical peak obtained at higher concentration in titration is attributed to the native lysozyme. Analysis of Binding Pattern of Lz-peptides with Lysozyme. NMR spectroscopy is one of the powerful tools for monitoring and characterizing intermolecular interactions.54 Using onedimensional 1H NMR spectra, it was observed that there are certain structural perturbations in peptide conformation upon addition of the native lysozyme. This was further confirmed by the broadening of the line shapes for Lz, LzK, and LzKW peptides upon addition of native lysozyme at low concentration (< 5 µM). The line broadening effect of the short peptide fragments in the presence of larger macromolecule clearly demonstrates that the peptide adopts similar T2 relaxation as the native lysozyme. In other words, there is a fast conformational exchange between the free and bound form of Lz-peptides in the presence of lysozyme (Figure 7, left panel). A similar pattern of broadening effect is also evident for indole ring protons of Trp residues (NεH) upon addition of macromolecule (data not shown). Importantly, the chemical shift perturbation reflective by the addition of macromolecule is minimal, as shown in Figure 7. This sample condition is ideal for transferred NOESY study to identify the magnetization transfer effect in probing short inter-nuclear distances between protons (< 5Å) to determine the three dimensional structural information of the peptide fragments in the presence of macromolecule/receptor.54 Unfortunately, we did not see large 21 ACS Paragon Plus Environment

Biomacromolecules

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

number of trNOE cross peaks for the three Lz-peptides (Figure 7, right panel) in the presence of lysozyme, suggesting that the secondary structure is not well defined in the presence of native lysozyme. These findings are in line with our CD results, which indicate that Lzpeptides remain as random coil conformation upon binding to macromolecule (Figure 6 and and Figure S5). In other words, the interaction between Lz-peptides and lysozyme monomer is not mediated with the adoption of any particular secondary structure. While the inhibition experiments using ThT assay or AFM were conducted at 65°C, it is worth mentioning here that the NMR experiments were performed at 25°C, or even lower at 15°C, to avoid the intermolecular chemical exchange between peptide amide and solvent water at higher temperature (Figure S6, Supporting Information) due to higher tumbling rate of the small peptide. In addition, the dynamics of protein-ligand interaction will be drastically different at 65°C compared to 25°C if the conformational exchange lies in the range of µs to ms NMR time scale. Furthermore, CD experiment was performed to obtain the secondary structural information of lysozyme at lower (25°C) as well as higher temperature (65°C) (Figure S7, Supporting Information). It is clear from CD spectra (Figure S7, Supporting Information) that the content of the disordered structure at higher temperature is increased compared to spectrum detected at lower temperature. This result is in good agreement with the one-dimensional 1H NMR spectrum of lysozyme at lower and higher temperature (Figure S6C of Supporting Information). We also found that the protein unfolding is reversible (Figure S8, Supporting Information). Taken together, the structural change for lysozyme at higher temperature is necessary to initiate the process of amyloid aggregation; it initiates the self-assembly by the interactions between lysozyme molecules. Therefore, we have performed NMR spectroscopy explicitly to explain the binding of Lz-peptides to human lysozyme and thus provide explanation for the observed differences in formation of fibril in the presence of studied Lz-peptides. 22 ACS Paragon Plus Environment

Page 22 of 39

Page 23 of 39

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

Biomacromolecules

Figure 7. The interaction of Lz-peptides with native lysozyme is investigated by one-dimensional 1H NMR spectra (left panel) and two-dimensional 1H- 1H trNOESY spectra (right panel). 1H NMR spectra of respective Lz-peptides in free solution (blue) and in presence of native lysozyme (red). trNOESY spectra are shown alongside (in green) for Lz (A), LzK (B), and LzKW (C), respectively.

