this view is supported by the large number of intrinsically unfolded 
 proteins that are the causative agents of amyloid diseases , such as 
 -synuclein , amyloid- peptide ( a ) , and amylin . in terms of folded protein precursors , a link between local or global 
 unfolding and the onset of aggregation has been documented . indeed , decreased native state stability and 
 a reduction in co - operativity have been linked with enhanced amyloidogenicity 
 of several proteins , including human lysozyme , transthyretin , prion protein , superoxide dismutase ( sod ) , and 2-microglobulin ( 2 m ) . these findings have guided theoretical approaches 
 to predict aggregation - prone regions of folded proteins by combining 
 the intrinsic propensity of a protein sequence to self - assemble with 
 its probability to become exposed ( e.g. , by increased predicted hydrogen - exchange 
 rates ) . along similar lines , strategies to stabilize 
 native - like conformations by the use of small molecules , aptamers , molecular chaperones or via other protein  protein interactions all reduce amyloid formation . however , a quantitative link between 
 the conformational properties and dynamics of individual partially 
 folded species and amyloid propensity remains elusive , in part because 
 of difficulties in identifying and characterizing amyloid intermediates 
 in atomic detail . in addition , the complexity of the energy landscape 
 of proteins , which involves many potentially amyloidogenic species , 
 exacerbates the intricacy of the system . in this study we investigate 
 the structural , kinetic , and thermodynamic 
 properties of sparsely populated intermediates of human and murine 
 2 m ( h2 m , m2 m , 
 respectively ) and link their properties to the known , very different 
 amyloid propensities of these proteins . m forms amyloid fibrils in the joints of patients 
 undergoing hemodialysis , in a pathological condition known as dialysis - related 
 amyloidosis . previous studies have examined 
 the link between the folding pathway of 2 m and its 
 aggregation propensity . these studies showed that h2 m does not aggregate at neutral ph unless additives such as cu , trifluoroethanol ( tfe ) , or other cosolvents , which partially unfold the protein , are added . as h2 m contains a thermodynamically unfavorable cis prolyl - peptide 
 bond at position 32 , protein folding involves a slow folding phase 
 which is attributed to trans cis proline isomerization of this bond . a native - like 
 intermediate containing a trans - pro 32 ( it ) accumulates during folding of h2 m , which can 
 be trapped by removal of the n - terminal six residues of the protein , 
 to create the variant n6 . at neutral 
 ph , the concentration of it correlates directly with the 
 rate of amyloid formation of h2 m , suggesting that formation of this on - pathway native - like 
 folding intermediate is a key determinant of amyloid formation . this 
 supposition is confirmed by the ability of n6 to aggregate 
 readily at ph 6.2 . on the other hand , 
 m2 m , despite being 70% identical in sequence to 
 h2 m and also containing a cis - x 
 pro 32 bond , does not form amyloid fibrils at neutral ph , or even 
 when unfolded under acidic conditions ( unless high concentrations 
 of salt are added ) ( figure 1a and 1b ) . the basis of such dramatically different amyloid propensities despite 
 the sequence and structural similarities of these two proteins remained 
 unclear . ( a ) the 
 nmr structure of monomeric h2 m ( gray-2xks ) overlaid with the crystal structure of m2 m bound to the mhc - i complex ( green-1lk2 ) or with the solution structure of n6 ( red-2xku ) . ( b ) aggregation assay of 80 m h2 m ( gray ) , m2 m ( green ) , or n6 ( red ) 
 in 10 mm sodium phosphate buffer , ph 6.2 ( three replicates for each 
 protein ) . negative stain electron micrographs , color - coded with the 
 same scheme , are shown on the right ( scale bar = 500 nm ) . ( d ) correlation between experimental 
 rdcs measured for m2 m in 10 mg / ml phage pf1 and 
 those back - calculated from the crystal structure 1lk2 ( r = 0.85 ) . here , we set out to investigate the molecular origins 
 of the reduced 
 amyloidogenicity of m2 m . we characterize the stability , 
 structure , and dynamics of the native protein and show that despite 
 its inability to form amyloid , m2 m is kinetically 
 and thermodynamically less stable than its human counterpart . the 
 folding pathway of m2 m is then explored using real - time 
 nmr , taking advantage of the power of nonuniformly sampling ( nus ) 
 methods to reveal detailed information on the energy landscape of 
 m2 m folding . combined with other biophysical methods , we show that while m2 m also folds through an it state , this species is relatively more flexible than its 
 h2 m counterpart and in conformational exchange with 
 other , less - structured non - native states . the molten globule - like 
 characteristics of the m2 m folding intermediate 
 reduce the lifetime of the structured , well - folded 
 it state , which now represents only a minor substate in 
 the structural ensemble . our findings confirm the vital role of it ( and hence a highly structured , yet non - native species ) in 
 determining the aggregation of 2 m . moreover , the 
 results highlight the importance of defining the energy landscape 
