Emerging Methods for Structural Analysis of Protein Aggregation PDF 2017

Send Orders for Reprints to [email protected] 331 Protein & Peptide Let...

Send Orders for Reprints to [email protected] 331 Protein & Peptide Letters, 2017, 24, 331-339 REVIEW ARTICLE ISSN: 0929-8665 eISSN: 1875-5305 Impact Factor: 1.069 Emerging Methods for Structural Analysis of Protein Aggregation BENTHAM Editor-in-Chief: SCIENCE Ben M. Dunn Eshan Khan, Subodh K. Mishra and Amit Kumar* Centre for Biosciences and Biomedical Engineering, Indian Institute of Technology, Indore 452020, Madhya Pradesh, India Abstract: Protein misfolding and aggregation is a key attribute of different neurodegenerative dis- eases. Misfolded and aggregated proteins are intrinsically disordered and rule out structure based drug design. The comprehensive characterization of misfolded proteins and associated aggregation pathway is prerequisite to develop therapeutics for neurodegenerative diseases caused due to the protein aggregation. Visible protein aggregates used to be the final stage during aggregation mecha- ARTICLE HISTORY nism. The structural analysis of intermediate steps in such protein aggregates will help us to discern the conformational role and subsequently involved pathways. The structural analysis of protein ag- Received: October 4, 2016 Revised: December 19, 2016 gregation using various biophysical methods may aid for improved therapeutics for protein misfold- Accepted: January 8, 2017 ing and aggregation related neurodegenerative diseases. In this mini review, we have summarized different spectroscopic methods such as fluorescence spectroscopy, circular dichroism (CD), nuclear DOI: 10.2174/09298665246661702061 magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and Ra- 23150 man spectroscopy for structural analysis of protein aggregation. We believe that the understanding of invisible intermediate of misfolded proteins and the key steps involved during protein aggregation mechanisms may advance the therapeutic approaches for targeting neurological diseases that are caused due to misfolded proteins. Keywords: Neurodegenerative diseases, protein aggregation, NMR spectroscopy, fluorescence spectroscopy, amyloid like pro- Protein & Peptide Letters teins, circular dichroism, raman spectroscopy. INTRODUCTION NUCLEAR MAGNETIC RESONANCE SPECTROS- COPY Neurodegenerative diseases such as Huntington’s disease (HD), Parkinson’s disease (PD), Alzheimer’s disease (AD), Amyloid fibrils of different aggregated proteins generally Prion diseases and Amyotrophic Lateral Sclerosis (ALS) share most of the features of protein aggregation. Various share various cellular mechanisms like inclusion bodies and methods have been employed to infer the structural and protein aggregation formation. These protein aggregates are mechanistic details of amyloid formation but the goal is still usually composed of misfolded protein (Figure 1) fibers with not achieved. Cryo-electron microscopy, X-ray studies of a β-sheet conformation are termed as amyloid which are dis- small fragments and electron tomography have helped to tributed in different regions of brain. Protein aggregates are characterize the cross β-structure of amyloid at atomic reso- formed either due to misfolding of specific proteins which lution. NMR parameters such as longitudinal relaxation serve as seeds for aggregation or de novo misfolding of en- rate (R1) and transverse relaxation rate (R2) play a vital role dogenous proteins. However, all of these diseases share during protein aggregation studies. Transverse relaxation rate common toxic protein gain/or loss of function. Compelling is fast while longitudinal relaxation rate is slow and the evidences suggest that protein aggregation involves a variety product of both R1R2 increases with the rigid limit during of mechanisms and each of the mechanism can be shared protein aggregation. with others during aggregation process of a single product. Another step in NMR spectroscopy based protein aggre- Understanding the thorough mechanisms of protein aggrega- gation studies is Low Field NMR spectroscopy. The particu- tion will aid to develop therapeutic strategies against these lar technique along with other biophysical techniques has devastating neurodegenerative diseases. The section herein been performed to study the denaturation and aggregation of will summarize some emerging methods for structural analy- β-lactoglobulin (β-LG). The spin-spin relaxation (T2) is af- sis of protein aggregation. fected at high concentration of protein only and does not change even after heating at 90 °C at low concentration of protein which concludes that the changes in spin-spin relaxa- *Address correspondence to this author at Centre for Biosciences and Bio- tion measurements (T2) were caused due to aggregation and medical Engineering, Indian Institute of Technology, Indore 452017, Mad- hya Pradesh, India; Tel: +91-731-2438767; Fax: +91-731-2364182; not due to the unfolding of protein. In the same manner E-mail: [email protected] the time dependent NMR aggregation assay reveals the trans 1875-5305/17 $58.00+.00 © 2017 Bentham Science Publishers 332 Protein & Peptide Letters, 2017, Vol. 24, No. 4 Khan and Kumar Native Unfolded Aggregate Amorphous Protein Protein Initiation Aggregate Figure 1. Schematic representation of protein aggregation. to cis equilibrium shift in the R3-S316P peptide bond during The oligomer can be probed directly in real time using 19 the growth phase of Tau protein aggregation. The current F NMR due to its high sensitivity and large chemical shift study states that the presence of hexapeptide along with the range. Suzuki et al studied the human islet-associated poly- correct turn conformation is also a very necessary event dur- peptide (IAPP) protein aggregation with the help of 19F ing tau fibril formation. NMR and concluded that the fibril assembly is a two-step process which does not involve any intermediate species Solid state NMR studies of transthyretin amyloid aggre-. Soluble amyloid aggregates can also be studied using gation and its amyloidogenic intermediates revealed the dis- sedimented solute NMR (SedNMR) in which highly concen- order of the AB loop region during amyloid formation. In trated sample is centrifuged using centrifugal force of magic- another study NMR spectroscopy revealed that 2, 2, 2- angle (MAS) rotor or the MAS rotor can be placed in an ul- trifluoroethanol (TFE) disrupted the well-ordered aggregates of Glucagon-like peptide-1, involved in amyloid formation, tra- centrifuge before NMR analysis providing the sharp 13C and 15N NMR lines [18, 23]. MAS NMR revealed the atomic into helical self-aggregates. These helical self-aggregates are structure of AβM01-42 amyloid fibrils in which Aβ42 dimer dimeric in nature which is an intermediate state during ag- along with the four β-strands forms a core fibril with two gregation. hydrophobic cores. NOE (Nuclear Overhauser Effect), J Relaxation dispersion (RD) experiment using NMR spec- coupling and chemical shifts suggest that the intermolecular troscopy has the