in most cells , proteostasis 
 involves continuous regulation of protein 
 synthesis and degradation , but the eye lens is a notable exception . during the final stages of maturation , lens cells 
 eject all organelles , preventing normal protein turnover .
the lens therefore provides a unique opportunity 
 to observe the effects of aging in relation to damage accumulation 
 in long - lived proteins , which are increasingly a subject of interest .
they constitute 
 approximately 3540% of the total soluble mass of the lens 
 and are composed of a 3:1 ratio of a to b . while a is found almost exclusively in 
 the lens , b has been found throughout the body including heart , 
 glia , muscle , kidney , lung , and skin cells .
gene knockout studies revealed that a mouse lens can develop normally 
 without b but not without a , which led to prompt cataract 
 formation .
nevertheless , the chaperone activity of 
 heteropolymers formed from a 3:1 ratio of a to b is 
 higher than either homopolymer , suggesting that the combination of 
 both proteins is optimal .
beta and gamma 
 crystallins are the two other major subgroups of crystallins in the 
 lens .
they act in conjunction with the -crystallins to maintain 
 lens structure and transparency and to achieve a suitable refractive 
 index . to behave properly as chaperones , 
 -crystallin monomers must 
 interact with each other to form polydisperse oligomers .
a 
 and b form dimers that then assemble into larger complexes 
 ranging from 15 to 50 monomers , with the subunits dynamically intermixing 
 and exchanging .
removing 
 the n- and c - terminal extensions allows for determination of partial 
 crystal structures .
when crystallins do not interact 
 with each other properly , they become water - insoluble and begin to 
 aggregate .
the influence of many post - translational modifications 
 ( ptms ) on this process has been investigated , including deamidation , 
 oxidation , disulfide formation , and truncation .
importantly , all of these ptms lead to mass shifts that are easily 
 detectable by mass spectrometry ( ms ) .
more subtle modifications 
 that also increase with age , such as 
 isomerization and epimerization , do not lead to easily detectable 
 mass shifts and have been significantly less studied .
methods for detecting isomerization in peptides ( meaning either 
 inversion of the chiral center of an amino acid to produce an epimer 
 or conversion of aspartic acid to isoaspartic acid ) include stereoselective 
 enzymatic digestion , ion mobility , and ms .
previous studies have 
 shown that aspartic acid and serine are the two amino acids most susceptible 
 to spontaneous isomerization .
deamidation 
 of asparagine residues generates aspartic acid , and this transformation 
 can also be accompanied by isomerization .
the mechanism by which l - asp converts into l - isoasp , d - isoasp , and d - asp has been studied previously and is known to proceed via 
 nonenzymatic formation of a succinimide ring , which can yield four 
 isomers , as shown in scheme 1 .
enol tautomerism and then hydrolyzed to form d - asp or d - isoasp . the mechanism 