Identification of Probable Binding Orientation of Lz-peptides using STD NMR. In-depth structural analysis was performed using saturation transfer difference (STD) NMR, which provides information regarding the localization of Lz-peptides over the macromolecules.42 STD NMR is a powerful and specific tool that helps in identifying the epitope of ligand (Lzpeptides in this case) that are in close proximity of a macromolecule (native lysozyme). More specifically, this NMR technique allows to detect transfer of selective saturation of the receptor to the ligand protons that are in close proximity via spin diffusion mechanism, without affecting the ligand signal.42 This infers spectrum containing ‘off’ resonance and ‘on’ resonance information, whereby the difference between off and on resonance spectra provides the distance information (ligand binding epitope) with respect to the macromolecule.55 Excess ligand relative to the receptor is thus used to achieve the 23 ACS Paragon Plus Environment

Biomacromolecules

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

magnetization transfer from receptor to the ligand in its bound state.42 On the contrary, no such STD effects must be obtained in the similar experimental set up in the absence of native lysozyme, which is also reflected in this study. One-dimensional STD NMR spectra are presented in Figure 8, which provide binding epitope-related information for Lz-peptides with respect to native lysozyme. It was identified that ring protons of Trp residues are in close proximity to the macromolecule, as evident by large STD effect, demonstrating more prominent STD signals in case of Lz, LzK and LzKW peptides (Figure 8). In particular, the 2H, 4H, 5H, 6H, and 7H of indole ring (W) are clearly indicative in the epitope mapping. Specific residues that are common to all three peptides, such as V4 (γH) and N8 (βH), are found to be in close proximity to the macromolecule. In a similar fashion, the side chain protons of K2 in LzK and LzKW are found to be close to the native lysozyme. A severe signal overlap is found for the side chain protons of Lys and Arg residues. R1 in all peptides is found to display prominent magnetization transfer with native lysozyme. The visualized confirmation of binding epitope as revealed by STD NMR was further analyzed with the help of molecular modelling.


Page 24 of 39

Page 25 of 39

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

Biomacromolecules

Figure 8. 1H STD NMR spectra for Lz (A), LzK (B) and LzKW (C) in conjunction with their interactions with native lysozyme. Reference spectra (blue) of Lz-peptides were recorded in presence of macromolecule (molar ratio of Lz-peptide: native lysozyme = 1:500). STD NMR spectra of the same sample representing the resonance transfer within the binding epitope are shown in red.


Biomacromolecules

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

Elucidating the Binding Model with Molecular Modelling. Our next aim was to find a probable interacting model of Lz-peptides with lysozyme. The derived inference regarding random coil structure for the three peptides was investigated with the help of simulated annealing and cluster analysis (see Figure S9, Supporting Information). In particular, the implicit generalized born condition was used for conformational sampling that allows the peptide to adopt a suitable 3-D geometry based on energy penalties.56 Analysis of the conformational snapshots was carried out using the k-mean hierarchical clustering technique. The selection criteria for peptide conformation was set with a cut-off value of population >200 in each cluster group. The rationale behind such criteria is that more population states are reflective of retaining similar three-dimensional state for each peptide in cluster group with higher population numbers. Details of population state and representative peptides conformation are presented in Supporting Information (Figure S9 and S10, Supporting Information).


Page 26 of 39

Page 27 of 39

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

Biomacromolecules

Figure 9. Docking model of Lz (A), LzK (B), and LzKW (C) with native lysozyme carried out using NMR restraints in HADDOCK. Scatter plot (middle panel) showing the docked conformations obtained from HADDOCK, among which the marked conformation (higher score, lower i-I-RMSD) is represented (left panel). Correlation of STD NMR with docked model is shown for Lz, LzK, and LzKW, where the identified protons in epitope binding region are shown as yellow spheres (right panel). The red surface of lysozyme protein indicates hydrophobic patch to which Lz-peptides are binding (right panel).