advantage over other techniques as it ren- interactions present in hydrophobic regions in Aβ42 peptides ders the structural characterization at the atomic level. The primarily influence the aggregation process. Hoop et al conformational changes during protein aggregation cause recently described the structural aspects of htt exon 1 core peak broadening, signals dephasing and increased relaxation using advanced MAS solid state NMR (ssNMR) and con- rates. The increased relaxation rate can be extrapolated with cludes that the aggregation was caused due to the collapse of R2observed = R2° + Rex, where R2° is the intrinsic relaxation rate poly Q domain. Magic-angle-spinning solid state NMR during RD experiment. Variable spin-lock irradiation and spectroscopy of transthyretin concludes that DAGH β-sheet CPMG refocusing pulses suppress the relaxation and the loses its stability due to the conformational changes which dispersion profile obtained can be fitted using dynamic mod- were caused by the interaction of AB loop regions with the els containing either two or more exchange states. strand A of DAGH β-sheet. These fitted data values provide thermodynamic data and Dark exchange saturation transfer (DEST) is an unusual structural information. Recent advancement in relaxation dispersion experiments based on pulse scheme along with NMR method to study the NMR of free molecules in solution and temporarily bound with high molecular weight other methodologies help to resolve the three-dimensional entity. However, DEST approach is a suitable method to structure based on chemical shift differences and char- study protein aggregation as the equilibrium between free acterize invisible species at atomic level. However, and invisible species can be monitored with experimental CPMG experiments also have limitations as it requires a very conditions. Fawzi et al recently applied the DEST NMR to high signal to noise (S/N) ratio as well as pertinent time scale and hence is not a good choice to study protein aggregation illustrate the exchange phenomenon of free and invisible state in amyloid-β (Aβ) monomers and polydisperse, protofi- that occurs too rapidly. The kinetics of folding and re- brils. DEST is helpful to unveil important information folding of the amyloidogenic precursor can be monitored in of the surface catalyzed secondary nucleation or oligomer real time in the NMR tube. Balbach et al. have devel- formation during amyloid formation that is a very cru- oped a simple mixing device to perform the 1D and 2D real- cial clue to reveal the paradigm behind the amyloid assem- time NMR experiments. Unfolded protein and refolding buffer is separated using an air bubble in the NMR tube in- bly. side the magnet. Later both are mixed applying the pressure Nonetheless, only NMR could reveal the presence of. Earlier, only one-dimensional spectrum could be stud- heterogeneous nature of aggregates during assembly. Al- ied but the recent advancement makes it feasible to though NMR spectroscopy is a very valuable technique to study the two-dimensional as well as three-dimensional spec- study the heterogeneous nature of protein aggregation, NMR tra. Various high resolution 3-D structure or structural mod- spectroscopy has also many challenges. NMR spectroscopy els for different aggregation proteins like tau , human requires a high concentration of protein samples that is not CA150 , transthyretin , amyloid β , HET-s prion feasible as highly concentrated protein leads to oligomerisa- and poly Q have been studied using NMR spec- tion hindering the proper analysis of NMR spectrum. An- troscopy and other biophysical method (Figure 2). other challenge in NMR spectroscopy for intrinsically disor- Emerging Methods for Structural Analysis of Protein Aggregation Protein & Peptide Letters, 2017, Vol. 24, No. 4 333 a. b. c. d d. Figure 2. Amyloid Structures resolved using NMR Spectroscopy (PDB ID: a. 2LMP b. 1IYT c. 2LFM d. 2MJ1) dered proteins, is the absence of rigid 3D structure that sub- amyloid (Aβ) peptides aggregation in different microenvi- sequently results in diminished chemical shift differences ronments. Later on, the Cast film method was applied to and overlapping of spectral signals, notably protons, that study the structural transformation of other peptides or pro- precludes the sequence specific assignment of resonances teins in amyloid-like aggregates. In the cast file method,. Other major conundrums during NMR spectroscopy are the sample is incubated on cuvette for evaporation to form sample quality and modulating the aggregation propensity. the thin protein film and far-UV spectra is recorded using the CD spectropolarimeter. Although, the cast film method is a Although there are many challenges associated with conducive method to study protein aggregation, the macro- NMR spectroscopy during protein aggregation studies, NMR spectroscopy is a sole technique which portrays the dynamic scopic anisotropy was a conundrum until universal chi- roptical spectrophotometer (UCS-1) was used to study the non-native along with invisible species. The author believes Aβ(1-40) and Aβ(1-42) peptides eliminating the anisotropic that the NMR including various other methods such as sin- artifacts. The ssCD study reveals the structural trans- gle-molecule Förster resonance energy transfer (FRET), EPR formation of α-Syn and the plasminogen activator inhibitor-1 (electron paramagnetic resonance), EM (electron micros- (PAIRC) peptide into β-sheet structure which further copy) will aid to unveil our quest of why, and how, a func- tional protein is converted into protein aggregate. changed to amyloid-like aggregates. Vibrational Circular Dichroism (VCD) is extension of CIRCULAR DICHROISM Circular Dichroism which can be used to study the chirality in amyloid fibrils. Amyloid fibril formation typically Protein secondary structure can be studied using Circular exhibits the enhanced VCD spectrum and a downshift of Dichroism (CD) Spectroscopy. In protein aggregation stud- amide. Measey et al, using the insulin and two other pep- ies, CD spectroscopy can be used either as far-UV CD in tides, have shown that the VCD intensity enhancement is place of size exclusion chromatography (SEC) or as thermal largely due to intersheet interaction rather than intrasheet scanning CD for high throughput screening. Thermal scan- interactions. Protein misfolding and aggregation in- ning CD is complementary to size exclusion chromatography duced by heat often leads to the formation of an amyloid-like that requires less time and very less amount of sample as structure. This amyloid-like structure formation can easily be compared to SEC to provide analogous efficiency. Far- analyzed in real time with less amount of sample in a very UV CD provides useful structural information of amyloid short time, using Circular Dichroism. In this method, the proteins using the g- factor algorithm, an implicit factor that turbidity and 90° light scattering during thermal fold- is affected by only molecular type rather than the pathlength ing/unfolding transition can be studied simultaneously that of light. makes it convenient for protein aggregation studies. Solid-state circular dichroic spectroscopy (ssCD) was During the course of protein aggregation research CD initially used to study the structural changes during β- spectroscopy has emerged with a variety of advancements. 