by which l - ser epimerizes to d - ser in proteins is 
 not well - established , although proposals have been made .
alpha and beta carbons are 
 labeled to highlight the difference between l - asp and l - isoasp , respectively . although isomerization 
 might appear to be a
ptm , studies have shown it can cause major perturbations in protein 
 structure . noguchi and coworkers successfully crystallized hen egg - white 
 lysozyme with an isoaspartic acid substitution at asp101 that caused 
 backbone deflection of nearly 90 relative to the native structure .
crystal structures of a modified ribonuclease 
 revealed that isoasp-32 induces conversion of an -helix to 
 a u - shaped loop .
isomerization not only 
 perturbs 3d structure but also can affect physical properties such 
 as solubility and bioactivity .
for example , isomerization of asp92 
 in immunoglobulin 2 ( igg2 ) leads to deactivation of the antigen - binding 
 region .
large - scale ( i.e. , proteomic ) 
 identification of single amino acid 
 isomerism within peptides is challenging because there is no change 
 in mass .
however , differences in fragment intensities following ms / ms 
 analysis can be used to detect isomers .
this method was first applied 
 to stereoisomers by tao and coworkers , who reported differences as 
 a ratio of relative abundances of a pair of fragment ions differing 
 most between l- and d- enantiomers ( rchiral = rd / rl ) .
it has been shown that 
 radical - directed dissociation ( rdd ) yields 
 the best chiral discrimination for analysis of peptide epimers .
previous work on the detection of isomerization 
 and epimerization in the sheep lens using tandem lc  ms revealed 
 novel sites of isomerization in the crystallins .
the present study focuses on changes in isomerization 
 and epimerization 
 for a , b , and b3 crystallin between water - soluble 
 ( ws ) and water - insoluble ( wi ) protein fractions from cow , sheep , and 
 pig eye lenses . to determine which regions of these proteins are most 
 susceptible to isomerization , enzymatic digestion into peptides
ms / ms analysis using both collision - induced 
 dissociation ( cid ) and rdd .
importantly , we found several regions 
 in the well - ordered crystallin domain that are disproportionately 
 isomerized in the wi fractions .
specific isomerization  hotspots  
 were identified and correspond to regions with serine or aspartic 
 acid repeats .
potential explanations for the isomerization of these 
 sites are offered , and comparison with other long - lived and problematic 
 proteins reveals that these sequence motifs are common .
cow , pig , and sheep 
 eye lenses were acquired as discarded tissue from corona cattle , inc . 

the approximate ages for each of the animals were 1218 
 months for the cow , 56 months for the pig , and 68 
 months for the sheep .
the supernatant was separated from the precipitate following 
 centrifugation at 15,100 g for 20 min at 4 c . 

the precipitate 
 ( wi ) was solubilized in 6 m urea and purified by dialysis against 
 6 m urea . for ws digestion ,
the protein was dissolved in 50 mm nh4hco3 buffer , ph 7.8 ; disulfide bonds were then 
 reduced with 100 mm dithiothreitol ( dtt ) at 95 c for 5 min . 
 after returning to room temperature ,
reduced cysteine residues were 
 capped using 100 mm iodoacetamide in the dark for 20 min .
finally , 
 the proteins were digested with trypsin overnight at 37 c using 
 a 50:1 protein to enzyme ratio . for the wi digestion , the proteins 
 were dissolved using 6 m 50 mm tris - hcl , ph 8.0 .
disulfide bonds were 
 cleaved using 200 mm dtt in tris - hcl , ph 8.0 at 37 c for 20 
 min . following this ,
200 mm iodoacetamide in tris - hcl , ph 8.0 was 
 added and the mixture was incubated in the dark for 1 h. to consume 
 unreacted iodoacetamide , 200 mm dtt was added and incubated for 1 
 h in the dark .
next , the urea concentration was diluted to < 0.6 
 m using 50 mm tris - hcl , 1 mm cacl2 , ph 7.6 .
the proteins 
 were digested using trypsin with a 50:1 protein to enzyme ratio for 
 16 h at 37 c . for the iodobenzoic acid modification
, the digested 
 peptides were desalted and cleaned using a peptide trap ( michrom bioresource ) . 

