alzheimer s disease ( ad ) is the 
 most common form of dementia , 
 affecting over 44 million people worldwide . ad is a progressive brain disorder that damages synapses and brain 
 cells and causes decline of memory , loss of cognitive and executive 
 functions , and eventually death . there 
 is no known cure for ad , and the etiology of the disease is not well 
 understood . previous studies have shown that amyloid -protein 
 ( a ) plays an important role in ad pathogenesis . a is , in reality , not one but a group of peptides 
 derived from the type-1 transmembrane protein , amyloid -protein 
 precursor ( app ) , through cleavage by - and -secretases . a exists in vivo primarily as 40 or 42 
 amino acid long peptides with a40 constituting 90% and a42 
 9% of all a species . even 
 though a42 is a relatively minor constituent , it has been found 
 to be the primary component of amyloid plaques , which are an important 
 pathologic hallmark of ad . in the plaques formation 
 of a fibrils in vitro and in vivo is a complex process involving multiple intermediate oligomeric 
 species , which are highly neurotoxic and are believed to be the proximal 
 neurotoxins acting in ad . immediately upon dissolution in vitro , a42 forms small oligomers , including dimers and tetramers , 
 as well as paranuclei ( pentamers and hexamers ) that self - associate 
 to form decamers and dodecamers . among these species , 
 the 56 kda dodecamer has been identified as a plausible cause of memory 
 deficits in the ad brain and in transgenic 
 mice . thus , targeting and remodeling 
 a42 oligomers is a primary therapeutic strategy for ad . one strategy for ad treatment and prevention 
 is preventing a 
 formation , which could be achieved by inhibiting or modulating the 
 - and/or -secretase enzymes . however , this approach has been problematic because both secretases 
 cleave substrates other than app , which are important in other functional 
 biological processes . another potential strategy is enhancing clearance 
 of a oligomers and aggregates from the brain . this can be accomplished either by facilitating degradation 
 of a by proteases or other clearance 
 mechanisms , or by directly remodeling the aggregation of a 
 into clearance - prone structures using suitable peptides or small molecules . many natural proteins , peptides , and small molecules have been 
 discovered to interact with a and modulate a self - assembly . among them , small molecules are particularly 
 attractive as a direct therapeutic strategy for the treatment of ad . for instance , biologically active molecules from green - tea ( ( )epigallocatechin-3-gallate , 
 egcg ) or the indian spice turmeric ( curcumin ) have been found to prevent 
 a aggregation and inhibit a-induced toxicity . inositol stereoisomers have been found to interact with a 
 and attenuate its neurotoxic effects . z - phe - ala - diazomethylketone 
 ( padk ) , which acts as a lysosomal modulator up - regulating the expression 
 of cathepsin b in lysosomes , has been shown to interact with a42 
 directly and modulate a42 oligomerization . c - terminal fragments of a42 and many polyphenol 
 molecules have been shown to inhibit a oligomerization , aggregation , 
 and toxicity , and molecules have been specially designed to inhibit not only aggregation 
 of a but also its metal binding and oxidation . molecular tweezers ( mts ) , which possess a torus - shaped 
 cavity with 
 a surrounding belt of alternating aromatic and aliphatic rings , were 
 designed to serve as host molecules binding specifically to lysine 
 and to a lesser extent to arginine residues . mts were shown 
 to be modulators of the aggregation of a and other amyloidogenic 
 proteins and effective inhibitors of the toxicity of these proteins . a lead mt derivative , clr01 ( figure 1a ) , was shown to inhibit the toxicity of multiple 
 amyloidogenic proteins in cell viability assays using cell lines and 
 primary cell cultures and protected synaptic integrity and function of hippocampal and 
 cortical neurons against the synaptotoxicity of a42 . in addition , peripheral administration of clr01 
 in transgenic mice led to a decrease in amyloid plaques , neurofibrillary 
 tangles , and brain inflammation , suggesting that it is a promising 
 candidate for therapeutic development . mechanistic investigation showed that disruption of a 
 self - assembly 
 is mediated by clr01 binding to the two lysine and the single arginine 
 residues in a. dynamic light scattering 
 ( dls ) and electron microscopy ( em ) experiments suggested that clr01 
 does not prevent oligomer formation but rather modulates a 
 self - assembly into formation of structures that are neither amyloidogenic 
 nor toxic . interestingly , by the relatively 
 low resolution of dls and em , these structures were similar in size 
 to the toxic and amyloidogenic oligomers of a alone , suggesting 
 that subtle conformational changes in a might account for the 
 apparent loss of amyloidogenic potential and toxic activity . however , 
 the low - resolution methods could not provide information about what 
 these changes might be . in addition , whether clr01 binds a 
 monomers , oligomers , and/or larger aggregates has not been demonstrated 
 directly . in several previous studies , a derivative called clr03 
 ( figure 1b ) was used as a negative control . this compound shares the polar bridgehead structure with clr01 but 
 lacks the hydrophobic arms , and therefore is not expected to bind 
 specifically to lysine or arginine . consequently , clr03 indeed acted 
 as a negative control and was not found to inhibit the aggregation 
 and/or toxicity of amyloidogenic proteins . nonetheless , how clr03 
 interacts with a and whether it has any effect on early a 
 oligomerization of a remains to be uncovered . to address 
 all these questions , here we used ion mobility spectrometry 
 coupled mass spectrometry ( ims - ms ) to investigate 
 the effect of clr01 and clr03 on a assembly . ims - ms has been 
 utilized successfully in the past to study a and amyloid assembly and the effects of various small molecules on the assembly process . full - length 
 a40 
 and a42 were synthesized by n-9-fluorenylmethoxycarbonyl 
 ( fmoc ) chemistry . the peptides were purified by reverse - phase hplc 
 and their integrity validated by mass spectrometry and amino acid 
 analysis as described previously . the samples were prepared in 10 mm ammonium acetate , and the ph was 
 adjusted to 7.4 . samples contained 10 m a42 and molecular 
 tweezers at different concentration ratios . an a42 sample without 
 mts microscopic 
 analysis was performed using a fei t-20 transmission electron microscope 
 operating at 200 kv . the a samples with and without molecular 
 tweezers were prepared using the same procedure as that for the mass 
 spectrometry analysis . the samples were kept at 4 c for 2 weeks . 