334 Protein & Peptide Letters, 2017, Vol. 24, No. 4 Khan and Kumar The use of near and far UV-CD along with other spectros- the lifetime of fluorophores(s) during protein aggregation. copy techniques such as NMR have shown the aggregation Fluorescence correlation spectroscopy (FCS) can be em- of RNase A due to heat denaturation. Vermeer et al ployed to study the various dynamic parameters such as dif- have studied that the unfolding and aggregation of a multi- fusion time, conformation and concentration of fluorescent domain protein immunoglobulin is strongly affected by al- molecule. Sahoo et al. described the stepwise protocol to tered temperature and pH conditions as the CD spectrum and study the molecular complex formation during aggregation other calorimetric parameters were largely affected by these. factors. The A4 or β-peptide (39 to 43 amino acid resi- &+ &+ dues) primarily involved in protein aggregation in Alz- 1 2 &+ 2 heimer’s disease. Circular Dichroism study of synthetic pep-  6 1 &+ 2+ tide namely β-(1-39) and β-(1-42), whereas β-(1-28) and β- 2 (29-42) adopts different conformations in different condi- +& 6 &+ 1 1+ +&O tions like temperature and pH. Intermolecular structures 2 play a vital role to discern the conformational properties of amyloid fibrils. The combination of circular dichroism (CD) theory and Vacuum-ultraviolet circular dichroism (VUVCD) 7KLRIODYLQ 7 1LOH5HG $16 spectroscopy has been implicated to understand the intermo- 2 lecular structures of β2-microglobulin (β2m) core fragments 2 of amyloid fibrils. The study concludes that the backbone as 1+ 1+ 1 2+ 2 2 well as side chain affects the conformational properties of +2 6 6 2+ 2 2 2 amyloid fibrils. 2 2 Synchrotron radiation circular Dichroism (SRCD) spec- %LV$16 0)& troscopy complements the small-angle X-ray scattering and infrared spectroscopy for protein folding studies. SRCD is Figure 3. Chemical structure of different extrinsic fluorescent advantageous over conventional CD as it provides high sig- probes to study protein aggregation. nal to noise ratio, fast data acquisition, time resolved meas- urements and also the high-throughput screening. SRCD spectroscopy study of trehalose response on α-synuclein con- Time-resolved Förster resonance energy transfer (FRET) formation states that the low concentration of trehalose pre- based study in α-synuclein aggregation explains the presence vents the aggregation process and helps in proper unfolding of multiple oligomeric intermediates. Single molecule. In another study, SRCD spectroscopy characterizes two Förster Resonance Energy Transfer based study of wild type different protein fibrils populations. Furthermore, the SRCD and mutants A53T, A30P, E46K of α-synuclein revealed that based database of dried proteins spectra helps to discriminate each oligomer has inherent tendency to form the beta-sheet between different types of aggregated proteins and super- structure. Furthermore, FRET based study of wild type α- secondary structures. Secondary structure prediction for synuclein and different mutated forms states that the proper- protein aggregates is a challenging task due to the lack of the ties of oligomers during the aggregation rather than absolute calibrated and standardized CD reference data for the protein concentration is pertinent for neurodegeneration. The aggregates. Micsonai et al. has developed an algorithm, β- change in NADH fluorescence and emission can be corre- structure selection (BeStSel), which can easily characterize lated with the aggregation process. The fraction of bound the parallel and antiparallel β-sheets using CD spectroscopy NADH significantly increases as well as emission maxima. Circular Dichroism is a suitable method to analyze the sifted towards the shorter wavelength during α-synuclein conformational changes in the secondary and tertiary struc- aggregation. Aggregation is a multistep process so to ture of a protein and hence Circular Dichroism has tremen- study the aggregation process multiparametric techniques are dous application in the protein aggregation studies. needed. Fourier transform infrared spectroscopy (FTIR), intrinsic fluorescence; stationary fluorescence anisotropy and FLUORESCENCE SPECTROSCOPY other imaging methods have been employed to study the thermal aggregation of lysozyme. The study describes the Owing largely to the advent of various methods, fluores- indispensable features of aggregation involved in the addi- cence spectroscopy has emerged as one of the most powerful tion and fusion of intermediates proteins clusters. techniques to study the protein aggregation. Proteins contain their natural fluorophores, tryptophan and tyrosine residues, The APP (Amyloid precursor protein) proteolysis forms as well as the addition of external fluorophores like ANS (1, αβ and the accumulation of the same consequently results in 8-anilinonaphthalene-sulfonate), bis-ANS, MFC (2-(2-furyl)- the development of neurodegeneration. Florescence based 3-hydroxychromone), and others (Figure 3) in proteins using studies have shown that aggregated A-beta is more harmful either mutagenesis or chemical modification makes them a than its monomeric form as it affects the membrane structure suitable choice for fluorescence spectroscopic studies. An- and properties. Recently Ding et al. reported that the other advantage of fluorescence spectroscopy relies on its hydroxypropyl methylcellulose (HPMC) at high concentra- broad application, conducive methodology, the smaller tion (50% HPMC) induces the aggregation in collagen. amount of sample and high signal to noise ratio. Fluo- However, pyrene fluorescence and fluorescence anisotropy rescence spectroscopy is a multiparametric technique that studies state that at low concentration of collagen, aggrega- can measure the different valuable changes like fluorescence tion is inhibited but the high concentration of collagen in- intensity, spectral shifts, fluorescence anisotropy as well as duces the aggregation. Confocal single molecules detec- Emerging Methods for Structural Analysis of Protein Aggregation Protein & Peptide Letters, 2017, Vol. 24, No. 4 335 tion is a fluorescence based technique which utilizes FRET- Tosatto et al. have described a new single molecule in- labeled samples to deduce the anisotropy, fluorescence life- termolecular FRET (smFRET) approach to delve into the time diffusivity, and Förster transfer efficiencies simultane- oligomerisation process. In this method sample is incubated ously. This method helps us to comprehend the segregation at 37 °C with shaking after labeling with AlexaFluor 488 or of subpopulation of folded and misfolded protein during ag- AlexaFluor 594. Oligomers have high fluorescence intensity gregation. Duy et al. have studied the fluorescence as compared