 of amyloidogenic proteins in detail to allow prediction of their amyloid 
 propensity . the findings presented also suggest that targeting a defined 
 non - native species should be a successful means of controlling the 
 fate of assembly of 2 m and , in principle , that of 
 other amyloidogenic proteins which aggregate via a specific , non - native 
 precursor . the pink plasmid containing the 
 h2 m , m2 m , or n6 gene was 
 transformed into e. coli cells of the bl21 de3 plyss- 
 strain . starter cultures were generated by inoculating 100 ml of lb 
 medium with cells containing the relevant gene and 50 g / ml 
 carbenicilin and 50 g / ml chloramphenicol and incubating overnight 
 at 37 c , 200 rpm . ( 1 
 g / l n - nh4cl , 2 g / l c - glucose ) 
 medium were inoculated with 10 ml of starter culture . cells were incubated 
 at 37 c , 200 rpm until they reached an od600 of 0.6 
 and then the expression of 2 m was induced by the 
 addition of isopropyl -d-1-thiogalactopyranoside ( iptg - final 
 concentration of 1 mm ) . expression was allowed to continue overnight 
 at 37 c , and cells were harvested next morning using a heraus 
 continual action centrifuge performing at 15  000 rpm . the cell 
 pellet containing 2 m as inclusion bodies was chemically 
 lysed by the addition of 50100 ml of lysis buffer ( 100 g / ml 
 lysozyme , 50 g / ml dnase i , 50 g / ml phenylmethylesulfonyl 
 fluoride ( pmsf ) , 10 mm tris - hcl ph 8.0 ) . further cell disruption was 
 performed using a constant cell disrupter system ( constantsystems ) 
 at a high pressure of 20.0 kpsi . inclusion bodies were separated using 
 centrifugation ( 15  000 rpm using a sorvall ss34 rotor ) in a 
 beckman centrifuge for 40 min at 4 c , and the inclusion body 
 pellet was washed with 10 mm tris - hcl ph 8.0 buffer four times . finally , 
 2 m was solubilized in 1020 mm tris - hcl ph 
 8.0 ( h2 m , n6 ) or 1020 mm tris - hcl 
 ph 8.5 ( m2 m ) containing 8 m urea ( mp biomedicals ) 
 and refolded by dialysis ( 3000 mw cutoff ) against 25 the refolded protein was centrifuged 
 for 30 min at 15000 rpm ( sorvall ss34 rotor ) to pellet insoluble material , 
 and the supernatant was loaded on a q - sepharose ( ge healthcare ) column 
 already equilibrated with 2 column volumes of 20 mm tris - hcl ph 8.0 
 ( h2 m , n6 ) or 20 mm tris - hcl ph 8.5 ( m2 m ) for anion exchange purification . bound protein was eluted 
 with a gradient of 0400 mm nacl ( in the same buffer ) over 
 800 ml and was freeze - dried after dialysis in dh2o or concentrated 
 using 3000 mw cutoff centricons ( avanti ltd ) . freeze - dried protein 
 was resuspended in 10 mm sodium phosphate buffer ph 7.0 ( h2 m , n6 ) and 10 mm sodium phosphate buffer ph 8.2 ( m2 m ) filtered through 0.2 m filters ( fisher scientific ) 
 and gel - filtered using a hiload superdex-75 prep column ( amersham 
 biosciences ) , calibrated with a standard gel filtration calibration 
 kit ( ge healthcare ) . the monomer peak was collected , concentrated , 
 aliquoted , and stored at 80 c or freeze - dried . assignments of the backbone atoms 
 of m2 m were obtained using samples of 500 or 750 
 m uniformly labeled ( n , c ) protein 
 in 10 mm sodium phosphate buffer ph 6.2 , 83.3 mm nacl , 0.02% ( w / v ) 
 nan3 , and 10% ( v / v ) d2o . three - dimensional ( 3d ) 
 nmr experiments were recorded at 25 c using varian inova spectrometers 
 ( agilent ) operating at proton frequencies of 500 mhz ( hnca , hnco , 
 cbca(co)nh , hn(ca)co ) and 750 mhz ( hncacb ) , equipped with a room temperature 
 or cryogenic probe , respectively . samples for h / d exchange experiments 
 were prepared in 10 mm sodium phosphate buffer ph 6.2 and then freeze - dried . freeze - dried protein was dissolved in 100% ( v / v ) d2o containing 
 83.3 mm nacl and placed into the nmr tube after manual mixing . the 
 loss of intensity of amide proton resonances was then monitored by 
 sofast h n hsqc spectra ( 510 min each ) at 25 c . residual 
 dipolar coupling ( rdc ) experiments ( j modulated series ) were carried 
 out using a sample of 200 m n - m2 m in 10 mm sodium phosphate ph 8.2 and aligned in 11 mg / ml bacteriophage 
 pf1 ( asla scientific ) . rdc data were back - calculated from crystal 
 structures using pales . for relaxation 
 experiments , a sample of 80 m n - m2 m was prepared in 10 mm sodium phosphate buffer at ph 6.2 with 10% 
 ( v / v ) d2o , 0.02% ( w / v ) nan3 , 83.3 mm nacl . t2 experiments were perfomed with 2048 and 128 complex points 
 in the direct and indirect dimension using 18.8 , 37.7 , 56 . 6 , 75.5 , 
 94.4 , 113.2 , 132,1 , 151.0 ms as relaxation delays . the relaxation 
 delays for the t1 experiments were 0.0.16 , 0.32 , 0.48 , 
 0.64 , 0.80 , 0.96 , 1.12 , 1.44 s. for h n noe cross - relaxation experiments saturation of amide protons was 
 achieved with a train of 120 pulses for 3.5 s prior to the experiment . all relaxation and rdc experiments were performed at 25 c , using 
 a 600 mhz varian inova spectrometer equipped with a room tempreature 
 probe . direct carbon detection experiments were performed using a 
 sample of c , n m2 m at 
 1.3 mm on a 950 mhz bruker spectrometer equipped with a cryogenic 
 tci probe ( 1024 and 160 complex points in the direct and indirect 
 dimensions ) . for real - time refolding experiments two refolding 
 protocols were followed : ( 1 ) protein samples were made in 10 mm sodium 