 for tem measurements , 10 l aliquots of samples were spotted 
 on glow - discharged , carbon - coated copper grids ( ted pella , inc . ) . the samples were stained with 10 mm sodium metatungstate for 10 min 
 and gently rinsed twice with deionized water . samples were analyzed 
 on a home - built ion mobility spectrometry - mass 
 spectrometer which is composed of a nanoelectrospray 
 ionization ( nano - esi ) source , an ion funnel , a temperature - controlled 
 drift cell , and a quadrupole mass filter followed by an electron multiplier 
 for ion detection . briefly , for ion - mobility measurements , ions 
 are generated continuously by a nano - esi source , focused and stored 
 in the ion funnel . a pulse of ions is injected into a temperature - controlled 
 drift cell filled with 35 torr helium gas , where they gently 
 pass through under the influence of a weak electric field . the injection 
 energy can be varied from 20 to 150 ev , but it is 
 usually kept as low as possible to minimize thermal heating of the 
 ions during the injection process . the ions exiting the drift cell 
 are mass analyzed with a quadrupole mass filter , detected by the conversion 
 dynode and channel electron multiplier , and recorded as a function 
 of their arrival time distribution ( atd ) . the ions in the drift 
 cell experience a constant force from the 
 electric field , e. this force is balanced by a retarding 
 frictional force due to collisions with the buffer gas , resulting 
 in a constant drift velocity , vd . the 
 drift velocity is proportional to the electric field:1here , the proportionality constant k is termed the 
 ion mobility . the absolute ion mobility 
 is dependent on the temperature ( t ) and the pressure 
 ( p ) of the buffer gas , so it is typically converted 
 to the reduced mobility k0:2the ions exiting the drift cell are mass analyzed 
 and detected as a function of the arrival time , ta . the reduced mobility k0 can be determined from the instrument parameters by using eq 3 and plotting ta versus p / v3 in eq 3 , l is the length of the drift cell 
 ( 4.503 cm ) , v is 
 the voltage across the drift cell , and t0 is the time the ions spend outside the drift cell before hitting 
 the detector . all of these quantities are either known constants or 
 are measured for each experiment . the reduced ion mobility k0 can be 
 related to the collision cross section  using kinetic theory4here , q is the ion charge , n is the buffer gas number density 
 at stp ,  is the 
 reduced mass of the ion  he collision , and kb is the boltzmann constant . the measured reduced mobility 
 ( k0 ) and the collision cross section (  ) 
 provide information about the three - dimensional configurations of 
 the ions . for peptide and protein ions , the secondary and tertiary 
 structural information and the oligomeric states experimental arrival time distributions 
 can be fitted by calculating 
 the flux of ions exiting the drift tube using ion transport theory . the ion packet is taken as a periodic delta 
 function , and the flux is given by eq 5:5here z is the ion charge , r0 is the radius of the initial ion packet , a is the area of the exit aperture , dl and dt are the longitudinal and 
 transverse diffusion coefficients , s is the initial 
 ion density , and  is the loss of ions due to reactions in the 
 drift cell . the fitted feature represents the theoretical atd for 
 one species with a given cross section . if a feature in the experimental 
 atd is broader than the fitted one , then the feature possibly represents 
 a family of structures , rather than a single structure . for the ims - ms experiments performed here , a and the molecular 
 tweezers were prepared in ammonium acetate buffer , in contrast to 
 previous experiments in which they were studied in sodium phosphate 
 buffer . this change in condition was not expected to cause major changes 
 in a assembly or its inhibition . to verify this expectation 
 experimentally , we assessed samples of a42 in the absence or 
 presence of clr01 or clr03 by tem . a42 was incubated with clr01 
 at 1:1 or 1:10 concentration ratio , respectively , and with clr03 at 
 1:10 concentration ratio . as shown in figure s1 ( supporting information ) , a42 shows long 
 fibrils in the absence of tweezers or in 
 the presence of clr03 , as observed previously in sodium phosphate 
 buffer . in the presence of an equimolar 
 concentration of clr01 , the a42 sample shows a few protofibril - like 
 structures and amorphous structures . at 1:10 a42:clr01 concentration , these tem results show 
 that clr01 inhibits the fibril formation by a42 , whereas clr03 
 does not , which is consistent with previous studies in sodium phosphate 
 buffer . these results indicate that the change of buffer has a minimal 
 effect on a fibrillogenesis and the way it is impacted by clr01 
 or clr03 , supporting the comparison between the data shown below and 
 previous biophysical investigations of these systems . mass spectra of a42 samples 