to monomers and those can be utilized to de- emission spectra of α-amylases during unfolding and aggre- duce the oligomer size as AlexaFluor 488 and AlexaFluor gation with respect to thermal and GndHCl treatment and 594 is a FRET pair. Recent advancement in smFRET observed the change in the red shift. (single molecule Förster resonance energy transfer), like fast flow microfluidic incorporation, made the data acquisition Infrared nanospectroscopy (nanoIR) is a combination of atomic force microscopy (AFM) and infrared spectroscopy fast resulting in the rescue of free diffusion of particles from the confocal volume. that can be applied to study protein aggregation. The first method can investigate the morphology and mechanical Recently, Iban et al. have studied the insulin aggregates properties during the course of protein aggregation , using nano-FTIR and elucidated the presence of a large while later is helpful to study the conformational changes in amount of α-helical structure in the fibrils which result in a protein secondary structures. high degree of protein organization. FTIR spectroscopy has been extensively used in numerous neurodegenerative Fluorescence spectroscopy using fluorescent dyes like diseases to study the protein misfolding and aggregation. In ThT, ANS, DCVJ, and bis-ANS have also been used to Alzheimer’s disease, amyloid-beta (Abeta), a small peptide, study the structural and physicochemical features of the is converted into aggregated fibrillary structure through oli- Transthyretin (TTR) protein aggregates formed during vari- gomeric intermediate. The specific alignment of β-sheet ous amyloid diseases such as familial amyloidotic polyneu- ropathy and senile systemic amyloidosis. Different fluo- strands within Abeta fibrils has been studied using FTIR. Furthermore, Petty and coworkers have studied the align- rescence spectroscopy methods help to delineate the steps ment of Abeta fibrils and have concluded the antiparallel involved in aggregation, making fluorescence spectroscopy arrangement of β-sheets across all strands using isotopically- an indispensable technique during protein aggregation stud- labeled Abeta. ies. Furthermore, various high-resolution fluorescence mi- croscopic techniques are suitable to visualize the protein α-synuclein protein is a major component of Lewy bod- aggregates formation and maturation; for example, total in- ies, abnormal protein aggregates formed in Parkinson's dis- ternal reflection fluorescence microscopy has been used to ease. FTIR and CD spectroscopy studies revealed that the α- see fibril growth. FRET-based imaging has been em- synuclein fibrils are basically antiparallel β-sheet structures ployed to study the aggregation and fibril formation in living like fibrillar Abeta in which N- and C- termini of α- cells. Fluorescence spectroscopy has truly revolution- synuclein takes part in intermolecular interactions. An- ized the protein aggregation studies; however, in near future other neurodegenerative disease in which protein aggrega- involvement of cell biology and fluorescence microscopy, tion is pronounced is Huntington disease. FTIR spectroscopy fluorescent amino acids and protein engineering as well as results have showed that the Huntington protein matures into fluorescent probe chemistry is also being anticipated for fur- fibrils through protofibrillar intermediates. ther advancement in protein aggregation studies. Protein misfolding and aggregation study using FTIR is not limited to in vitro study, but can also be used within the FOURIER TRANSFORM INFRARED SPECTROS- cells and tissue. Protein aggregation studies are very onerous COPY (FTIR) as protein aggregates are very small, initial oligomers in Fourier transform infrared spectroscopy (FTIR) is an- nanoscale range while larger in μM size. The spectral differ- other technique which is being employed to study the protein ences are also very slight which is another conundrum and aggregation. Each of the protein has C=O, C/N stretching requires spectra with the high signal as compared to the vibration, and N/H bending which corresponds to the Amide background. Synchrotron, with the infrared source, provides I (~1650 cm−1) and Amide II (~1540 cm−1) bands in FTIR, high brightness which makes it suitable to combat with the respectively. Amide I band corresponds to α-helix, β- aforesaid conundrum and has been employed to study aggre- sheet, turn, and unordered conformations while aggregated gation within diseased tissues of subjects suffering from proteins have a frequency around 1620–1625 cm−1 which is Huntington’s disease , Alzheimer’s disease [69, 79], attributed due to the specific hydrophobic environment. Parkinson’s disease , and scrapie [74a-c]. FTIR data interpretation has been very well described by FTIR spectroscopy as well as microspectroscopy has Nilsson et al. ; briefly, the primary event in FTIR spec- advanced the information on the mechanisms of protein mis- trum interpretation proceeds with examination of primary folding and aggregation in different neurodegenerative dis- sequence of a peptide. Commonly Asn and Gln amino acids eases including Parkinson's disease, Alzheimer's disease, are largely present in amyloid and contains the IR vibrations Huntington's disease, amyotrophic lateral sclerosis and prion which overlaps with the amide I band. Later the curve fitting diseases. However, new advancement in existing methodol- and subsequently analyzing the 2nd derivative provides the ogy such as in vivo imaging technologies will help us to un- further information. The interpretation of FTIR data ad- derstand further the protein aggregation mechanisms and vanced with the integration of the area of each band which therapeutic strategies. corresponds to the relative percentage of secondary structure present. 336 Protein & Peptide Letters, 2017, Vol. 24, No. 4 Khan and Kumar RAMAN SPECTROSCOPY edge of each step and key factors involved in each of the steps will provide the better understanding of mechanisms Raman Spectroscopy is another technique to study the involved. Various techniques which involve the structural secondary structure changes at various stages of protein ag- analysis of protein aggregation at the different level of pro- gregation and fibril formation. Deep UV Raman spec- tein aggregation will help in the advancement of protein ag- troscopy (DUVRR) coupled with hydrogen-deuterium ex- gregation studies. Furthermore, structural features of amy- change have added advantages over the conventional meth- loid at soluble and insoluble phase have been studied using ods as it helps to explore the insight fibril core structural different biophysical studies but studies on intermediary organization and backbone dihedral angle [81b, 82]. During phases of protein aggregation are still a conundrum at this protein aggregation, secondary structures are affected by the time. In the present minireview, we have contributed a brief local environment around aromatic amino acids, for example summary about the methods for structural analysis of protein tyrosine and phenylalanine