We have used residue information obtained from STD NMR as active residues for Lzpeptides (ligand). For the receptor part (lysozyme), calculation of solvent exposed surface area was performed, and surface residues were provided as active residues for receptor.43 Docking calculation was executed in an iterative manner using various conformations for three peptides, as outlined in the Experimental Section.41 Retention of final docked complex was based on highly correlated conformation (Figure 9, left panel) with high HADDOCK 27 ACS Paragon Plus Environment

Biomacromolecules

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

score, -62 a.u. (Lz), -79 a.u. (LzK), and -65 a.u. (LzK); and lowest i-I-rmsd value of 0 Å in all cases (Figure 9, middle panel). Similar analysis of the obtained complex was performed with comparative analysis of SASA, where a reduction in values for solvent exposure surface area (Å2) is found, upon binding with Lz-peptides (data not shown).57 Figure 9 represents the docked conformation of Lz, LzK, and LzKW with lysozyme. The graph of the computed docked states is indicative of the least i-I-rmsd and high HADDOCK score, which represents the favorable binding conformation. Furthermore, the docked state of all Lz-peptides correlates well with the STD NMR results. Protons found to depict the epitope binding via magnetization transfer are shown as yellow spheres (Figure 9, right panel). Briefly, residues at the N-terminal regions of Lz, LzK, and LzKW peptides such as R1, A2/K2, W3, V4 are found to be in close proximity to lysozyme, which matches with STD NMR results (Figure 8A, 8B, and 8C). On the other hand, with the help of SASA analysis, it was revealed that the interaction of Lz-peptides with lysozyme was mediated via a hydrophobic interaction as the driving force behind this interaction. Figure 9 (right panel) indicates that the binding region of peptides to lysozyme comprises largely of hydrophobic residues (red color). These residues mainly include Trp, Leu, Ile, Val, and Ala. Interestingly, it was found that the binding region of LzK peptide in receptor mainly comprises of Trp and Val residues (W109, W112, V121, V125, and V130) that could be responsible for reductioninthe amount of fibrillar aggregates. Probable binding mode of Lz-peptides with lysozyme to interfere fibrillization process. The hot-spot residues of lysozyme responsible for inducing amyloid structural conversion along with positioning of Lz-peptides are shown in Figure 10A. Note that the positioning of LzK and LzKW peptides differ from that of Lz, which is attributed to the presence of Lys and Trp residues. Close inspection of the docking structure of Lz peptide in lysozyme reveals that the positioning of the peptide fragment is in close proximity (4 Å) to hot spot residue T70, 28 ACS Paragon Plus Environment

Page 28 of 39

Page 29 of 39

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

Biomacromolecules

which is involved in hydrophobic interaction (Figure 9A, right panel and 10C). Similarly, another hot-spot residue W112 is in close proximity (4 Å) to the binding position of LzK and LzKW, respectively, and is involved in hydrophobic interaction (Figure 9B, right panel and 10E; 9C, right panel and 10G). The involvement of Lz-peptides to these hot-spot residues can thus be attributed to the property of amyloid inhibition of lysozyme. Two-dimensional TOCSY spectrum was carried out with 1:1 concentration of lysozyme and Lz-peptide to further understand this phenomenon at an atomic resolution with the help of chemical shift perturbation. In particular, it was observed that the γ-proton of T70 disappeared upon addition of Lz peptide, which can be attributed to spin relaxation effect. On the other hand, a sharp chemical shift perturbation was observed for CαH-proton of Lz, as shown in Figure 10B. The two other peptides, LzK and LzKW, were found to be interacting with W112, which was indicated by chemical shift change for the indole ring (NεH) proton. The chemical shift perturbation of W112 of lysozyme is shown in Figure 10D and 10F by LzK and LzKW, respectively indicating that the short LzK peptides stabilize the lysozyme monomer and hence inhibit the fibrillization. Importantly, we have observed in this study that the binding phenomena of Lz, LzK (modified with a charged residue), and LzKW (modified with charged and aromatic residues) peptides with lysozyme monomer were almost comparable in terms of blue shift, quenching, and anisotropicfree energy of binding. The “hot-spot” residues responsible for lysozyme fibrillization are lysozyme variants like I56T, F57I, W64R, F57I/T70N and W112R/T70N.7 These residues mainly belong to hydrophobic and/or aromatic class of amino acids. Based on the reported evidence, it is realized that such “hot-spot” regions are the key target area, which can be traced with hydrophobic interaction, for the development of anti-amyloid agents.7,31,58,59 Interestingly, the probable binding models elucidated with the help of NMRrestrained docking method in the present study also highlight the importance of hydrophobic 29 ACS Paragon Plus Environment