 in the absence or presence of mts are shown in figure 1 . in the mass spectrum of a42 alone ( figure 1e ) , there are four peaks , which correspond to a42 
 species with charge states z / n = 
 4 , 3 , 5/2 , and 2 ( where z represents charge and n represents oligomer order ) , 
 respectively , as described previously . in the mass 
 spectrum of a 1:10 mixture of a42 and clr01 ( figure 1c ) , there are three sets of peaks which correspond 
 to 4 , 5 , and 6 charge states of the complexes 
 of a42 with one , two , three , or four clr01 molecules bound . as the mass spectrometry study was conducted in negative ion mode , 
 the binding form of clr01 is with loss of sodium ions , which results 
 in producing a42 and clr01 complexes with higher charge states . note that no 5/2 a42 peak ( m / z = 1805 ) , which represents dimer or higher order oligomers , 
 is observed . this indicates that clr01 disrupts the formation of a42 
 dimers and higher order oligomers . no peaks of uncomplexed a42 
 are observed in the mass spectrum , suggesting that clr01 binds to 
 a42 directly with high affinity and there are no clr01-free 
 a42 species present in solution . ( a , 
 b ) molecular structures of clr01 and clr03 compounds ; ( c  e ) 
 mass spectra of a42 samples : ( c ) 1:10 mixture of a42 
 and clr01 ; ( d ) 1:10 mixture of a42 and clr03 ; ( e ) a42 
 alone . each species is noted in brackets where the first number is 
 the number of a42 molecules and the second number represents 
 the number of bound small molecules . in contrast , the spectrum of a 
 1:10 mixture of a42 and clr03 
 ( figure 1d ) shows four peaks corresponding 
 to the 4 , 3 , 5/2 , and 2 a42 
 species , similarly to a42 alone ( figure 1e ) . there are two additional small peaks tailing the 3 and 
 4 a42 peaks corresponding to the 3 and 4 
 complex species of a42 with one clr03 bound , respectively . the intensities of these two complex peaks are much lower than those 
 with clr01 bound , suggesting that the affinity of clr03 binding to 
 a42 is much lower than that of clr01 . to better understand 
 the effects of clr01 on a42 , the mass 
 spectra of a42 with different ratios of clr01 ( 1:1 , 1:2 , 1:5 , 
 and 1:10 ) were recorded . the mass spectrum of a 1:1 mixture is shown 
 in figure 2d as an example , and others are 
 provided in figure s2 ( supporting information ) . the mass spectrum of the 1:5 mixture of a42 and clr01 is 
 similar to that of the 1:10 mixture ( figure 1c ) with peaks corresponding to complex species a42 and clr01 
 with charge states 4 , 5 , and 6 . as the concentration 
 of clr01 decreases ( 1:2 and 1:1 ratios ) , the complex species of a42 
 and clr01 with lower charge states ( 4 , 3 , 5/2 ) 
 are observed in the mass spectra . one possible explanation is that 
 clr01 itself is slightly basic in aqueous solution and the observed 
 binding form of clr01 is clr01 with loss of sodium ions ; therefore , 
 the complexes tend to carry more charges in the presence of high concentration 
 clr01 . note that no z / n = 5/2 
 clr01-free a42 dimer peak is observed in any of the mixtures . however , in the low - ratio mixtures ( 1:1 and 1:2 , see figure 2d and figure s2 , supporting 
 information ) , the 5/2 complex peaks of a42 oligomers 
 with clr01 molecules bound are observed ( m / z = 1950 , 2096 , and 2241 representing [ 2 + 1 ] , 
 [ 2 + 2 ] , and [ 2 + 3 ] a42clr01 
 complexes , respectively ) . these results suggest that clr01 not only 
 binds to a42 monomers but also to small a42 oligomers 
 with relatively high affinity , thereby disrupting the formation of 
 larger a42 oligomers even at 1:1 ratio . as the concentration 
 of clr01 increases , the a42 oligomers decrease in abundance 
 or altogether disappear . atds of ( a ) z / n = 5/2 a42 
 ( m / z = 1805 ) in an a42 sample 
 without clr01 ; ( b ) z / n = 5/2 
 a42 and clr01 complex ( m / z = 1950 ) ; and ( c ) z / n = 5/2 
 a42 and clr01 complex ( m / z = 2096 ) in a 1:1 mixture of a42 and clr01 . each atd is fit 
 with multiple features using the procedure described in the experimental methods section , and the oligomer order 
 ( n ) is noted for each feature . ( d ) a mass spectrum 
 of the 1:1 mixture of a42 and clr01 is shown as an example . each species is noted in brackets where the first number is the number 
 of a42 molecules and the second number represents the number 
 of bound clr01 molecules . mass spectra of mixtures of a42 and clr01 
 with different ratios are shown in figure s2 ( supporting information ) . ( e ) cross sections of dimer and tetramer 
 in the uncomplexed or clr01-complexed 5/2 a42 . the 
 error for the cross sections reported here is between 0 and 1% . to better understand the effects of clr01 
 on a42 oligomerization no atds for the z / n = 5/2 
 a42 peak ( m / z = 1805 ) could 
 be recorded , as it was not observed in any of the mixtures of a42 
 and clr01 ( figures 1c and 2d and figure s2 , supporting information ) . the atds of the [ 2 + 1 ] and [ 2 + 2 ] complex 
 peaks were recorded and are shown in figure 2b and c ( the signal of [ 2 + 3 ] complex species was too weak to obtain 