that can be studied using aggregation using different biophysical techniques. The un- DUVRR. Protein aggregates acquire different morphologies derstanding of structural changes that occur during protein and secondary structures at different conditions like pH and aggregation using Fluorescence spectroscopy, FTIR, NMR, temperature that results into fibril polymorphism. Ra- CD and Raman spectroscopy will help us in the therapeutics man Spectroscopy along with different microscopic methods development for several diseases that are caused due to the such as scanning electron microscopy (SEM) and atomic protein aggregation. Thus, the information obtained using force microscopy (AFM) is a very helpful tool to study this these techniques along with other multidisciplinary ap- fibril polymorphism [84, 85]. proaches will advance our understanding and knowledge of Protein aggregation is a sequential process in which low protein aggregation and misfolding and associated neurode- energy β-sheet oligomers aggregate into multiple assemblies generative disease therapeutics. with a nucleus which further converts into filamentous and subsequently fibrils when a critical concentration of CONFLICT OF INTEREST nucleus is achieved. The detection of these prefibrills is ex- tremely challenging as these fibrils are very unstable and The authors confirm that this article content has no found in exceedingly low concentration. Shashilov et al used conflict of interest. two-dimensional correlation spectroscopy (2DCoS) along with DUVRR to study the human white egg lysozyme ACKNOWLEDGEMENTS (HEWL) correlation between Cα– H band and the amide I Authors thank to the Department of Science & Technol- band at different time points during the protein aggregation ogy, Govt. of India for their support through grant [82b]. Protein aggregation in insulin has also been studied [SR/FT/LS-29/2012]. Authors thank to Department of using DUVRR spectroscopy. However, DUVVRR is not Biotechnology, GoI for their support through SRF to Mr. able to detect the β -sheet rich prefibrillar so improved Eshan Khan [DBT/JRF/14/AL/234]. methods to study this β-sheet rich prefibrillar are required. Surface-enhanced Raman spectroscopy (SERS) is an- REFERENCES other ultrasensitive technique based on the fact that Raman Sawaya, M.R.; Sambashivan, S.; Nelson, R.; Ivanova, M.I.; Sievers, signal amplification increases drastically if any molecule is S.A.; Apostol, M.I.; Thompson, M.J.; Balbirnie, M.; Wiltzius, J.J.; within the range of 1-2 nm of noble nanostructures. Insulin McFarlane, H.T.; Madsen, A.O.; Riekel, C.; Eisenberg, D. Atomic aggregates were studied using SERS at different stages of structures of amyloid cross-beta spines reveal varied steric zippers. Nature, 2007, 447(7143), 453-457. protein aggregation at pH 1.6. Protein aggregates were cen- Bernadó, P.; Åkerud, T.; García de la Torre, J.; Akke, M.; Pons, M. trifuged and the supernatant was adsorbed using gold Combined Use of NMR Relaxation Measurements and Hydrody- nanoparticle assuming each adsorbed oligomers have spe- namic Calculations To Study Protein Association. Evidence for cific ‘SERS fingerprints’ characterized by amide I band in Tetramers of Low Molecular Weight Protein Tyrosine Phosphatase 1665 – 1680 cm region. Kurouski et al in their study con- in Solution. J. Am. Chem. Soc., 2003, 125(4), 916-923. Kneller, J.M.; Lu, M.; Bracken, C. An Effective Method for the cluded that the number of insulin aggregates increase ini- Discrimination of Motional Anisotropy and Chemical Exchange. J. tially and then decrease slowly. Tip-enhanced Raman Am. Chem. Soc., 2002, 124(9), 1852-1853. spectroscopy (TERS) is another sensitive technique to study Indrawati, L.; Stroshine, R.L.; Narsimhan, G. Low-field NMR: A protein aggregation and AFM-based TERS has been reported tool for studying protein aggregation. J. Sci. Food Agric., 2007, 87(12), 2207-2216. to study the structural organization of insulin fibrils at the Jiji, A.C.; Shine, A.; Vijayan, V. Direct Observation of Aggrega- nanoscale and the amino acids composition of amyloid fi- tion-Induced Backbone Conformational Changes in Tau Peptides. brils. Angew. Chemie (International ed. in English), 2016, 55(38), 11562- 11566. Lim, K.H.; Dasari, A.K.; Hung, I.; Gan, Z.; Kelly, J.W.; Wemmer, CONCLUSION D.E. Structural Changes Associated with Transthyretin Misfolding Therapeutic development requires thorough understand- and Amyloid Formation Revealed by Solution and Solid-State NMR. Biochemistry, 2016, 55(13), 1941-1944. ing of the cause and the mechanisms involved for any dis- Chang, X.; Keller, D.; O’Donoghue, S.I.; Led, J.J. NMR studies of ease and the same is true for the neurodegenerative diseases. the aggregation of glucagon-like peptide-1: formation of a symmet- Different therapeutic approaches targeting the neurodegen- ric helical dimer. FEBS Lett., 2002, 515(1–3), 165-170. erative disease caused by protein aggregation are known Carver, J.P.; Richards, R.E. A general two-site solution for the chemical exchange produced dependence of T2 upon the carr- albeit the lack of meticulous knowledge of protein aggrega- Purcell pulse separation. J. Magnet. Resonance (1969), 1972, 6(1), tion mechanism makes these approaches insignificant. As 89-105. protein aggregation is a multi-step process, hence the knowl- Emerging Methods for Structural Analysis of Protein Aggregation Protein & Peptide Letters, 2017, Vol. 24, No. 4 337 Korzhnev, D.M.; Salvatella, X.; Vendruscolo, M.; Di Nardo, A.A.; Colvin, M.T.; Silvers, R.; Ni, Q.Z.; Can, T.V.; Sergeyev, I.; Rosay, Davidson, A.R.; Dobson, C.M.; Kay, L.E. Low-populated folding M.; Donovan, K.J.; Michael, B.; Wall, J.; Linse, S.; Griffin, R.G. intermediates of Fyn SH3 characterized by relaxation dispersion Atomic Resolution Structure of Monomorphic Abeta42 Amyloid NMR. Nature, 2004, 430(6999), 586-590. Fibrils. J. Am. Chem. Soc., 2016, 138(30), 9663-9674. Shen, Y.; Lange, O.; Delaglio, F.; Rossi, P.; Aramini, J.M.; Liu, G.; Roche, J.; Shen, Y.; Lee, J.H.; Ying, J.; Bax, A. Monomeric Eletsky, A.; Wu, Y.; Singarapu, K.K.; Lemak, A.; Ignatchenko, A.; Abeta(1-40) and Abeta(1-42) Peptides in Solution Adopt Very Arrowsmith, C.H.; Szyperski, T.; Montelione, G.T.; Baker, D.; Bax, Similar Ramachandran Map Distributions That Closely Resemble A. Consistent blind protein structure generation from NMR chemi- Random Coil. Biochemistry, 2016, 55(5), 762-775. cal shift data. Proc. Nat. Acad. Sci. USA, 2008, 105(12), 4685- Hoop, C.L.; Lin, H.K.; Kar, K.; Magyarfalvi, G.; Lamley, J.M.; 4690. Boatz, J.C.; Mandal, A.; Lewandowski, J.R.; Wetzel, R.; van der Hansen, D.F.; Vallurupalli, P.; Kay, L.E. Using relaxation disper- Wel, P.C. Huntingtin exon 1 fibrils feature an interdigitated beta- sion NMR spectroscopy to determine structures of excited, invisible hairpin-based polyglutamine core. 2016, 113(6), 1546-1551. protein states. J. Biomol. NMR, 2008, 41(3), 113-120. Paravastu, A.K.; Leapman, R.D.; Yau, W.M.; Tycko, R. Molecular Baldwin, A.J.; Walsh, P.; Hansen, D.F.; Hilton, G.R.; Benesch, structural basis for polymorphism in Alzheimer's beta-amyloid fi- J.L.; Sharpe, S.; Kay, L.E. Probing dynamic conformations of the brils. Proc. Nat. Acad. Sci. USA, 2008, 105(47), 18349-18354. high-molecular-weight alphaB-crystallin heat shock protein ensem- Crescenzi, O.; Tomaselli, S.; Guerrini, R.; Salvadori, S.; D'Ursi, ble by NMR spectroscopy. J. Am. Chem. Soc., 2012, 134(37), A.M.; Temussi, P.A.; Picone, D. Solution structure of the Alz- 15343-15350. heimer amyloid beta-peptide (1-42) in an apolar microenvironment. Neira, J.L. NMR as a tool to identify and characterize protein fold- Similarity with a virus fusion domain. FEBS, 2002, 269(22), 5642- ing intermediates. Arch. Biochem. Biophys., 2013, 531(1–2), 90-99. 