 phosphate buffer ph 6.2 and freeze - dried . unfolding was performed 
 by dissolving the freeze - dried protein ( 23 mg ) in 3060 
 l of the same buffer containing 8 m urea at 37 c for 
 1 h , and the protein was then refolded by rapid 10-fold dilution in 
 10 mm sodium phosphate ph 6.2 , 10% ( v / v ) d2o , and 0.02% 
 ( w / v ) nan3 ; ( 2 ) protein samples were made in 250 l 
 of 10 mm sodium phosphate and 10% ( v / v ) d2o , and the ph 
 was adjusted to 2.0 ( or ph 3.6 ) using tris - hcl . refolding was then 
 initiated by addition of 50 l of 300 mm sodium phosphate buffer , 
 ph 7.2 ( final ph 6.16.3 ) . both refolding protocols were found 
 to give rise to similar spectra of the intermediate species . this 
 observation demonstrates that the increased flexibility of the murine 
 intermediate ( see results ) is not the result 
 of the residual 0.8 m urea present at the end of the first refolding 
 protocol . the refolding from it to native h2 m was monitored by a series of sofast h n hsqc spectra at 25 c , with 
 80 increments in the indirect dimension , two scans per increment and 
 512 complex points , resulting in a total acquisition time of 45 s. to assign the i1 state of m2 m , refolding 
 was monitored by continuous acquisition of nus nmr spectra . n best - trosy were collected on separate 
 samples ( 800 mhz bruker avance iii hd spectrometer with 3 mm tci cryoprobe ) . n best - trosy , having the highest 
 sensitivity , was chosen as a reference spectrum to guide 3d spectra 
 multidimensional decomposition ( mdd ) coprocessing with a sliding time 
 frame window , resulting in a temporal resolution of a few minutes ( see supplementary methods ) . to aid assignment of the real - time spectra , two samples 
 were prepared 
 in which the early intermediate of m2 m was highly 
 populated . the first consisted of 600 m of uniformly labeled c , n m2 m in 10 mm sodium phosphate 
 and 10 mm sodium acetate ph 3.6 , and the second consisted of 250 m 
 of uniformly labeled c , n m2 m in 10 mm sodium phosphate ph 6.2 with 1 m urea added . both samples 
 gave rise to hnca spectra that closely resembled the real - time hnca 
 spectrum of the early intermediate of m2 m . additional 
 3d spectra were performed using these samples including hnca , hnco , 
 and cbcaconh utilizing a 600 mhz varian inova spectrometer equipped 
 with a room tempreature probe . talos+ uses h , nh , co , c , and c backbone chemical shifts 
 to calculate the random coil chemical shift index which is then converted 
 to backbone s. aggregation assays performed on these samples 
 confirmed that the early folding intermediate of m2 m is not aggregation - prone . samples containing 
 60 m protein 
 in 10 mm sodium phosphate buffer , ph 6.2 , or 10 mm sodium phosphate , 
 with 10 mm sodium acetate ph 3.6 , or in 10 mm sodium phosphate buffer , 
 ph 6.2 , with 1 m urea , with the appropriate amount of nacl added to 
 give a total ionic strength of 100 mm ) , 0.02% ( w / v ) nan3 and 10 m thioflavin t ( tht ) were incubated at 37 c 
 in sealed 96 well plates ( thermo scientific ) with agitation at 600 
 rpm . fluorescence was monitored at 480  10 nm after excitation 
 at 440  10 nm using a fluorostar optima microplate reader ( bmg 
 labtech ) . urea stock solutions containing 
 75 mm sodium phosphate buffer , ph 6.2 , and either no urea or 10.5 
 m urea were made , and the exact concentration of urea was determined 
 using the measured refractive index ( ceti refractometer ) . the stock 
 solutions were used to make samples of protein containing 010 
 m urea in 0.2 m increments , with a final protein concentration of 
 0.2 mg / ml . samples were incubated at 25 c for 12 h before analysis 
 using tryptophan fluorescence . fluorescence was excited at 295 nm , 
 and the emission was monitored at 340 nm using a photon technology 
 international qm-1 spectrofluorimeter ( pti ) . the data were then globally 
 fit to a two - state model:1where guf ( kj mol ) is the equilibrium stability , muf is the equilibrium m - value , a and c represent the denaturant - dependence 
 of the folded and unfolded signal intensities , respectively , and b 
 and d are the signal intensities of the folded and unfolded states , 
 respectively , in the absence of denaturant . carbon coated copper grids 
 were prepared by the application of a thin layer of formvar with an 
 overlay of carbon . samples were centrifuged ( 14  000 g , 10 min ) , and the pellets were resuspended in fresh 10 
 mm sodium phosphate buffer , ph 6.2 , diluted to a final protein concentration 
 of 12 m with deionized water and then applied to the grid in 
 a dropwise fashion . the grid was then carefully dried with filter 
 paper before it was negatively stained by the addition of 18 l 
 of 2% ( w / v ) uranyl acetate . experiments were performed 
 using an applied - photophysics sx1.8mv stopped - flow fluorimeter . the 
 temperature was held constant at 37 c ( 0.1 c ) using 
 a neslab circulating water bath system . experiments were performed 
 in buffered solutions containing 10 mm sodium phosphate ( ph 6.2 ) and 
 83.3 mm sodium chloride with or without 0.4 m sodium sulfate . refolding 
 experiments were performed by 1:10 dilution of 80 m protein 
 in buffer containing 8 m urea , into buffered solutions with final 
 urea concentrations in the range 0.758.0 m. the final urea 
 concentration ranged from 3.0 to 8.0 m for unfolding experiments . 