 a reliable atd ) . the atd of the 5/2 a42 peak of pure 
 a42 ( figure 2a ) shows four features 
 with arrival times of 712 , 680 , 620 , and 540 s , which 
 were previously assigned as a42 dimer , tetramer , hexamer , and 
 dodecamer , respectively , based on their cross section values ( see 
 refs ( 12 ) and ( 45 ) for a detailed discussion 
 of the 5/2 peak assignment ) . in contrast , in the 1:1 mixture 
 of a42 and clr01 , the atd of the m / z = 1950 peak ( figure 2b , labeled 
 as [ 2 + 1 ] species ) shows only two features with arrival 
 times of 690 and 640 s , which are assigned as dimer and tetramer , 
 respectively . this indicates there is one clr01 molecule bound to 
 the a42 dimer and two clr01 molecules bound to the a42 
 tetramer , respectively . no features at lower arrival times are observed , 
 suggesting there are no a42 hexamers , dodecamers , or other 
 larger oligomers formed in the presence of clr01 . the atd of the m / z = 2096 peak ( figure 2c , labeled as [ 2 + 2 ] species ) also shows two dominant 
 features with arrival times of 700 and 660 s , which 
 correspond to a42 dimer and tetramer , respectively . this indicates 
 that there are two clr01 molecules bound to the a42 dimer and 
 four clr01 molecules bound to the a42 tetramer . again , the 
 highest oligomers with clr01 bound observed are tetramers and absence 
 of features at lower arrival times indicates no hexamer , dodecamer , 
 or higher order oligomer formation . these results indicate that clr01 
 not only binds to a42 monomers but also to small oligomers 
 and inhibits the formation of hexamer and higher order oligomers . interestingly , the cross sections of dimers with 
 one or two clr01 molecules bound are significantly smaller than those 
 of the dimer with no ligands attached , even though their mass has 
 increased . this result suggests clr01 induces more interaction between 
 the two monomers , leading to a compact conformation and overall size 
 reduction . similarly , the cross sections of tetramers with two or 
 four clr01 molecules bound are smaller than those of the tetramer 
 with no clr01 bound . in addition , the tetramer atd peaks with clr01 
 bound ( figure 2b , c ) are much narrower than 
 in wild type a42 . this indicates there is little structure 
 variation in the clr01 bound tetramer while in wt a42 the tetramer 
 family of structures is both larger and more varied . the unbound a42 
 tetramer normally adopts a family of structures that have a bent arrangement 
 ( 120 angle ) . it is likely 
 that the tetramers with clr01 bound adopt either a more closed square 
 ring structure or a pyramidal structure accounting for the fact they 
 are smaller than clr01-free a42 tetramers and that dimer can not 
 be added to form hexamer . a similar effect prevents a40 from 
 growing beyond tetramer and explains its greatly reduced toxicity 
 relative to a42 . to explore 
 whether clr01 can remodel the early oligomerization of a42 
 not only immediately upon dissolution but also after the oligomers 
 have already formed , a42 was incubated for 4 h on ice , following 
 which clr01 was added to the samples . the samples were incubated at 
 a low temperature to allow quasi - equilibrium of small oligomers to 
 be reached but avoid extensive aggregation , which happens at higher 
 temperatures and leads to clogging of the nano - esi capillaries , preventing 
 further analysis . ( a ) mass 
 spectrum of a42 alone with 4 h of incubation on ice ; 
 ( b ) mass spectrum of the a42 sample immediately after the addition 
 of 1:1 clr01 . each species is noted in brackets where the first number 
 is the number of a42 and the second number represents the number 
 of bound clr01 molecules . ( c ) 
 atd of the z / n = 5/2 a42 
 peak for the a42 in the absence of clr01 after 4 h 
 of incubation on ice . ( d ) atd of the z / n = 5/2 a42 peak after addition of 1:1 clr01 . ( e and 
 f ) atds of 5/2 a42 oligomer complexes after addition 
 of 1:1 clr01 to the preaggregated a42 sample . each atd is fit 
 with multiple features using the procedure described in the experimental methods section , and the oligomer order following incubation , the mass spectrum of a42 ( figure 3a ) shows four peaks with charge states of z / n = 4 , 3 , 5/2 , 
 and 2 . the atd of the 5/2 peak shows four features 
 corresponding to dimer , tetramer , hexamer , and dodecamer , similar 
 to figure 2a . upon addition of clr01 at 1:1 
 concentration ratio , new peaks appeared corresponding to z / n = 4 , 3 monomer complexes and z / n = 5/2 oligomer complexes ( figure 3b ) . overall , the spectrum was similar to the one 
 obtained in the inhibition study ( figure 2d ) , 
 with the exception that no peaks were observed with 3 or 4 clr01 molecules 
 bound . the data indicate that clr01 binds to a42 monomers and 
 preformed a42 oligomers directly , mostly with 1:1 or 1:2 stroichiometry , 
 whereas binding of additional clr01 molecules is less likely after 
 incubation . this suggests the existence of two main binding sites 