5648. Zeeb, M.; Balbach, J. Protein folding studied by real-time NMR Vivekanandan, S.; Brender, J.R.; Lee, S.Y.; Ramamoorthy, A. A spectroscopy. Methods (San Diego, Calif.), 2004, 34(1), 65-74. partially folded structure of amyloid-beta(1-40) in an aqueous envi- (a) Balbach, J.; Forge, V.; van Nuland, N.A.; Winder, S.L.; Hore, ronment. Biochem. Biophys. Res. Commun., 2011, 411(2), 312-316. P.J.; Dobson, C.M. Following protein folding in real time using Fonar, G.; Samson, A.O. NMR structure of the water soluble NMR spectroscopy. Nat. Struct. Biol., 1995, 2(10), 865-70; (b) Bal- Abeta17-34 peptide. Biosci. Reports, 2014, 34(6), e00155. bach, J.; Forge, V.; Lau, W.S.; van Nuland, N.A.; Brew, K.; Dob- Fawzi, N.L.; Ying, J.; Torchia, D.A.; Clore, G.M. Probing exchange son, C.M. Protein folding monitored at individual residues during a kinetics and atomic resolution dynamics in high-molecular-weight two-dimensional NMR experiment. Science (New York, NY), 1996, complexes using dark-state exchange saturation transfer NMR spec- 274(5290), 1161-1163. troscopy. Nat. Protoc, 2012, 7(8), 1523-1533. Sillen, A.; Leroy, A.; Wieruszeski, J.M.; Loyens, A.; Beauvillain, Fawzi, N.L.; Ying, J.; Ghirlando, R.; Torchia, D.A.; Clore, G.M. J.C.; Buee, L.; Landrieu, I.; Lippens, G. Regions of tau implicated Atomic-resolution dynamics on the surface of amyloid-beta proto- in the paired helical fragment core as defined by NMR. Chembio- fibrils probed by solution NMR. Nature, 2011, 480(7376), 268-272. chem, 2005, 6(10), 1849-1856. (a) Knowles, T.P.; Waudby, C.A.; Devlin, G.L.; Cohen, S.I.; Ferguson, N.; Becker, J.; Tidow, H.; Tremmel, S.; Sharpe, T.D.; Aguzzi, A.; Vendruscolo, M.; Terentjev, E.M.; Welland, M.E.; Krause, G.; Flinders, J.; Petrovich, M.; Berriman, J.; Oschkinat, H.; Dobson, C.M. An analytical solution to the kinetics of breakable Fersht, A.R. General structural motifs of amyloid protofilaments. filament assembly. Science (New York NY), 2009, 326(5959), 1533- Proc. Nat. Acad. Sci., 2006, 103(44), 16248-16253. 7; (b) Cohen, S.I.A.; Linse, S.; Luheshi, L.M.; Hellstrand, E.; Jaroniec, C.P.; MacPhee, C.E.; Bajaj, V.S.; McMahon, M.T.; Dob- White, D.A.; Rajah, L.; Otzen, D.E.; Vendruscolo, M.; Dobson, son, C.M.; Griffin, R.G. High-resolution molecular structure of a C.M.; Knowles, T.P.J. Proliferation of amyloid-β42 aggregates oc- peptide in an amyloid fibril determined by magic angle spinning curs through a secondary nucleation mechanism. Proc. Nat. Acad. NMR spectroscopy. Proc. Nat. Acad. Sci. USA, 2004, 101(3), 711- Sci., 2013, 110(24), 9758-9763; (c) Buell, A.K.; Galvagnion, C.; 716. Gaspar, R.; Sparr, E.; Vendruscolo, M.; Knowles, T.P.J.; Linse, S.; Petkova, A.T.; Ishii, Y.; Balbach, J.J.; Antzutkin, O.N.; Leapman, Dobson, C.M. Solution conditions determine the relative impor- R.D.; Delaglio, F.; Tycko, R. A structural model for Alzheimer's tance of nucleation and growth processes in α -synuclein aggrega- beta -amyloid fibrils based on experimental constraints from solid tion. Proc. Nat. Acad. Sci. USA, 2014, 111(21), 7671-7676. state NMR. Proc. Nat. Acad. Sci. USA, 2002, 99(26), 16742-16747. (a) Dyson, H.J.; Wright, P.E. Nuclear magnetic resonance methods (a) Siemer, A.B.; Arnold, A.A.; Ritter, C.; Westfeld, T.; Ernst, M.; for elucidation of structure and dynamics in disordered states. Riek, R.; Meier, B.H. Observation of highly flexible residues in Methods Enzymol., 2001, 339, 258-70; (b) Hennig, M.; Bermel, W.; amyloid fibrils of the HET-s prion. J. Am. Chem. Soc., 2006, Spencer, A.; Dobson, C.M.; Smith, L.J.; Schwalbe, H. Side-chain 128(40), 13224-13228; (b) Siemer, A.B.; Ritter, C.; Steinmetz, conformations in an unfolded protein: chi1 distributions in dena- M.O.; Ernst, M.; Riek, R.; Meier, B.H. 13C, 15N Resonance As- tured hen lysozyme determined by heteronuclear 13C, 15N NMR signment of Parts of the HET-s Prion Protein in its Amyloid Form. spectroscopy. J. Mol. Biol., 1999, 288(4), 705-723; (c) Liu, A.; J. Biomol. NMR, 2006, 34(2), 75-87. Riek, R.; Wider, G.; von Schroetter, C.; Zahn, R.; Wüthrich, K. Eftekharzadeh, B.; Piai, A.; Chiesa, G.; Mungianu, D.; Garcia, J.; NMR experiments for resonance assignments of 13C, 15N doubly- Pierattelli, R.; Felli, I.C.; Salvatella, X. Sequence Context Influ- labeled flexible polypeptides: Application to the human prion pro- ences the Structure and Aggregation Behavior of a PolyQ Tract. tein hPrP(23–230). J. Bbiomol. NMR, 2000, 16(2), 127-138; (d) Biophys. J., 2016, 110(11), 2361-2366. Zhang, O.; Forman-Kay, J.D.; Shortle, D.; Kay, L.E. Triple- Suzuki, Y.; Brender, J.R.; Hartman, K.; Ramamoorthy, A.; G. resonance NOESY-based experiments with improved spectral reso- Marsh, E.N. Alternative Pathways of Human Islet Amyloid Poly- lution: applications to structural characterization of unfolded, par- peptide Aggregation Distinguished by (19)F NMR-Detected Kinet- tially folded and folded proteins. J. Biomolecular NMR, 1997, 9(2), ics of Monomer Consumption. Biochemistry, 2012, 51(41), 8154- 181-200. 8162. Joshi, V.; Shivach, T.; Yadav, N.; Rathore, A.S. Circular Dichroism (a) Bertini, I.; Luchinat, C.; Parigi, G.; Ravera, E. SedNMR: on the Spectroscopy as a Tool for Monitoring Aggregation in Monoclonal edge between solution and solid-state NMR. Acc. Chem. Res., 2013, Antibody Therapeutics. Anal. Chem., 2014, 86(23), 11606-11613. 46(9), 2059-69; (b) Tycko, R. Progress towards a molecular-level (a) McPhie, P. CD studies on films of amyloid proteins and poly- structural understanding of amyloid fibrils. Curr. Opin. Struct. peptides: quantitative g-factor analysis indicates a common folding Biol., 2004, 14(1), 96-103; (c) Laws, D.D.; Bitter, H.-M.L.; Liu, K.; motif. Biopolymers, 2004, 75(2), 140-7; (b) Baker, B.R.; Garrell, Ball, H.L.; Kaneko, K.; Wille, H.; Cohen, F.E.; Prusiner, S.B.; R.L. g-Factor analysis of protein secondary structure in solutions Pines, A.; Wemmer, D.E. Solid-State NMR Studies of the Secon- and thin films. Faraday Discuss, 2004, 126(0), 209-222. dary Structure of a Mutant Prion Protein Fragment of 55 Residues (a) Shanmugam, G.; Jayakumar, R. Structural analysis of amyloid That Induces Neurodegeneration. Proc. Nat. Acad. Sci. USA, 2001, beta peptide fragment (25-35) in different microenvironments. Bio- 98(20), 11686-11690; (d) Siemer, A.B.; Ritter, C.; Ernst, M.; Riek, polymers, 2004, 76(5), 421-34; (b) Shanmugam, G.; Polavarapu, R.; Meier, B.H. High-resolution solid-state NMR spectroscopy of P.L. Structure of Aβ(25–35) Peptide in Different Environments. the prion protein HET-s in its amyloid conformation. Angewandte Biophys. J., 2004, 87(1), 622-630. Chemie (International ed. in English), 2005, 44(16), 2441-2444. 