 to obtain refolding data at 0 m urea , a ph jump was performed by a 
 1:10 dilution of 80 m protein in 10 mm phosphate ( ph 2.5 ) into 
 80 mm sodium phosphate ( ph 6.2 ) . data were normalized to the signal 
 of the folded and unfolded protein in 0 and 8 m urea , respectively . 
 at each urea concentration at least seven kinetic traces were obtained , 
 averaged , and fitted to a single exponential function using the manufacturer s 
 software . gui1 was 
 determined by plotting the fluorescence at the end point of a 20 s 
 kinetic trace of folding against urea concentration and by plotting 
 the fluorescence of the unfolded state against urea concentration 
 ( in the latter case , the values at low urea concentration were obtained 
 by linear extrapolation from the values at high urea concentration ) . the fluorescence of the it state decreased with increasing 
 urea concentration until it approached the fluorescence of the unfolded 
 state . to estimate gui1 at 0 m urea , data were also recorded in the presence of 0.4 m na2so4 and the data in 0 and 0.4 m na2so4 were fitted globally to eq 1 . as well as being 70% identical in sequence , m2 m and h2m/n6 have similar structures 
 ( backbone rmsd  1.5  ) ( figure 1a ) . however , only n6 is able to aggregate 
 into amyloid fibrils at ph 6.2 as monitored by the increase in tht 
 fluorescence and by negative stain em ( figure 1b ) , in agreement with previous studies . to enable nmr studies of m2 m , chemical shift assignments were obtained for the native monomeric 
 protein using a range of 3d nmr experiments ( bmrb 19772 ) ( 85% of backbone 
 atoms were assigned ; see methods ) . n hsqc spectrum of native m2 m at ph 6.2 shows a single set of well - dispersed intense peaks , 
 characteristic of a folded protein in solution that undergoes limited 
 chemical exchange on the ms time scale ( figure 1c ) . the only available structure of 
 m2 m is the crystal structure of the protein bound 
 to the heavy chain of the major histocompatibility complex ( mhc - i ) . in the case of h2 m , binding 
 to the heavy chain causes conformational changes particularly in the 
 ab loop and the d strand of the protein . to determine whether the crystal structure of m2 m bound to the mhc - i complex constitutes a good representation for 
 the structure of the monomeric protein in solution , residual dipolar 
 couplings ( rdc ) were measured . figure 1d shows that there is excellent agreement ( r = 0.85 ) between the measured rdcs and those 
 back - calculated from the crystal structure of mhc - i - bound m2 m ( figure 1d ) , confirming the identity of the solution structure of monomeric 
 m2 m with that bound to mhc - i . therefore , differences 
 in the structures of the native proteins can not explain the different 
 amyloid potential of the two variants . we next assessed whether 
 differences in thermodynamic and/or kinetic 
 stability between m2 m and h2m/n6 
 could rationalize their different amyloid propensity . equilibrium 
 denaturation experiments revealed that m2 m is less 
 stable than h2 m ( gun = 12.4 kj / mol ) ( figure 2a , 2b and supplementary table 1 ) . remarkably , m2 m is less stable than the aggregation prone n6 ( figure 2a , 2b ) demonstrating that thermodynamic instability can not explain 
 the inability of m2 m to form amyloid . notably , n6 
 shows a reduced m - value compared with h2 m and m2 m , consistent with exposure of hydrophobic residues that are 
 normally buried in the core of h2 m ( 10 of the 17 
 core residues become more exposed in n6 ) . n hsqc spectra revealed that m2 m is also kinetically less stable than h2 m , with 
 amide protons exchanging with the solvent more rapidly than h2 m , while n6 is the least kinetically stable of the 
 three proteins studied here ( figure 2c , figure s1a  c , and figure s2 ) . nmr relaxation experiments on native m2 m also showed no regions of increased dynamics on the ps ns 
 time scale , apart from the de loop which is known to be flexible in 
 all 2 m variants ( figure s2d thus , there is no correlation between thermodynamic or kinetic 
 stability and amyloidogenicity of these different 2 m variants . ( a ) equilibrium denaturation curves for m2 m , h2 m , and n6 monitored using tryptophan 
 fluorescence ( 75 mm sodium phosphate buffer ph 6.2 , 25 c ) . ( b ) 
 unfolding free energies obtained by fitting data in ( a ) to a two state 
 model ( see table s1 ) . ( c ) representative 
 hydrogen exchange profiles for the amide hydrogen of residue 83 in 
 h2 m , m2 m , and n6 at 25 
 c and ph 6.2 ( see figure s2 ) . we next investigated 
 whether the folding pathway of m2 m also involves 
 transient formation of an intermediate containing trans x - pro 32 ( known as the it state ) , the accumulation of which has been shown to 
 correlate directly with the rate of amyloid formation of the human 
 protein . for these experiments , m2 m was unfolded either by incubating the protein at ph 2.0 
 or by the addition of 8 m urea at ph 6.2 . refolding was then initiated 
 by dilution to a buffer of ph 7.2 or to a buffer lacking urea ( see methods , figure s3 ) , 
 and nmr spectra were collected in real time to track the refolding 
 reaction in residue - specific detail . in the case of h2 m , and in accordance with previous studies , a well - dispersed spectrum was observed 3 min after refolding was 
 initiated , in which only small chemical shift differences are detected 
 in comparison with those of the native protein ( figure 3a and 3b ) . these results 
 indicate that a native - like intermediate ( the it state ) 