 for clr01 on a , lys16 , and lys28 . interestingly , the atd of the z / n = 5/2 unbound a42 peak ( figure 3d ) showed only three features representing dimer , tetramer , and a 
 small amount of hexamer after the addition of equimolar clr01 . the 
 feature representing a42 dodecamer was eliminated after the 
 addition of clr01 , suggesting that the binding of clr01 dissociated 
 the preformed a42 dodecamer . moreover , the relative intensity 
 of the hexamer decreased significantly compared to that before clr01 
 addition ( figure 3c ) , suggesting clr01 began 
 to also dissociate hexamers . the atds of the z / n = 5/2 a42:clr01 oligomer complex peaks 
 ( figure 3e , f ) showed only the two features 
 corresponding to dimer and tetramer with clr01 molecules bound , as 
 observed immediately upon mixing of a42 and clr01 ( figure 2b and c ) , suggesting that binding of clr01 to a42 
 dimers or tetramers changes their structure so that additional dimers 
 can not be added to form hexamers . the atds of the z / n = 5/2 peaks were monitored again after 
 1 day of incubation at 4 c , and the results ( see figure s3 , supporting information ) were similar to those 
 obtained following 4 h of incubation , suggesting that clr01 maintained 
 the distribution of a42 oligomers , in which dodecamers were 
 excluded and hexamers were a minor species . to test the effect 
 of the a42:clr01 concentration ratio 
 on the remodeling of a42 oligomerization , higher ratios of 
 clr01 ( 1:5 and 1:10 , respectively ) the mass spectrum of the 1:5 mixture is shown in figure s4c ( supporting information ) ( the result of the 1:10 
 ratio was similar to that of the 1:5 mixture and therefore is not 
 shown ) . the mass spectrum of the 1:5 mixture showed mostly complexes 
 of a42 monomer with clr01 molecules . no z / n = 5/2 peak of clr01-free or clr01-complexed a42 
 was observed . taken together , these results 
 indicate that clr01 remodels a42 oligomerization both at low 
 and high concentration ratios . the atds of z / n = 3 of 
 a42 monomer in the absence or presence of clr01 ( 1:1 ) are shown 
 in figure s5 ( supporting information ) . 
 in the atd of the 3 monomer peak of a42 alone , there 
 are two dominant peaks with arrival times of 640 and 680 
 s , previously identified as a solvent - free conformer and a 
 solution - like conformer , respectively . the atd for the 3 peak of a42 complexed with one 
 clr01 molecule shows two similar features with arrival times of 668 
 and 712 s . by analogy , these are assigned as the solvent - free 
 and solution - like conformers of a42 monomer with one clr01 
 bound , respectively . the atds of z / n = 4 and 5 complexes of a42 with 
 one , two , three , or four clr01 molecules bound in a 1:5 mixture , respectively , 
 are shown in figure s6 ( supporting information ) . those for z / n = 4 have 
 two features in their atds , and as the number of bound clr01 molecules 
 increases , the intensity of the compact , shorter - time feature increases , 
 relative to the more extended , longer - time feature . overall , there 
 are no features with shorter arrival times detected which indicates 
 that only monomer with clr01 complexed is present in solution . the 
 cross sections of the a42:clr01 monomer complexes are shown 
 in figure s6c ( supporting information ) . addition of each clr01 ligand increases the size of the complex by 
 an amount comparable to the size of clr01 , suggesting that no major 
 structural transitions occur in the monomers upon clr01 binding . a40 
 has an identical sequence to that of a42 except for absence 
 of ile41 and ala42 residues at the c - terminus but has very different 
 assembly and pathological properties . thus , it is interesting to examine 
 how clr01 affects its early oligomerization in comparison to a42 . the mass spectra of a40 alone and a40 mixed with clr01 
 at different ratios are provided in figure s7 ( supporting information ) . the mass spectrum of a40 alone 
 shows three peaks which correspond to z / n = 4 , 3 , and 5/2 , similar to the spectrum 
 of the a42 . the mass spectra of mixtures of a40 and 
 clr01 at different ratios show sets of peaks at charge states 3 , 
 4 , 5 , and 6 corresponding to a40:clr01 
 complexes . up to four clr01 molecules are observed bound to a40 . 
 at lower clr01 concentration ( 1:1 ratio , figure s7b , supporting information ) , there are three z / n = 5/2 peaks at m / z = 1876 , 2021 , and 2167 , corresponding to [ 2 + 1 ] , [ 2 + 2 ] , and [ 2 + 3 ] oligomer complexes 
 of a40 and clr01 . at higher concentrations of clr01 ( 1:5 and 
 1:10 ratios ) , no 5/2 oligomer complexes were detected , suggesting 
 that no dimer or higher - order oligomers formed . the atds of 
 these 5/2 oligomer peaks are shown in figure 4 ( the signal of the [ 2 + 3 ] complex was too weak to obtain a 
 reliable atd and therefore is not shown ) . the 5/2 peak of 
 a40 ( m / z = 1731 , figure 4a ) shows two features with arrival times of 690 
 and 620 s which previously were assigned as a40 
 dimer and tetramer ( see ref ( 12 ) for a detailed discussion of the 5/2 peak assignment ) . 