Biomacromolecules

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

interaction (Figure 9, right panel). In addition to the fibrillization event, lysozyme is also known to have antimicrobial property. Based on the above discussion to the binding of Lzpeptide and analysis of this binding epitope, we have also performed a compare and contrast study for anti-amyloid proposition and antimicrobial activity (Figure S11 and S12, Supporting Information).

Figure 10. (A) Representation of Lz-peptides positioning and hot-spot residues responsible for lysozyme amyloidogenesis. Sequential representation of R107-R115 in the globular structure of Lysozyme is shown in red color, where comparison of Lz-peptides positioning with respect to positioning of hot-spot residues in lysozyme is shown. Depiction of interacting amino acids from lysozyme responsible for hydrophobic interaction with Lz-peptides, as obtained from HADDOCK is shown in stick representation for Lz (C), LzK (E), and LzKW (G). The hot-spot residues of lysozyme (red contours) responsible for amyloidogenesis were identified to interact with Lz-peptides through chemical shift perturbation in TOCSY spectrum (green contours), shown in (B, D, F) for Lz, LzK, and LzKW.

The obtained results suggest that inhibitory effect observed for Lz-peptide is due to interaction between lysozyme and charged or nonpolar groups present in short peptide. In the LzK-peptide, the number of positive charge is increased, leading to a slightly higher inhibitory efficiency compared to Lz-peptide. Significantly different effect was observed in the case of LzKW-peptide which contains the same number of charges as LzK, but amino 30 ACS Paragon Plus Environment

Page 30 of 39

Page 31 of 39

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

Biomacromolecules

acid (Ala) with non-polar side chain is replaced by non-polar aromatic amino acid containing indole (Trp). Taken together, this short peptide has no significant effect on the lysozyme amyloid fibrillization. This result is quite interesting, as the presence of aromatic residues usually cause improvement of the anti-amyloid properties of small molecules. It is also worth mentioning that the binding site for Lz-peptide was different from LzK/LzKW, which was supported by NMR experiments. From the molecular modelling it is clear that the ligands (Lz-peptides) interact with the “hot-spot” residues of lysozyme residues, which are responsible for fibril formation. There are several factors such as hydrogen bonding or electrostatic interaction and hydrophobic interaction that govern the stabilization of the complex. Thus, this manuscript describes our first experimental results, and more robust analysis that direct towards the significance of lysozyme amino acids participating in the amyloidogenesis spectacle is planned in future studies for the purpose of finding potential amyloid inhibitory agents. Although several studies confirm the anti-amyloid effects of key candidates using NMR and computational tools, precise information related to lysozyme amino acids involved in interaction has not been revealed.8,9,31,58 Thus, more robust analysis that directs towards the significance of lysozyme amino acids participating in the amyloidogenesis spectacle is necessary. Such understanding will be helpful in strengthening the research field oriented towards finding potential amyloid inhibitory agents.