 accumulates during folding of h2 m , consistent with 
 previous results . n sofast hsqc spectrum of native 
 h2 m in 10 mm sodium phosphate ph 6.2 . n sofast hsqc spectrum of h2 m collected 3 min after refolding was initiated by urea dilution . peaks that are already in their native positions are colored gray , 
 while peaks with non - native chemical shifts are shown in red . n hsqc spectrum of n6 
 closely resembles the spectrum of the real - time folding intermediate 
 of h2 m . an overlay of the spectra shown in b and 
 c is shown as an inset . n hsqc 
 spectrum of native m2 m in 10 mm sodium phosphate 
 ph 6.2 . n best - trosy - hsqc 
 of m2 m collected 3 min after refolding was initiated 
 by urea dilution . peaks that are already in their native positions 
 are colored green while peaks with non - native chemical shifts are 
 shown in purple . n hsqc 
 spectrum of m2 m at ph 3.6 closely resembles the 
 spectrum of the real - time folding intermediate of m2 m . as previously reported , the spectrum 
 of n6 is very similar 
 to that of the human it state ( figure 3c ) . in marked 
 contrast with the behavior of h2 m , however , the h n hsqc spectrum of m2 m 3 min after refolding is initiated revealed only 20 intense 
 peaks with limited chemical shift dispersion in the h 
 dimension which coexist with the most intense peaks of the native 
 state ( figure 3d and 3e ) . interestingly , the partially folded state of 
 m2 m at ph 3.6 gives rise to a h n hsqc spectrum that closely resembles the spectrum collected 
 at ph  6.2 , 3 min after refolding was initiated ( figure 3f ) . these results 
 suggest that partially folded species are significantly populated 
 during the folding of m2 m , but that these species 
 differ in structure and/or dynamics compared with their human counterparts . in order to assign the real - time spectrum of the intermediate state 
 of m2 m , continuous , nus nmr spectra ( 2d - best - trosy 
 hsqc , 3d - hnca+ , and 3d - hnco+ ) were collected during refolding and 
 the whole data set was coprocessed together , resulting in a temporal 
 resolution of a few minutes . importantly , 
 and in contrast with other real - time studies of protein folding which 
 consist of acquisition of sequential stand - alone spectra , this nus approach requires the acquisition of 
 only a single spectrum . therefore , it does not require prior knowledge 
 about the folding reaction in order to decide on the length of each 
 individual experiment , since the time resolution can be determined 
 in the processing step . additionally , increased sensitivity is achieved 
 by coprocessing less sensitive 3d nmr spectra ( e.g. , hnca ) with 2d 
 experiments ( e.g. , hsqc ) . the same 
 set of intense resonances shown in figure 3e were observed in 3d real - time spectra , 
 consistent with these residues being flexible in the folding intermediate 
 of m2 m . as only 20 spin systems are present in the 
 real - time hnca spectrum further backbone assignment experiments were performed on m2 m at ph 3.6 ( figure 3f ) and m2 m at ph 6.2 with 1 m urea added , 
 both of which gave rise to spectra similar to those of the kinetic 
 intermediate ( figure 3f and data not shown ) , removing the need for rapid data acquisition 
 ( see methods ) . the residue - type specific information 
 of the c atoms from these equilibrium experiments greatly facilitated 
 the assignment of the real - time hnca spectrum . the assignment 
 walk on the c resonances of m2 m 5 min after 
 folding was initiated is shown in figure 4a . the assignment revealed 
 that all of the intense peaks shown in figure 3e correspond to residues located in the n - terminal 
 region , the a strand , and the ab loop of m2 m ( figure 4b and 4c ) , regions of the polypeptide chain whose dynamics have been 
 implicated in the initiation of the aggregation of the human protein . the c - terminal four residues of the protein were also detected with 
 chemical shifts that are different ( h+n > 
 2 ppm ) from those in the native structure . the backbone assignments 
 ( n , nh , c , c , co atoms ; supplementary table 2 ) allow the accurate prediction of the order parameter 
 s and , therefore , an assessment of protein dynamics . figure 4b and 4c show that the 20 n - terminal residues show significantly 
 increased dynamics in the folding intermediate in comparison with 
 the native state , while the c - terminal four residues , while visible , 
 have conformational dynamics similar to those of the native protein . ( a ) the 
 assignment walk on the c atoms of the real - time best - hnca spectrum 
 of m2 m collected 5 min after refolding was initiated 
 by urea dilution at ph 6.2 . the s parameters of the native 
 state are shown as light blue bars and as a red line . ( c ) residues 
 observed in the real - time spectrum of the folding intermediate are 
 highlighted in red in the structure of m2 m . ( d ) far - uv cd spectra of native m2 m at ph 6.2 ( green ) and of the folding intermediate 3 min 
 after folding was initiated by a ph jump from ph 2.0 to ph 6.2 ( red ) . ( e ) normalized fluorescence signal at the end of each stopped - flow 
 transient ( 20 s ) as a function of urea concentration for m2 m in the absence ( red ) or presence ( black ) of 0.4 m sodium 
 sulfate . the data were fitted globally to a two - state model ( see methods ) . the molten - globule - like behavior of the intermediate state 
 ( named 
 here i1 ) prevents the direct observation of the majority 
 of the resonances ( apart from the 20 most flexible ) in h - detected experiments , as they are in conformational / solvent exchange . 