 for the 1:1 mixture of a40 and clr01 , the atds of 5/2 
 [ 2 + 1 ] and [ 2 + 2 ] oligomer complexes ( figure 4b and c ) show two primary features , which can be 
 assigned as dimer and tetramer based on their cross sections . these 
 results indicate that there are one or two clr01 molecules bound to 
 a40 dimers and two or four clr01 molecules bound to a40 
 tetramers . no features at shorter arrival times were observed , indicating 
 that there are no hexamer or larger oligomers formed . interestingly , 
 the intensities of the tetramer feature for [ 2 + 1 ] and 
 [ 2 + 2 ] complex species ( figure 4b and c ) are lower than that of the 5/2 , clr01-free a40 
 tetramer feature , which indicates that the formation of tetramer is 
 slower in the presence of clr01 in the a40 sample than in its 
 absence . ( a ) atd of z / n = 5/2 a40 
 ( m / z = 1731 ) for a40 alone ; 
 ( b and c ) atds of z / n = 5/2 
 a40 and clr01 complexes ( m / z = 1876 and 2021 ) in the 1:1 mixture of a40 and clr01 . each 
 atd is fit with multiple features using the procedure described in 
 the experimental methods section , and the 
 oligomer order ( n ) is noted for each feature . note 
 the atds with clr01 bound , panels b and c , are significantly narrower 
 than wild type , panel a. the atds of monomer complexes with charge states z / n = 3 , 4 , 5 , 
 or 6 
 are shown in figures s8 and s9 ( supporting information ) . the z / n = 4 species 
 ( figure s9a , supporting information ) show 
 two features in their atds corresponding to the solvent - free conformer 
 and the solution - like conformer of a40 complexed with clr01 . 
 taken together , these results indicate that clr01 binds to a40 
 with relatively high affinity and inhibits its early oligomerization . the cross sections of a40 monomer complexes are shown in figure 
 s9b ( supporting information ) . similarly 
 to the a42 case , addition of each clr01 ligand increases the 
 size of the monomer complex by an amount comparable to the size of 
 clr01 , suggesting that no major structural transitions occur in the 
 monomers upon clr01 binding . as noted above , clr03 has been 
 used as a negative - control compound , 
 which was not expected to inhibit a oligomerization or aggregation . hence , we felt it was important to do similar experiments that are 
 reported here . time - dependent ion mobility study of the effects of clr03 
 on a42 
 early oligomerization . ( a  c ) atds of the 5/2 a42 
 peak ( m / z = 1805 ) for the 1:10 mixture 
 of a42 and clr03 at different time points ; ( d  f ) atds 
 of the 5/2 a42 peak ( m / z = 1805 ) for a42 alone at different time points . each atd 
 is fit with multiple features using the procedure described in the experimental methods section . the oligomer order 
 ( n ) and cross section are noted for each feature . a time - dependent study of the 
 atds of the 5/2 a42 
 peak ( m / z = 1805 ) of a42 
 alone and the 1:10 mixture of a42 and clr03 is shown in figure 5 . the atd of the 5/2 a42 peak for 
 the 1:10 mixture of a42 and clr03 at 30 min ( figure 5a ) shows four features that can be assigned as a42 
 dimer , tetramer , hexamer , and dodecamer based on their cross sections , 
 which is similar to the results of a42 alone at 30 min ( figure 5d ) . interestingly , the intensity of the dodecamer 
 feature of the 5/2 peak for the mixture of a42 and 
 clr03 is relatively higher than other features , whereas the intensity 
 of the dodecamer feature of a42 alone at 30 min is relatively 
 lower than other features , suggesting that the growth of dodecamer 
 in the presence of clr03 is faster than in its absence . after up to 
 24 h of incubation , the dodecamer in the a42 and clr03 mixture 
 becomes an even more dominant feature in the atds ( figure 5b and c ) , whereas the clr01-free a42 sample 
 does not change substantially and appears to be in a state of quasi - equilibrium 
 ( figure 5e and f ) . the observation of dodecamer 
 and the significant rapid growth of dodecamer in the a42 sample 
 in the presence of clr03 suggest that clr03 not only does not inhibit 
 the formation of a42 dodecamer but actually facilitates the 
 dodecamer formation . atds of z / n = 3 and 4 
 peaks for a42 alone and a 1:1 mixture of a42 and clr03 
 are shown in figure 6 . the atd of the z / n = 3 a42 peak for the 
 mixture of a42 and clr03 ( figure 6b ) 
 shows two features at 640 and 680 s corresponding 
 to the compact and extended conformers , respectively , which is similar 
 to those of a42 alone ( figure 6a ) . remarkably , 
 there is another feature at a substantially shorter arrival time ( 500 
 s ) as noted by the arrow in addition to the two monomer features , 
 which is not observed for a42 alone . this indicates the presence 
 of relatively large oligomers ( n  2 ) formed 
 in the presence of clr03 . a similar feature is observed in the atd 
 of the z / n = 3 peak of a42 
 with one clr03 bound , which corresponds to large oligomers ( n  2 ) . in figure 6e and f , 
 dominant features with shorter arrival times are observed in the atds 