CONCLUSIONS In summary, the present study is based on the hypothesis of targeting the native lysozyme as a model for inhibiting amyloidogenesis propagation. Identification of suitable anti-amyloid agents has currently been one of the most popular strategies. The importance of such approach lies in inhibition of the accelerated amyloid progression of the native proteins. Note that the toxicity of fibrillar structures is lower compared to the intermediate proto-fibrillar 31 ACS Paragon Plus Environment

Biomacromolecules

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 oligomeric conformations.18 Moreover, the chances of protein refolding into the native structure after the disintegration of fibril are least probable. Both of these statements indicate the limitation and/or drawback of the approach for targeting fibrillar structure. Our assumption is that agents having binding affinity towards native protein can be promising candidates for developing anti-amyloid therapeutics. The present study has focused on human lysozyme as a model amyloid protein, and the important findings of this work are as follows. First, using AFM and ThT assay, we have identified that peptide fragments derived from lysozyme protein have the ability to inhibit lysozyme fibril formation at different extents. We have also estimated that there is no conformational change in the macromolecular structure upon binding with the Lz-peptides. Estimation for the absence of any secondary structural perturbation was identified using CD spectroscopy. Similar biophysical analysis of the binding pattern was made using the intrinsic fluorescence of Trp residues. Interestingly, it was also revealed using the fluorescence experiments that Lz-peptides have comparable affinity towards lysozyme. Based on blue shift measurement, quenching-based Stern-Volmer constant, and anisotropy-based dissociation constant, all peptides were found to have a comparative binding affinity towards lysozyme. These data indicate that hydrophobic interaction is the main driving force for mediating the binding of Lz-peptides to lysozyme. Next, the structural aspects of interaction phenomenon were revealed using one-dimensional and two-dimensional NMR experiments that demonstrate random coil structure of peptides. This data also correlate with other experimental findings from CD spectroscopy. Additional information related to peptide location over the macromolecule was identified using STD NMR. The epitope mapping pattern reveals the residues Trp, Val, Arg, and Asn to be in close proximity of the macromolecule. Finally, the insights at the atomistic level were elucidated using HADDOCK, where restraint files were input based on STD NMR results. Interestingly, SASA and binding 32 ACS Paragon Plus Environment

Page 32 of 39

Page 33 of 39

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

Biomacromolecules

site analysis reveal that Lz-peptides have favorable docked conformation in the hydrophobic region of lysozyme. Further studies on Lz-peptides are worthy in the future for identifying the kinetics involved in the amyloidogenesis pathway. Likewise, further applications of Lzpeptides to other amyloid-prone proteins such as amyloid beta (Aβ40/42), hIAPP, and αsynuclein are underway in our laboratory. Overall, identification of similar suitable agents having the affinity towards both fibrillar and native protein may prove to be worthy in future drug design approaches.60

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:XXXX ThT fluorescence spectrum for lysozyme monomer and fibril and kinetic profile of amyloid fibrillization of 10 µM human lysozyme detected using Thioflavin T fluorescence assay (Figure S1); AFM images of human lysozyme fibrils, and Lzpeptides (Figure S2); CD spectrum of lysozyme monomer and fibril (Figure S3); Growth curves of lysozyme fibrillization in the absence and presence of Lz peptides (Figure S4); The content of secondary structure of Lysozyme (60 µM) in the absence and presence of Lz peptides, obtained from deconvolution of the CD spectra using CDNN software (Table S1); CD spectrum displaying secondary structure change for Lz-peptides with increment addition of native lysozyme (Figure S5); One-dimensional 1

H NMR spectra of Lz peptides and human lysozyme (Figure S6); CD spectra at lower

as well as higher temperature (Figure S7); Temperature induced refolding study (Figure S8); Cluster analysis and representative model of Lz-peptides (Figure S9, S10); Binding site representation for LPS head group over lysozyme (Figure S11); Binding 33 ACS Paragon Plus Environment

Biomacromolecules

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

site of Lz- peptides, responsible for anti-amyloid activity, in comparison with the binding site of carbohydrate moieties of LPS to lysozyme (Figure S12).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ; [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the research grant from Institutional fund (Plan Project-II to A.B.), the Slovak Grant Agency VEGA 2/0181/13 and ESF 26220220005 (to ZG and ZB) and RSF grant 14-23-00199 (to NEN). AB also would like to acknowledge DBT, Government of India for infrastructure development fund (BT/PR3106/INF/22/138/2011) to Bose Institute for purchasing 700 MHz NMR spectrometer with cryo-probe. AG and RKK thank CSIR, Govt. of India for senior research fellowship. Central Instrument Facility (CIF) of Bose Institute is greatly acknowledged for peptide synthesis, CD, Fluorescence and NMR experiments.