 to overcome this problem direct c - detection experiments 
 were performed on m2 m at ph 3.6 in order to obtain 
 information about the structural properties of the rest of the protein 
 in the i1 state . these experiments revealed 80 
 resonances , 20 of which have sharp lines which correspond to the 20 
 n - terminal residues assigned above . other resonances show broader 
 lines suggesting that they correspond to residues with a higher degree 
 of folding ( figure s5 ) . the far - uv cd spectrum 
 of m2 m obtained 3 min after the ph jump from ph 
 2.0 to ph 6.2 shows that in i1 73% of native -sheet 
 structure is already formed , as quantified by the ratio of the intensities 
 at 219 nm ( figure 4d ) . these results show that m2 m at ph 3.6 is partially 
 structured , with the majority of residues being in a -sheet 
 conformation , potentially native - like . these residues undergo chemical 
 exchange on the ms time scale ( or exchange rapidly with solvent ) and , 
 therefore , can not be observed in the h n spectrum shown in figure 3e . in order to estimate the stability of the i1 state , 
 stopped - flow fluorescence experiments were used to determine the fluorescence 
 intensity of m2 m 20 s after folding was initiated 
 ( figure 4e ) . these 
 experiments revealed that , by contrast with the it state 
 of h2 m for which the gun is 9.57  0.54 kj / mol at 37 c , the i1 state of m2 m ( gun =   4.8 
 kj / mol at 37 c ) is only marginally stable in solution , in accordance 
 with the real - time nmr data ( figure 4e ) . overall , the data show that m2 m folds through a flexible / molten globule - like intermediate state 
 in which the n - terminus and the a strand are dynamic and detached 
 from a native - like -sandwich fold ( i1 ) ( figure 4c ) . the amyloid 
 fibrils of h2 m are composed of parallel 
 in register -strands , while in the native 
 monomer the -strands are all antiparallel ( figure 1a ) . thus , a major conformational 
 change has to occur on the pathway to fibrils . detachment of the a 
 strand might represent a first step toward the remodeling of the native 
 protein , and therefore , the early intermediate of m2 m , i1 , might be expected to be highly amyloidogenic . to 
 test this hypothesis , aggregation assays were performed using n6 
 at ph 6.2 as a mimic of the highly aggregation - prone state it , and m2 m at ph 3.6 , conditions which favor the 
 less structured intermediate state ( i1 ) of m2 m . consistent with previous results , these experiments showed 
 that n6 aggregates rapidly at ph 6.2 with a lag time of 30 
 h , resulting in the formation of amyloid fibrils ( figure 5a ) . in marked contrast , no 
 increase in tht fluorescence was observed for m2 m at ph 3.6 ( figure 5a ) . indeed , the majority of the murine protein remained soluble after 
 100 h of incubation , while n6 was quantitatively converted 
 into amyloid fibrils ( figure 5b and 5c ) . interestingly , the small 
 amount of m2 m that was not found in the supernatant 
 also formed short fibrils 300 nm in length ( figure 5c ) . these results show that 
 the partially folded state of m2 m is not highly 
 aggregation prone . on the other hand , the specific structural features 
 of the native - like it intermediate of h2 m are crucial for assembly . ( a ) tht fluorescence assays of 
 60 m 
 n6 in 10 mm sodium phosphate buffer ph 6.2 ( red ) or 60 m 
 m2 m in 10 mm sodium phosphate , 10 mm sodium acetate 
 ph 3.6 ( green ) . ( b ) sds - page 
 gel of the end points shown in ( a ) . 20 l of the reaction were 
 spun down using a benchtop centrifuge for 20 min and a sample of the 
 total reaction ( w ) or of the supernatant ( s / n ) after centrifugation , 
 was analyzed by sds - page . ( c ) afm image of 60 m m2 m after 95 h of incubation ( green in a , note that fibrils 
 correspond to < 5% of the protein added ) and electron micrograph 
 of 60 m n6 ( fibrils correspond to > 95% of the protein 
 added ) after 95 h of incubation ( red in a ) . the data presented 
 above demonstrate that the conformational properties of the dynamic 
 i1 state of m2 m are different from those 
 the native - like h2 m it state . however , 
 additional states could be populated after the formation of i1 and prior to the formation of native m2 m . indeed , as the folding time progresses a third set of peaks ( apart 
 from the native and the flexible intermediate states ( figure 3 ) ) emerges in the real - time h these peaks show small chemical shift differences 
 compared with the native m2 m resonances and are 
 generally broad ( figure s4 ) suggesting 
 that additional , more native - like states are populated at later times 
 during the folding of the protein . this observation presumably reflects 
 an ordered assembly mechanism , in which the initially highly dynamic 
 intermediate ( i1 ) folds to the native state via a transiently 
 populated more structured ( it - like ) state . to investigate 
 this possibility further , partially folded m2 m at 
 ph 3.6 ( which mimics the i1 state figures 3e and 3f ) was allowed to fold by a rapid ph jump to ph 6.2 and folding was 
 monitored in real time using nmr . n hsqc spectrum collected 3 min after refolding was initiated showed 
 a well dispersed spectrum ( figure 6a ) in striking and marked contrast with the molten 
 globule - like spectrum of the i1 state shown in figure 3e . indeed , the spectrum 
 obtained 3 min after the ph jump is reminiscent of that of native 
 m2 m with significant chemical shift differences 
 being limited to residues in the n - terminal region ( residues 16 ) , 
 the bc , de , and fg loops ( figure 6a and 6b ) . moreover , the peaks 
 that show chemical shift differences from the native protein are remarkably 
 similar to the third set of peaks observed in figure s4b , consistent with folding from the partially folded 
 state ( i1 ) to a more native - like intermediate ( presumably 
 it ) . importantly , residues that show significant chemical 
 shift changes are surrounded by residues whose resonances are not 
 detected in the real - time nmr spectrum ( figure 6b ) . these areas reside in close spatial proximity 
 to pro 32 in the native structure and , therefore , are exchange - broadened . 