approximately 5 nmoles of the digestion mixture , 15 l of 15 
 mm 4-iodobenzoic acid nhs - activated ester in dioxane , and 5 l 
 of borate buffer ( ph 8.6 ) were combined and incubated for 1 h at 37 
 c .
important : dimethyl sulfoxide should not be substituted 
 for dioxane in this step because it can cause aspartic acid isomerization .
the modification side products at arginine and tyrosine side chains 
 were removed by incubating the reaction mixture in 1 m hydroxylamine , 
 ph 8.5 .
these procedures have been previously determined not to yield any 
 detectable isomerization / epimerization in control experiments . to quantify 
 isomer identification , an rchiral value 
 approach ,
, risomer represents the ratios of the relative intensities of a pair of fragments 
 that varies the most between two isomers ( ra / rb ) . if risomer = 1 , then there the two tandem ms spectra are indistinguishable 
 and the species are likely not isomers .
if risomer > 1 , then a larger number indicates a higher probability 
 that two unique molecules are represented . to confidently identify 
 each of these isomers by ms / ms , we use a threshold that was determined 
 by performing a t test on the risomer values obtained by performing cid and rdd on a mixture 
 of synthetic peptides separated by lc
ms . using 99% confidence 
 intervals , the risomer threshold for cid 
 is > 1.9 and for rdd it is > 2.4 .
we have previously 
 described a 
 modified bottom - up strategy for detecting sites of isomerization , 
 including epimerization , in proteins . in brief , proteolytically digested peptides are separated by reverse - phase 
 column chromatography and analyzed by ms with minimal time exclusion 
 windows to favor repeated analysis of the same m / z .
for example , the n - terminal fragment of a crystallin 
 from the wi protein fraction of sheep lens , acetyl - mdiaiqhpwfk , 
 elutes at four different times , each separated by 2 min , as 
 shown in figure 1a . 
 to establish that these peaks represent isomers , repeated ms / ms analysis 
 of each eluting peak is required ( both cid and rdd are used in separate 
 runs).figure 1b shows cid spectra from each of the lc peaks 
 in figure 1a .
although 
 the spectra are similar , suggesting that they originate from the same 
 peptide sequence , the relative intensities of certain peaks change 
 noticeably .
for example , the intensities of y9 , b7 , and y10 ions 
 vary considerably between lc peaks 1 and 2 .
these differences can 
 be quantified into risomer values , which are provided in figure 1c . for cid analysis ,
risomer values above 1.9 represent statistically significant 
 differences , meaning that all four peaks in figure 1a are different isomers of acetyl - mdiaiqhpwfk . 

mass spectra alone do not provide information about which isomer corresponds 
 to the native l - asp peptide , but in this case , the largest 
 peak constitutes > 90% of the total relative abundance and likely 
 represents 
 the unmodified l - asp isomer . if the wi fraction is highly 
 isomerized , in which case identification of the l - asp isomer 
 becomes ambiguous , then comparison with the less isomerized ws digest 
 or an authentic standard is used to identify the l - asp isomer .
( a ) lc 
 chromatogram for the separation of the four isomers of ac - mdiaiqhpwfk 
 in the wi sheep lens digest .
( b ) cid spectra from each of the lc peaks . labeled fragments 
 are those used to determine risomer values . 

the degree of peptide isomerization from the ws and wi protein 
 fractions of sheep a crystallin is summarized in figure 2 .
all isomers were confirmed 
 by comparison of either cid or rdd ms / ms spectra , as described above . 

for example , the two isomers of aipvsr were indistinguishable by cid but could be confidently detected 
 using rdd ( see si figure s1 ) . additionally , 
 some isomers coelute , preventing quantitation of the relative abundance 
 of each form .
the first downward bar on the left side of figure 2a ( 111 ) represents the data from figure 1 .
this peptide , acetyl - mdiaiqhpwk , 
 was found to be 9% isomerized , with error bars representing standard 
 deviations from three technical replicates .
the color coding of the 
 bars corresponds to the three distinct structural regions present 
 in -crystallins , the n - terminal disordered region , the crystallin - ordered 
 region , and the disordered c - terminal extension .
these regions are illustrated relative to a partial crystal 
 structure on the right side of figure 2 .
the full protein sequence 
 is provided at the bottom of figure 2 , color coded and with residues of interest marked 
 for easy location .
the trends in figure 2 illustrate that the average amount of isomerization 
 is significantly higher in the n and c termini than in the well - ordered 
 crystallin domain for both the ws and wi fractions .
these dynamic 
 regions play important roles in the assembly of -crystallins 
 into higher - order structures , but this 
 flexibility may also enable isomerization by facilitating more frequent 
 access to the pathways outlined in scheme 1 .
percent isomerization 
 of water - soluble ( ws ) a sheep versus 
 water - insoluble ( wi ) a sheep .
orange , disordered n - terminus ; 
 blue , structured -crystallin domain ; purple , disordered c - terminus . 