Page 34 of 39

Page 35 of 39

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

Biomacromolecules

REFERENCES

1. Knowles, T. P.; Vendruscolo, M.; Dobson, C. M. Nat Rev Mol Cell Biol 2014, 15 (6), 384-96. 2. Haass, C.; Selkoe, D. J. Nat Rev Mol Cell Biol 2007, 8 (2), 101-12. 3. Harrison, R. S.; Sharpe, P. C.; Singh, Y.; Fairlie, D. P. Rev Physiol Biochem Pharmacol 2007, 159, 1-77. 4. Liu, C.; Zhao, M.; Jiang, L.; Cheng, P. N.; Park, J.; Sawaya, M. R.; Pensalfini, A.; Gou, D.; Berk, A. J.; Glabe, C. G.; Nowick, J.; Eisenberg, D. Proc Natl Acad Sci U S A 2012, 109 (51), 20913-8. 5. Rambaran, R. N.; Serpell, L. C. Prion 2008, 2 (3), 112-7. 6. Horvath, I.; Weise, C. F.; Andersson, E. K.; Chorell, E.; Sellstedt, M.; Bengtsson, C.; Olofsson, A.; Hultgren, S. J.; Chapman, M.; Wolf-Watz, M.; Almqvist, F.; Wittung-Stafshede, P. J Am Chem Soc 2012, 134 (7), 3439-44. 7. Swaminathan, R.; Ravi, V. K.; Kumar, S.; Kumar, M. V.; Chandra, N. Adv Protein Chem Struct Biol 2011, 84, 63-111. 8. Gazova, Z.; Siposova, K.; Kurin, E.; Mučaji, P.; Nagy, M. Proteins 2013, 81 (6), 994-1004. 9. Antosova, A.; Chelli, B.; Bystrenova, E.; Siposova, K.; Valle, F.; Imrich, J.; Vilkova, M.; Kristian, P.; Biscarini, F.; Gazova, Z. Biochim Biophys Acta 2011, 1810 (4), 465-74. 10. Langkilde, A. E.; Vestergaard, B. FEBS Lett 2009, 583 (16), 2600-9. 11. Frare, E.; Mossuto, M. F.; de Laureto, P. P.; Tolin, S.; Menzer, L.; Dumoulin, M.; Dobson, C. M.; Fontana, A. J Mol Biol 2009, 387 (1), 17-27. 12. LeVine, H. Protein Sci 1993, 2 (3), 404-10. 13. Parthasarathy, S.; Inoue, M.; Xiao, Y.; Matsumura, Y.; Nabeshima, Y.; Hoshi, M.; Ishii, Y. J Am Chem Soc 2015, 137 (20), 6480-3. 14. Tycko, R. Neuron 2015, 86 (3), 632-45. 15. Pires, R. H.; Karsai, Á.; Saraiva, M. J.; Damas, A. M.; Kellermayer, M. S. PLoS One 2012, 7 (9), e44992. 16. Chatani, E.; Imamura, H.; Yamamoto, N.; Kato, M. J Biol Chem 2014, 289 (15), 10399-410. 17. Xu, J.; Zhang, J. Z.; Xiang, Y. J Phys Chem A 2013, 117 (29), 6373-9. 18. Buell, A. K.; Dhulesia, A.; Mossuto, M. F.; Cremades, N.; Kumita, J. R.; Dumoulin, M.; Welland, M. E.; Knowles, T. P.; Salvatella, X.; Dobson, C. M. J Am Chem Soc 2011, 133 (20), 7737-43. 19. Zhang, X.; Tian, Y.; Li, Z.; Tian, X.; Sun, H.; Liu, H.; Moore, A.; Ran, C. J Am Chem Soc 2013, 135 (44), 16397-409. 20. Härd, T.; Lendel, C. J Mol Biol 2012, 421 (4-5), 441-65. 21. Takahashi, T.; Mihara, H. Acc Chem Res 2008, 41 (10), 1309-18. 22. Sciarretta, K. L.; Gordon, D. J.; Meredith, S. C. Methods Enzymol 2006, 413, 273-312. 23. Wang, Q.; Liang, G.; Zhang, M.; Zhao, J.; Patel, K.; Yu, X.; Zhao, C.; Ding, B.; Zhang, G.; Zhou, F.; Zheng, J. ACS Chem Neurosci 2014, 5 (10), 972-81. 24. Viet, M. H.; Ngo, S. T.; Lam, N. S.; Li, M. S. J Phys Chem B 2011, 115 (22), 7433-46. 25. Viet, M. H.; Siposova, K.; Bednarikova, Z.; Antosova, A.; Nguyen, T. T.; Gazova, Z.; Li, M. S. J Phys Chem B 2015, 119 (16), 5145-55. 26. Cukalevski, R.; Boland, B.; Frohm, B.; Thulin, E.; Walsh, D.; Linse, S. ACS Chem Neurosci 2012, 3 (12), 1008-16. 27. Bayer, T. A.; Wirths, O. Acta Neuropathol 2014, 127 (6), 787-801. 28. Rajasekhar, K.; Chakrabarti, M.; Govindaraju, T. Chem Commun (Camb) 2015, 51 (70), 1343450. 29. Olubiyi, O. O.; Frenzel, D.; Bartnik, D.; Glück, J. M.; Brener, O.; Nagel-Steger, L.; Funke, S. A.; Willbold, D.; Strodel, B. Curr Med Chem 2014, 21 (12), 1448-57.