 for z / n = 4 peaks of a42 
 with and without clr03 bound , in addition to the monomer feature , 
 indicating the presence of large oligomers ( n  
 2 ) . these results are consistent with the results of the atds of the z / n = 5/2 a42 peak ( m / z = 1805 , figure 5 ) and further support the fact that clr03 facilitates self - assembly 
 of a42 . clr03 facilitates a42 oligomer formation : ( a ) atd 
 of the z / n = 3 peak for 
 a42 alone , 
 ( b and c ) atds of z / n = 3 
 a42 without and with clr03 bound for a 1:10 mixture of a42 
 and clr03 , ( d ) atd of the z / n = 
 4 peak for a42 alone , ( e and f ) z / n = 4 a42 without and with clr03 bound for 
 a 1:10 mixture of a42 and clr03 . each atd is fit with multiple 
 features using the procedure described in the experimental 
 methods section . the arrows indicate the 
 formation of oligomers ( n  2 ) in the mixture 
 of a42 and clr03 . the effect of 
 clr03 on a40 oligomerization is shown in figure 7 . the mass spectrum of a 1:10 mixture of a40 and clr03 
 shows peaks corresponding to z / n = 3 and z / n = 4 
 monomer with one clr03 bound but no clr03 attachment to the z / n = 5/2 peak ( figure s10b , supporting information ) . however , the atd of 
 the 5/2 a40 peak ( m / z = 1731 ) in the presence of clr03 ( figure 7b ) shows formation of both a40 hexamer and dodecamer based 
 on their cross sections . in figure 7c , the 
 cross sections for the dimers and tetramers of a40 are given 
 for a40 alone and for a40 mixed with clr01 and clr03 . note that clr03 significantly increases both cross sections , whereas 
 clr01 significantly decreases both cross sections . ( a , b ) atds of the 5/2 
 a40 peak ( m / z = 1731 ) for 
 the a40 samples in the absence or presence of clr03 . each atd 
 is fit with multiple features using the procedure described in the experimental methods section , and the oligomer order 
 ( n ) is noted for each feature . ( c ) cross sections 
 of 5/2 a40 oligomers for samples of a40 in the 
 absence or presence of clr01 or clr03 . the atds of z / n = 3 
 and 
 4 peaks of the 1:10 a40:clr03 mixture ( figure s11 , supporting information ) show features at arrival 
 times shorter than those of monomers , suggesting formation of large 
 oligomers ( n  2 ) in the presence of clr03 . our mass 
 spectrometry study of the lead molecular tweezer , clr01 , 
 and the related derivative , clr03 , provides novel observations that 
 could not have been obtained previously due to the low resolution 
 of the methods used . our investigation reveals that clr01 and clr03 
 bind to a with very different affinities . three clr01 molecules 
 bind with relatively higher affinity and a fourth weakly , but only 
 one clr03 molecule binds weakly to a. the a:clr01 
 stoichiometry found in our study is consistent 
 with previous data suggesting that there are three possible binding 
 sites for molecular tweezers on a at arg5 , lys16 , and lys 28 . possible explanations for our observation of 
 a fourth clr01 molecule weakly binding to a40 and a42 
 could simply stem from differences in instrumentation , or might reflect 
 nondiscriminating electrostatic and/or aromatic interactions between 
 a and clr01 molecules , which might have been broken under harsher 
 ionization conditions in the study by sinha et al . these nonspecific dispersive interactions could also explain 
 the weak binding of one clr03 molecule to a , which was not 
 observed within the limits of nmr detection in the previous study . the observation of three z / n = 
 5/2 oligomer complexes in the 1:1 and 1:2 mixtures of a42 
 and clr01 , respectively , suggests that clr01 not only binds to a42 
 monomers but also to small a42 oligomers ( dimers and tetramers ) . this is important , as dodecamers have been identified 
 as probable toxic agents in ad . understanding the mechanism of 
 how clr01 blocks dodecamer formation is crucial for developing a therapeutic 
 strategy for ad . remarkably , the cross sections of the dimer and tetramer 
 decreased substantially upon binding of clr01 ( see figure 2e ) . these results suggest that clr01 interacts with 
 a42 to change the folding of the monomer , which in turn changes 
 the binding interface in dimer and tetramer formation , resulting in 
 compact structures that resist further aggregation . once multiple 
 clr01 ligands bind to the monomer , even dimer formation is prevented . the ability of clr01 to remodel early a42 oligomerization 
 after the oligomers had an opportunity to form and reach a quasi - equilibrium 
 state for 4 h was assessed by ims as well . interestingly , even at 
 low concentration ( 1:1 ratio ) , clr01 was capable of removing preformed 
 a42 dodecamers and hexamers . at high concentrations ( 1:5 and 
 1:10 ratios ) these data are consistent with the inhibition results immediately 
 upon mixing a42 and clr01 . it is possible that clr01 binds 
 to a42 monomers and oligomers and redirects them into either 
 a slower aggregation process or an off - pathway set of structures . 