 a similar scenario has been observed for the real - time folding intermediate 
 of h2 m ( it ) . furthermore , n6 shows chemical shift differences to native 
 h2 m in these same regions . together , the results show that m2 m folds through 
 a native - like intermediate state that has similar structural properties 
 to the amyloidogenic it state of h2 m . 
 however , due to its decreased stability , this species is copopulated 
 with an ensemble of partially folded , flexible states in which the 
 n - terminal region is highly disordered ( i1 ) . n sofast hsqc 
 spectrum of m2 m collected 3 min after refolding 
 was initiated from the partially folded state at ph 3.6 ( blue ) , overlaid 
 with the h n sofast hsqc of native 
 m2 m ( collected after 90 min ) ( gray ) . n chemical 
 shift differences between the native and the it state of 
 m2 m , using the spectra displayed in ( a ) . blue dots residues that show chemical shift differences greater than 
 1 ppm ( dashed line ) are colored yellow , those that show chemical shift 
 differences less than 1 ppm are shown in gray , and residues that are 
 broadened beyond detection in the 3 min spectrum are colored red . 
 the strucure of m2 m colored in the same color scheme 
 is shown on the right . the results presented above highlight the 
 importance of determining 
 the precise details of the folding energy landscape of a protein in 
 order to elucidate whether one or more partially folded , or non - native 
 states , have the potential to initiate amyloid assembly . characterization 
 of ensembles of interconverting non - native species that not only are 
 lowly populated but also have a short lifetime , such as those involved 
 in protein folding and aggregation , is a challenging task , even for 
 the most advanced biophysical methods . while time - resolved nmr studies 
 on proteins can be highly informative , these studies usually suffer 
 from low resolution and poor sensitivity . by combining the use of 
 sparsely sampled nmr and coprocessing of more complicated / less sensitive 
 experiments with others that show increased sensitivity , we demonstrate 
 here the detection and atomic level depiction of the early molten 
 globule state , i1 , of m2 m , that is populated 
 for only 15 min , a task that would not have been possible 
 using standard nmr methodologies ( see supplementary methods ) . the power of the real - time nmr experiments allowed 
 us to identify conditions that stabilize the i1 state ( ph 
 3.6 or addition of 1 m urea at ph 6.2 ) and to perform more detailed 
 nmr experiments on the trapped intermediate state that led to a complete 
 description of the folding mechanism of the murine protein . the approach 
 is potentially applicable to other amyloidogenic proteins or proteins 
 that fold through the accumulation of transient intermediate states . the folding 
 pathway of h2 m has been investigated in detail over 
 the past decade using different protein variants and different techniques . together , these studies have shown that the folding 
 of h2 m involves the formation of a native - like intermediate 
 it that is kinetically trapped by virtue of the non - native trans x - pro 32 bond . this species is preceded 
 by a less well characterized species ( i1 ) that forms in the dead time of a stopped flow experiment 
 ( <3 ms ) and is less structured than the it state ( figure 7 ) . the fine details 
 of the exchange processes between these different folding intermediates 
 has a dramatic effect on the propensity to aggregate . for h2 m the structured aggregation - prone it state is 
 the most highly populated species during folding , accumulating , on 
 average , to 4% ( ph 7.0 , 37 c ) at equilibrium . by contrast , the flexible i1 state represents the most 
 highly populated intermediate state during the folding of m2 m . indeed , the most intense peaks of the i1 state 
 ( but not those of the it state ) are visible in the spectrum 
 of native m2 m , enabling estimation of its equilibrium 
 concentration to 7% ( figure s6 ) . 