35

ACS Paragon Plus Environment

Biomacromolecules

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

30. Wang, C.; Yang, A.; Li, X.; Li, D.; Zhang, M.; Du, H.; Li, C.; Guo, Y.; Mao, X.; Dong, M.; Besenbacher, F.; Yang, Y. Nanoscale 2012, 4 (6), 1895-909. 31. Vuong, Q. V.; Siposova, K.; Nguyen, T. T.; Antosova, A.; Balogova, L.; Drajna, L.; Imrich, J.; Li, M. S.; Gazova, Z. Biomacromolecules 2013, 14 (4), 1035-43. 32. Ghosh, A.; Datta, A.; Jana, J.; Kar, R. K.; Chatterjee, C.; Chatterjee, S.; Bhunia, A. Mol Biosyst 2014, 10 (6), 1596-612. 33. Smirnova, I.; Kasho, V.; Sugihara, J.; Kaback, H. R. Proc Natl Acad Sci U S A 2009, 106 (51), 21561-6. 34. Wang, L.; Clifford, B.; Graybeal, L.; Tolley, L.; McCarroll, M. E. J Fluoresc 2013, 23 (5), 881-8. 35. Moriz, M.; Bernd, M., Angew Chem Int Ed Engl 1999, 38 (12), 5. 36. Shah, S. H.; Kar, R. K.; Asmawi, A. A.; Rahman, M. B.; Murad, A. M.; Mahadi, N. M.; Basri, M.; Rahman, R. N.; Salleh, A. B.; Chatterjee, S.; Tejo, B. A.; Bhunia, A. PLoS One 2012, 7 (11), e49788. 37. Banerjee, V.; Kar, R. K.; Datta, A.; Parthasarathi, K.; Chatterjee, S.; Das, K. P.; Bhunia, A. PLoS One 2013, 8 (8), e72318. 38. Lindorff-Larsen, K.; Piana, S.; Palmo, K.; Maragakis, P.; Klepeis, J. L.; Dror, R. O.; Shaw, D. E. Proteins 2010, 78 (8), 1950-8. 39. Feig, M.; Karanicolas, J.; Brooks, C. L. J Mol Graph Model 2004, 22 (5), 377-95. 40. Lorenzen, K.; Schworer, M.; Troster, P.; Mates, S.; Tavan, P. Journal of Chemical Theory and Computation 2012, 8 (10), 3628-3636. 41. Dominguez, C.; Boelens, R.; Bonvin, A. M. J Am Chem Soc 2003, 125 (7), 1731-7. 42. Bhunia, A.; Bhattacharjya, S.; Chatterjee, S. Drug Discov Today 2012, 17 (9-10), 505-13. 43. de Vries, S. J.; van Dijk, M.; Bonvin, A. M. Nat Protoc 2010, 5 (5), 883-97. 44. Minati, L.; Edginton, T.; Bruzzone, M. G.; Giaccone, G. Am J Alzheimers Dis Other Demen 2009, 24 (2), 95-121. 45. Sacchettini, J. C.; Kelly, J. W. Nat Rev Drug Discov 2002, 1 (4), 267-75. 46. Tjernberg, L. O.; Näslund, J.; Lindqvist, F.; Johansson, J.; Karlström, A. R.; Thyberg, J.; Terenius, L.; Nordstedt, C. J Biol Chem 1996, 271 (15), 8545-8. 