338 Protein & Peptide Letters, 2017, Vol. 24, No. 4 Khan and Kumar Hu, H.Y.; Li, Q.; Cheng, H.C.; Du, H.N. beta-sheet structure forma- synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F al- tion of proteins in solid state as revealed by circular dichroism spec- pha-synuclein. J. Mol. Biol., 2005, 353(2), 357-372. troscopy. Biopolymers, 2001, 62(1), 15-21. Tosatto, L.; Horrocks, M.H.; Dear, A.J.; Knowles, T.P.; Dalla Harada, T.; Kuroda, R. Circular Dichroism Measurement of a Pro- Serra, M.; Cremades, N.; Dobson, C.M.; Klenerman, D. Single- tein in Dried Thin Films. Chem. Lett., 2002, 3(3), 326-327. molecule FRET studies on alpha-synuclein oligomerization of Park- Harada, T.; Kuroda, R. CD measurements of beta-amyloid (1-40) inson's disease genetically related mutants. Sci. Reports, 2015, 5, and (1-42) in the condensed phase. Biopolymers, 2011, 95(2), 127- 16696. 134. Plotegher, N.; Stringari, C.; Jahid, S.; Veronesi, M.; Girotto, S.; Hu, H.-Y.; Chen, Y.-N.; Xu, S.-Q.; Xu, G.-J. Structural Transfor- Gratton, E.; Bubacco, L. NADH fluorescence lifetime is an en- mation of the Peptide Fragments from the Reactive Center Loops of dogenous reporter of alpha-synuclein aggregation in live cells. Serpins: Circular Dichroic Studies. Chinese J. Chem., 2001, 19(10), FASEB J., 2015, 29(6), 2484-2494. 954-959. Giugliarelli, A.; Tarpani, L.; Latterini, L.; Morresi, A.; Paolantoni, (a) Bukauskas, V.; Šetkus, A.; Šimkienė, I.; Tumėnas, S.; M.; Sassi, P. Spectroscopic and Microscopic Studies of Aggrega- Kašalynas, I.; Rėza, A.; Babonas, J.; Časaitė, V.; Povilonienė, S.; tion and Fibrillation of Lysozyme in Water/Ethanol Solutions. J. Meškys, R. Solid surface dependent layering of self-arranged struc- Phys. Chem B, 2015, 119(41), 13009-13017. tures with fibril-like assemblies of alpha-synuclein. Appl. Surface Ding, C.; Zhang, M.; Li, G. Fluorescence study on the aggregation Sci., 2012, 258(10), 4383-4390; (b) Zibaee, S.; Fraser, G.; Jakes, R.; of collagen molecules in acid solution influenced by hydroxypropyl Owen, D.; Serpell, L.C.; Crowther, R.A.; Goedert, M. Human beta- methylcellulose. Carbohydrate Polymers, 2016, 136, 224-231. synuclein rendered fibrillogenic by designed mutations. J. Biol. Hillger, F.; Nettels, D.; Dorsch, S.; Schuler, B. Detection and analy- Chem., 2010, 285(49), 38555-38567. sis of protein aggregation with confocal single molecule fluores- (a) Measey, T.J.; Schweitzer-Stenner, R. Vibrational circular di- cence spectroscopy. J. Fluorescence, 2007, 17(6), 759-765. chroism as a probe of fibrillogenesis: the origin of the anomalous Duy, C.; Fitter, J. How Aggregation and Conformational Scram- intensity enhancement of amyloid-like fibrils. J. Am. Chem. Soc., bling of Unfolded States Govern Fluorescence Emission Spectra. 2011, 133(4), 1066-76; (b) Ma, S.; Cao, X.; Mak, M.; Sadik, A.; Biophys. J., 2006, 90(10), 3704-3711. Walkner, C.; Freedman, T.B.; Lednev, I.K.; Dukor, R.K.; Nafie, Adamcik, J.; Mezzenga, R. Study of amyloid fibrils via atomic L.A. Vibrational circular dichroism shows unusual sensitivity to force microscopy. Curr. Opin. Colloid Interface Sci., 2012, 17(6), protein fibril formation and development in solution. J. Am. Chem. 369-376. Soc., 2007, 129(41), 12364-12365. Sarroukh, R.; Goormaghtigh, E.; Ruysschaert, J.M.; Raussens, V. Measey, T.J.; Schweitzer-Stenner, R. Vibrational Circular Dichro- ATR-FTIR: a "rejuvenated" tool to investigate amyloid proteins. ism as a Probe of Fibrillogenesis: The Origin of the Anomalous In- Biochim. Biophys. Acta, 2013, 1828(10), 2328-2338. tensity Enhancement of Amyloid-like Fibrils. J. Am. Chem. Soc., Lindgren, M.; Sorgjerd, K.; Hammarstrom, P. Detection and char- 2011, 133(4), 1066-1076. acterization of aggregates, prefibrillar amyloidogenic oligomers, Benjwal, S.; Verma, S.; Röhm, K.-H.; Gursky, O. Monitoring pro- and protofibrils using fluorescence spectroscopy. Biophys. J., 2005, tein aggregation during thermal unfolding in circular dichroism ex- 88(6), 4200-4212. periments. Protein Sci., 2006, 15(3), 635-639. (a) Collins, S.R.; Douglass, A.; Vale, R.D.; Weissman, J.S. Mecha- Tsai, A.M.; van Zanten, J.H.; Betenbaugh, M.J.I. Study of protein nism of Prion Propagation: Amyloid Growth Occurs by Monomer aggregation due to heat denaturation: A structural approach using Addition. PLoS Biol., 2004, 2(10), e321; (b) Ban, T.; Yamaguchi, circular dichroism spectroscopy, nuclear magnetic resonance, and K.; Goto, Y. Direct observation of amyloid fibril growth, propaga- static light scattering. Biotechnol. Bioeng., 1998, 59(3), 273-280. tion, and adaptation. Accounts Chem. Res., 2006, 39(9), 663-670. Vermeer, A.W.; Norde, W. The thermal stability of immunoglobu- Roberti, M.J.; Bertoncini, C.W.; Klement, R.; Jares-Erijman, E.A.; lin: unfolding and aggregation of a multi-domain protein. Biophys. Jovin, T.M. Fluorescence imaging of amyloid formation in living J., 2000, 78(1), 394-404. cells by a functional, tetracysteine-tagged [alpha]-synuclein. Nat. Barrow, C.J.; Yasuda, A.; Kenny, P.T.; Zagorski, M.G. Solution Methods, 2007, 4(4), 345-351. conformations and aggregational properties of synthetic amyloid Haris, P.I.; Severcan, F. FTIR spectroscopic characterization of beta-peptides of Alzheimer's disease. Analysis of circular dichroism protein structure in aqueous and non-aqueous media. J. Mol. Catal. spectra. J. Mol. Biol., 1992, 225(4), 1075-1093. B Enzym., 1999, 7(1-4), 207-221. Matsuo, K.; Hiramatsu, H.; Gekko, K.; Namatame, H.; Taniguchi, Miller, L.M.; Wang, Q.; Telivala, T.P.; Smith, R.J.; Lanzirotti, A.; M.; Woody, R.W. Characterization of Intermolecular Structure of Miklossy, J. Synchrotron-based infrared and X-ray imaging shows β2-Microglobulin Core Fragments in Amyloid Fibrils by Vacuum- focalized accumulation of Cu and Zn co-localized with beta- Ultraviolet Circular Dichroism Spectroscopy and Circular Dichro- amyloid deposits in Alzheimer's disease. J. Struct. Biol., 2006, ism Theory. J. Phys. Chem. B, 2014, 118(11), 2785-2795. 155(1), 30-37. Wallace, B.A. Protein characterisation by synchrotron radiation Nilsson, M.R. Techniques to study amyloid fibril formation in vitro. circular dichroism spectroscopy. Quart. Rev. Biophys., 2009, 42(4), Methods (San Diego, Calif.), 2004, 34(1), 151-160. 317-370. Horrocks, M.H.; Li, H.; Shim, J.U.; Ranasinghe, R.T.; Clarke, Ruzza, P.; Hussain, R.; Biondi, B.; Calderan, A.; Tessari, I.; Bu- R.W.; Huck, W.T.; Abell, C.; Klenerman, D. Single molecule fluo- bacco, L.; Siligardi, G., Effects of Trehalose on Thermodynamic rescence under conditions of fast flow. Anal. Chem., 2012, 84(1), Properties of Alpha-synuclein Revealed through Synchrotron Ra- 179-185. diation Circular Dichroism. Biomolecules, 2015, 5(2), 724-734. Amenabar, I.; Poly, S.; Nuansing, W.; Hubrich, E.H.; Govyadinov, Nesgaard, L.W.; Hoffmann, S.V.; Andersen, C.B.; Malmendal, A.; A.A.; Huth, F.; Krutokhvostov, R.; Zhang, L.; Knez, M.; Heberle, Otzen, D.E. Characterization of dry globular proteins and protein J.; Bittner, A.M.; Hillenbrand, R. Structural analysis and mapping fibrils by synchrotron radiation vacuum UV circular dichroism. of individual protein complexes by infrared nanospectroscopy. Nat. Biopolymers, 2008, 89(9), 779-795. Commun., 2013, 4, 2890. Micsonai, A.; Wien, F.; Kernya, L.; Lee, Y.