 this reduces the population of the m2 m it state , with the effect that aggregation no longer occurs ( at least 
 on an experimentally tractable timescale ) ( figure 7 ) . thus , although the folding mechanisms 
 of human and murine 2 m are conserved ( the same species 
 are populated on the pathway to the native state ) , the precise balance 
 between these states reduces the population of the key amyloidogenic 
 precursor it for m2 m and thus defines 
 the course of amyloid formation . the native - like 
 it state is predominantly populated during the folding 
 of h2 m ( gray scheme , left ) . this allows the entrance 
 to the aggregation landscape ( red scheme ) as it shows enhanced 
 amyloidogenicity . in the case of m2 m ( right ) it represents only a minor conformation during folding , but 
 instead the flexible molten globule - like state in which the a strand 
 is detached from the -sandwich fold is the major non - native 
 species . as i1 is not aggregation - prone , m2 m is protected from misfolding and instead folds to the native state 
 ( the energy levels of the aggergation landscape are drawn for illustration 
 purposes only ) . interestingly , urea denaturation 
 experiments on a partially folded 
 state of h2 m formed at ph 3.6 have shown that the 
 n - terminal six residues and the a strand are the least stable regions , 
 while the rest of the protein forms a stable core . these results are consistent with the real - time nmr studies 
 on the i1 state of m2 m presented here , 
 showing that the conformational properties of the early partially 
 folded states of h2 m and m2 m are 
 similar . interestingly , neither of these states is able to form long , 
 straight fibrils characteristic of amyloid , but instead they form 
 short rod - like fibrils ( m2 m at ph 3.6 ) or worm - like 
 fibrils ( h2 m at ph 3.6 ) ( figure 5 ) . a direct link between decreased native state stability and increased aggregation propensity has been observed for several proteins including 
 lysozyme , transthyretin , and antibody light chains . accordingly , destabilizing mutations enhance the rate of exchange 
 between the native protein and partially folded non - native species , 
 which show increased amyloidogenicity compared with the native state . interestingly , m2 m is less thermodynamically and 
 kinetically stable than h2 m and , as a consequence , 
 the molten globule - like nonamyloidogenic i1 state is the 
 most abundant non - native species ( figure 7 ) . the increased conformational dynamics 
 results in a protein that is unstable yet protected from amyloid assembly , 
 since i1 is not able to form amyloid . these findings argue 
 against a simple link between native state stability and amyloidogenicity 
 ( at least for 2 m ) . instead , they highlight the importance 
 of the precise conformational properties of the native - like it state that are vital for assembly . together , the results 
 demonstrate that amyloid formation of 2 m at neutral 
 ph is initiated via the highly structured it state . hence , 
 from the myriad of potential non - native conformations 
 that could be populated during folding , only the it state 
 allows the entrance of 2 m to the aggregation landscape 
 ( figure 7 ) . such a 
 finding highlights the ordered specificity in the early stages of 
 assembly into amyloid and opens the opportunity to target a specific 
 non - native state in order to control the onset of aggregation , for 
 example through the development of antibodies , nanobodies , or small 
 molecules that specifically recognize this species . the results highlight 
 the importance of considering multiple factors in order to predict 
 amyloid formation . hydrophobicity , the propensity of the sequence 
 to aggregate , the stability of the native state , solubility , transient 
 exposure of aggregation - prone regions through protein dynamics , and 
 the stability of the polypeptide sequence within the fully assembled 
 fibril structure itself , may all contribute to enhanced amyloidogenicity . 
 although , these factors are well understood individually , the interplay 
 between them during aggregation remains poorly explained . the results 
 presented emphasize the importance of understanding the energy landscape 
 of aggregation in intricate detail , from both thermodynamic and kinetic 
 view points , in order to predict whether or not a protein will aggregate 
 and how / why minor alterations in solution conditions / amino acid sequence 
 can have a dramatic effect on the course of assembly , by small changes 
 in the relative populations of amyloidogenic versus nonamyloidogenic 
 states . in this study we have 
 used nus nmr methods to study the relationship 
 between protein folding and aggregation of a globular protein that 
 forms amyloid fibrils from a structured precursor state , using 2 m as the test protein . we show that the least thermodynamically 
 stable protein is the least aggregation - prone sequence of the family 
 of proteins studied here . analysis of the folding energy landscape 
 of the protein using real - time nmr revealed that the decreased stability 
 and decreased lifetime of a precise and well - defined native - like amyloidogenic 
 precursor ( it ) are sufficient to tip the balance from aggregation 
 to folding . the power of sparsely sampled nmr allowed us not only 
 to detect a dynamic intermediate state ( i1 ) of m2 m but also to structurally characterize this species in residue - specific 
 detail . the results reveal that the least stable protein ( m2 m ) populates predominantly a flexible intermediate ( i1 ) that is not aggregation - prone , while its more stable counterpart 
 ( h2 m ) folds through a native - like intermediate that 
 has enhanced amyloidogenicity . subtle changes in the folding energy 
 landscape thus lead to dramatic changes in the aggregation outcome . instead it is the precise balance and kinetic 
 partitioning of intermediate states that determines whether 2 m will fold to the native state or aggregate to form amyloid 
 fibrils .
the 
 balance between protein folding and misfolding is a crucial 
 determinant of amyloid assembly . 
 transient intermediates that are 
 sparsely populated during protein folding have been identified as 
 key players in amyloid aggregation . however , due to their ephemeral 
 nature , structural characterization of these species remains challenging . 
 here 
 , using the power of nonuniformly sampled nmr methods we investigate 
 the folding pathway of amyloidogenic and nonamyloidogenic variants 
 of 2-microglobulin ( 2 m ) in atomic 
 detail . despite folding via common intermediate states , 
 we show that 
 the decreased population of the aggregation - prone itrans state and population of a less stable , more dynamic species ablate 
 amyloid formation by increasing the energy barrier for amyloid assembly . 
 the results show that subtle changes in conformational dynamics can 
 have a dramatic effect in determining whether a protein is amyloidogenic , 
 without perturbation of the mechanism of protein folding .