47. Soto, C.; Kindy, M. S.; Baumann, M.; Frangione, B. Biochem Biophys Res Commun 1996, 226 (3), 672-80. 48. Soto, C.; Sigurdsson, E. M.; Morelli, L.; Kumar, R. A.; Castaño, E. M.; Frangione, B. Nat Med 1998, 4 (7), 822-6. 49. Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Lindman, S.; Minogue, A. M.; Thulin, E.; Walsh, D. M.; Dawson, K. A.; Linse, S. J Am Chem Soc 2008, 130 (46), 15437-43. 50. Louis-Jeune, C.; Andrade-Navarro, M. A.; Perez-Iratxeta, C. Proteins 2012, 80 (2), 374-81. 51. Chan, D. I.; Prenner, E. J.; Vogel, H. J. Biochim Biophys Acta 2006, 1758 (9), 1184-202. 52. Biedermann, F.; Uzunova, V. D.; Scherman, O. A.; Nau, W. M.; De Simone, A. J Am Chem Soc 2012, 134 (37), 15318-23. 53. Greenfield, N. J. Nat Protoc 2006, 1 (6), 2876-90. 54. Zuiderweg, E. R. Biochemistry 2002, 41 (1), 1-7. 55. Domadia, P. N.; Bhunia, A.; Sivaraman, J.; Swarup, S.; Dasgupta, D. Biochemistry 2008, 47 (10), 3225-34. 56. Rahaman, O.; Estrada, T. P.; Doren, D. J.; Taufer, M.; Brooks, C. L.; Armen, R. S. J Chem Inf Model 2011, 51 (9), 2047-65. 57. Nandi, S. K.; Chakraborty, A.; Panda, A. K.; Ray, S. S.; Kar, R. K.; Bhunia, A.; Biswas, A. PLoS Negl Trop Dis 2015, 9 (3), e0003661. 58. Morshedi, D.; Rezaei-Ghaleh, N.; Ebrahim-Habibi, A.; Ahmadian, S.; Nemat-Gorgani, M. FEBS J 2007, 274 (24), 6415-25. 59. Luo, J.; Wärmländer, S. K.; Gräslund, A.; Abrahams, J. P. Chem Commun (Camb) 2013, 49 (58), 6507-9.

36


Page 36 of 39

Page 37 of 39

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

Biomacromolecules

60. Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini, M.; Melchiorre, C. J Med Chem 2008, 51 (3), 347-72.

37


Biomacromolecules

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 of Contents (TOC)

38


Page 38 of 39

Page 39 of 39

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

Biomacromolecules

Table of Content 19x9mm (600 x 600 DPI)


Evidence for Inhibition of Lysozyme Amyloid Fibrillization by Peptide

Recommend Documents