-H.; Goto, Y.; Réfré- Ii, K. The role of beta-amyloid in the development of Alzheimer's giers, M.; Kardos, J. Accurate secondary structure prediction and disease. Drugs Aging, 1995, 7(2), 97-109. fold recognition for circular dichroism spectroscopy. Proc. Nat. (a) Kneipp, J.; Miller, L.M.; Spassov, S.; Sokolowski, F.; Lasch, P.; Acad. Sci. USA, 2015, 112(24), E3095-E3103. Beekes, M.; Naumann, D. Scrapie-infected cells, isolated prions, Munishkina, L.A.; Fink, A.L. Fluorescence as a method to reveal and recombinant prion protein: a comparative study. Biopolymers, structures and membrane-interactions of amyloidogenic proteins. 2004, 74(1-2), 163-7; (b) Kretlow, A.; Wang, Q.; Beekes, M.; Biochim. Biophys. Acta, 2007, 1768(8), 1862-85. Naumann, D.; Miller, L.M. Changes in protein structure and distri- Sahoo, B.; Drombosky, K.W.; Wetzel, R. Fluorescence Correlation bution observed at pre-clinical stages of scrapie pathogenesis. Bio- Spectroscopy: A Tool to Study Protein Oligomerization and Aggre- chim. Biophys. Acta, 2008, 1782(10), 559-65; (c) Kretlow, A.; gation In Vitro and In Vivo. Methods Mol. Biol. (Clifton, N.J.), Wang, Q.; Kneipp, J.; Lasch, P.; Beekes, M.; Miller, L.; Naumann, 2016, 1345, 67-87. D. FTIR-microspectroscopy of prion-infected nervous tissue. Bio- Kaylor, J.; Bodner, N.; Edridge, S.; Yamin, G.; Hong, D.P.; Fink, chim. Biophys. Acta, 2006, 1758(7), 948-59; (d) Susi, H.; Michael A.L. Characterization of oligomeric intermediates in alpha- Byler, D. Protein structure by Fourier transform infrared spectros- copy: Second derivative spectra. Biochem. Biophys. Res. Commun., Emerging Methods for Structural Analysis of Protein Aggregation Protein & Peptide Letters, 2017, Vol. 24, No. 4 339 1983, 115(1), 391-397; (e) Arrondo, J.L.; Muga, A.; Castresana, J.; amide I linewidths in native states and fibrils. J. Mol. Biol., 2003, Goni, F.M. Quantitative studies of the structure of proteins in solu- 330(2), 431-42; (b) Oladepo, S.A.; Xiong, K.; Hong, Z.; Asher, tion by Fourier-transform infrared spectroscopy. Prog. Biophys. S.A.; Handen, J.; Lednev, I.K. UV resonance Raman investigations Mol. Biol., 1993, 59(1), 23-56; (f) Hering, J.A.; Innocent, P.R.; of peptide and protein structure and dynamics. Chem. Rev., 2012, Haris, P.I. Neuro-fuzzy structural classification of proteins for im- 112(5), 2604-2628. proved protein secondary structure prediction. Proteomics, 2003, (a) Shashilov, V.A.; Lednev, I.K. Advanced Statistical and Numeri- 3(8), 1464-75; (g) Hering, J.A.; Innocent, P.R.; Haris, P.I. Towards cal Methods for Spectroscopic Characterization of Protein Struc- developing a protein infrared spectra databank (PISD) for pro- tural Evolution. Chem. Rev., 2010, 110(10), 5692-5713; (b) Xu, M.; teomics research. Proteomics, 2004, 4(8), 2310-2319. Shashilov, V.; Lednev, I.K. Probing the cross-beta core structure of Conway, K.A.; Harper, J.D.; Lansbury, P.T.Jr. Fibrils formed in amyloid fibrils by hydrogen-deuterium exchange deep ultraviolet vitro from alpha-synuclein and two mutant forms linked to Parkin- resonance Raman spectroscopy. J. Am. Chem. Soc., 2007, 129(36), son's disease are typical amyloid. Biochemistry, 2000, 39(10), 2552- 11002-11003. 2563. Shashilov, V.A.; Lednev, I.K. 2D correlation deep UV resonance Ulrih, N.P.; Barry, C.H.; Fink, A.L. Impact of Tyr to Ala mutations raman spectroscopy of early events of lysozyme fibrillation: kinetic on alpha-synuclein fibrillation and structural properties. Biochim. mechanism and potential interpretation pitfalls. J. Am. Chem. Soc., Biophys. Acta, 2008, 1782(10), 581-585. 2008, 130(1), 309-317. Poirier, M.A.; Li, H.; Macosko, J.; Cai, S.; Amzel, M.; Ross, C.A. Kurouski, D.; Lu, X.; Popova, L.; Wan, W.; Shanmugasundaram, Huntingtin spheroids and protofibrils as precursors in polyglu- M.; Stubbs, G.; Dukor, R.K.; Lednev, I.K.; Nafie, L.A. Is Su- tamine fibrilization. J. Biol. Chem., 2002, 277(43), 41032-41037. pramolecular Filament Chirality the Underlying Cause of Major Bonda, M.; Perrin, V.; Vileno, B.; Runne, H.; Kretlow, A.; Forró, Morphology Differences in Amyloid Fibrils? J. Am. Chem. Soc., L.; Luthi-Carter, R.; Miller, L. M.; Jeney, S. Synchrotron Infrared 2014, 136(6), 2302-2312. Microspectroscopy Detecting the Evolution of Huntington’s Dis- (a) Anderson, M.; Bocharova, O.V.; Makarava, N.; Breydo, L.; ease Neuropathology and Suggesting Unique Correlates of Dys- Salnikov, V.V.; Baskakov, I.V. Polymorphism and ultrastructural function in White versus Gray Brain Matter. Anal. Chem., 2011, organization of prion protein amyloid fibrils: an insight from high 83(20), 7712-7720. resolution atomic force microscopy. J. Mol. Biol., 2006, 358(2), (a) Choo, L.P.; Wetzel, D.L.; Halliday, W.C.; Jackson, M.; LeVine, 580-96; (b) Fandrich, M. On the structural definition of amyloid fi- S.M.; Mantsch, H.H. In situ characterization of beta-amyloid in brils and other polypeptide aggregates. CMLS, 2007, 64(16), 2066- Alzheimer's diseased tissue by synchrotron Fourier transform infra- 2078. red microspectroscopy. Biophys. J., 1996, 71(4), 1672-9; (b) Rak, Sorci, M.; Grassucci, R.A.; Hahn, I.; Frank, J.; Belfort, G. Time- M.; Del Bigio, M.R.; Mai, S.; Westaway, D.; Gough, K. Dense-core dependent insulin oligomer reaction pathway prior to fibril forma- and diffuse Abeta plaques in TgCRND8 mice studied with synchro- tion: cooling and seeding. Proteins, 2009, 77(1), 62-73. tron FTIR microspectroscopy. Biopolymers, 2007, 87(4), 207-17; Kurouski, D.; Sorci, M.; Postiglione, T.; Belfort, G.; Lednev, I.K. (c) Leskovjan, A.C.; Lanzirotti, A.; Miller, L.M. Amyloid plaques Detection and structural characterization of insulin prefibrilar oli- in PSAPP mice bind less metal than plaques in human Alzheimer's gomers using surface enhanced Raman spectroscopy. Biotechnol. disease. NeuroImage, 2009, 47(4), 1215-1220. Prog., 2014, 30(2), 488-495. Szczerbowska-Boruchowska, M.; Dumas, P.; Kastyak, M.Z.; (a) Kurouski, D.; Deckert-Gaudig, T.; Deckert, V.; Lednev, I.K. Chwiej, J.; Lankosz, M.; Adamek, D.; Krygowska-Wajs, A. Bio- Structure and Composition of Insulin Fibril Surfaces Probed by molecular investigation of human substantia nigra in Parkinson's TERS. J. Am. Chem. Soc., 2012, 134(32), 13323-13329; (b) disease by synchrotron radiation Fourier transform infrared mi- Kurouski, D.; Deckert-Gaudig, T.; Deckert, V.; Lednev, Igor K. crospectroscopy. Arch. Biochem. Biophys., 2007, 459(2), 241-248. Surface Characterization of Insulin Protofilaments and Fibril Poly- (a) Dong, J.; Wan, Z.; Popov, M.; Carey, P.R.; Weiss, M.A. Insulin morphs Using Tip-Enhanced Raman Spectroscopy (TERS). Bio- assembly damps conformational fluctuations: Raman analysis of phys. J., 2014, 106(1), 263-271.