 in either case , the resulting structures are nonamyloidogenic and 
 nontoxic . our data reveal for the first time that the loss 
 of amyloid - formation propensity and toxicity correlate with disruption 
 of the oligomerization process and compaction of oligomers formed 
 in the presence of clr01 . surprisingly , the related derivative , 
 clr03 , was found to facilitate 
 the early aggregation of a42 , especially promoting the formation 
 of hexamers and dodecamers . perhaps even more surprisingly , clr03 
 also facilitated the formation of hexamers and dodecamers in a40 , 
 which does not form these oligomers on its own . given the fact that clr03 is an organic phosphate , we 
 wondered whether it is possible the addition of clr03 has a simple 
  salting out-like effect , which would thereby promote 
 a aggregation . to address this possibility , an organic phosphate , p - nitrophenylphosphate ( pnpp ) , was added to a samples 
 and was found not to induce significant changes in a oligomerization 
 ( see figure s12 , supporting information ) . this result implies a simple  salting out-like 
 effect is not occurring for clr03 and that clr03 interacts with a 
 in a specific manner . a major difference between the structures 
 of clr01 and clr03 is 
 that clr01 has a torus - shaped cavity whereas clr03 does not have a 
 cavity structure . clr03 carries a bridge - like structure with negatively 
 charged phosphate groups on each side . it is possible that one of 
 the phosphate groups on clr03 interacts with positively charged residues 
 ( lys , arg , n - terminus ) of a through coulombic interactions . the other phosphate group on the other side of clr03 could then attract 
 a positively charged residue of another a or a oligomer . thus , by attracting positively charged residues in a , clr03 
 could facilitate a oligomerization and aggregation . alternatively , 
 clr03 may interact weakly with two positively charged 
 groups in a single a , resulting in no observation of peaks 
 for single a with multiple clr03 in the mass spectra ( figure 1d ) . as a consequence this is a form of salting out , and 
 it may be more effective than pnpp . however , the fact that assembly 
 is promoted to structures similar to wt a42 speaks against 
 salting out as a dominant assembly mechanism . clr01 , which also 
 carries two phosphate groups , however , does not 
 catalyze a oligomerization . this suggests that inclusion of 
 lysine or arginine inside the cavity of clr01 is of paramount importance 
 for clr01 s mode of action . the central hydrophobic region 
 of a is regarded to be important for the hydrophobic clustering 
 of a. recently , a macrocyclic inhibitor cucurbituril has 
 been shown to inhibit amyloid fibrillation by hydrophobic interactions 
 with nonpolar phenylalanine residues of a. clr01 , which has hydrophobic arms , is likely to have additional 
 hydrophobic interactions with lysine residues . the binding of clr01 
 to lysine residues , especially lys16 which is close to the central 
 hydrophobic region of a , may result in conformation change 
 of a and compaction of a oligomers . our data suggest 
 that binding of clr01 causes a monomers to either resist oligomer 
 formation altogether or to redirect them to nontoxic oligomer assembly . previous detailed analysis showed that a40 formed a nearly 
 closed planar tetramer that resisted further dimer addition . here , we found that adding clr01 to a40 
 significantly reduced the cross sections of both the dimer and tetramer , 
 leading to nearly isotropic assembly and reducing the likelihood of 
 even forming the tetramer , much less higher - order oligomers . on the 
 other hand , the presence of clr03 in the solution significantly extended 
 both the dimer and tetramer , yielding cross sections similar to those 
 of the corresponding a42 oligomers and leading to hexamer and 
 dodecamer formation . the essential features of these results 
 are given in cartoon style 
 in figure 8 . a42 wt rapidly forms dodecamer , 
 but addition of the molecular tweezer clr01 eliminates dodecamer formation 
 by inducing the dimer and tetramer to form compact species that can not 
 add additional a42 . a40 wt forms terminal compact tetramers , but addition of clr03 
 leads to open tetramer formation and eventual dodecamer formation . these contrary effects are potentially of great importance in a 
 assembly and require further study to reveal the details involved . these studies , which will include both high level molecular dynamics 
 modeling and additional direct sampling of structures of a 
 oligomers , are underway . oligomerization of ( a ) a42 wild type , ( b ) a42 with the 
 presence of clr01 , ( c ) a40 wild type , and ( d ) a40 with 
 the presence of clr03 . a42 and a40 are represented with 
 blue and red balls , respectively .
the early oligomerization of amyloid 
 -protein ( a ) 
 has been shown to be an important event in the pathology of alzheimer s 
 disease ( ad ) . 
 designing small molecule inhibitors targeting a 
 oligomerization is one attractive and promising strategy for ad treatment . 
 here we used ion mobility spectrometry coupled to mass spectrometry 
 ( ims - ms ) to study the different effects of the molecular tweezers 
 clr01 and clr03 on a self - assembly . 
 clr01 was found to bind 
 to a directly and disrupt its early oligomerization . 
 moreover , 
 clr01 remodeled the early oligomerization of a42 by compacting 
 the structures of dimers and tetramers and as a consequence eliminated 
 higher - order oligomers . 
 unexpectedly , the negative - control derivative , 
 clr03 , which lacks the hydrophobic arms of the tweezer structure , 
 was found to facilitate early a oligomerization . 
 our study 
 provides an example of ims as a powerful tool to study and better 
 understand the interaction between small molecule modulators and a 
 oligomerization , which is not attainable by other methods , and provides 
 important insights into therapeutic development of molecular tweezers 
 for ad treatment .