peptide 164173 
 does not contain error bars because it only appeared baseline - resolved 
 in one digest .
the full protein sequence is given below the plot , 
 with aspartic acid residues in bolded / black and serine residues in 
 underlined / black .
asp105 and asp106 are in bold red text in the amino 
 acid sequence and are shown explicitly in the crystal structure ( pdb 3l1f ) to highlight an 
 important region of isomerization .
stars indicate isomerized regions 
 where isomerization was identified , but quantitation was not possible 
 due to incomplete chromatographic separation .
interestingly , the degree of isomerization in the disordered 
 regions , 
 although slightly more abundant in the wi fraction , is similar in 
 both the ws and wi fractions .
this suggests that these modifications 
 do not significantly drive aggregation and loss of solubility , as 
 previously reported .
differences in degree 
 of isomerization are more notable within the crystallin region , where 
 isomerization is significantly more abundant in the wi fraction ( except 
 for the dramatically modified peptide 104112 that will be 
 discussed further below ) .
cumulatively , these results imply that modifications 
 to flexible regions may be more facile but also less consequential 
 in terms of altered functionality .
fewer peptides 
 are isomerized in b , and the average degree of isomerization 
 is less in both the ws and wi fractions compared with a . the 
 flexible n - terminal domain is highly isomerized , similar to a . 

interestingly , the c - terminal extension is not isomerized , which contrasts 
 with a and is largely due to the fact that the c - terminal extension 
 of b lacks any aspartic acid residues and contains only a single 
 serine .
the degree of isomerization observed in the ws versus wi fractions 
 also varies more dramatically compared with a , with greater 
 isomerization being observed in the wi fraction for all peptides . 

a and b share similar functionality and freely intermix 
 to form higher order structures , yet comparison of figures 2 and 3 illustrates significant differences in propensity and effects of 
 isomerization .
the sequence alignment of the two proteins is < 60% 
 ( figure s2 ) , but the tertiary structures 
 of a and b are very similar , as illustrated by the crystal 
 structure of truncated bovine b on the right side of figure 3 . despite the similarity , sequence variation appears to have 
 a significant effect on the ability of b to accommodate isomerization 
 and retain solubility , in agreement with previous observations .
another interesting difference between the two 
 -crystallins is the overall abundance of acidic amino acids . 

aspartic acid in a comprises 8.8% of residues and glutamic 
 acid contributes another 5.8% , whereas b contains 6.3% aspartic 
 acid and 8.0% glutamic acid . although isomerization of glutamic acid 
 is possible , the formation of the glutarimide intermediate is much 
 slower compared with the succinimide equivalent in aspartic acid .
therefore , even though both proteins contain 
 a similar percentage of acidic residues , a contains more residues 
 prone to isomerization , and these residues often reside in regions 
 where isomerization is facile .
isomerization of b from sheep , 
 which differs significantly 
 from what was observed for a ( see figure 2a , formatting is identical ) .
asp109 in the 
 amino acid sequence is in red bold text and shown explicitly in the 
 crystal structure ( pdb 3l1 g ) to highlight an important site of isomerization .
the same analysis that was performed 
 on the  crystallins 
 was also conducted on one of the  crystallins .
b3 is composed of 211 amino acids , 
 with two large structured domains connected by a flexible linker . 

the disordered n - terminal and c - terminal regions are much smaller 
 than those observed in the -crystallins .
the total amount of 
 isomerization detected in the ws and wi fractions of b3 is 
 lower than that in a and b , which is likely due to the 
 greater fraction of highly structured regions .
similar to b , 
 b3 is characterized by significant disparity between the degree 
 of isomerization in the ws versus wi fractions , with greater isomerization 
 being observed in the latter .
this suggests again that structural 
 perturbations in b3 are consequential and lead to rapid loss 
 of solubility .
the isomerized versions of several peptides , including slrplhidgpdhk and kmeivdddvpslw , are only detectable 
 in the wi fractions ( see figure s3 ) .
slrplhidgpdhk is part of 
 the structurally critical connecting peptide that joins the two domains 
 of the monomer , denoted by the green bar in figure 4 .
kmeivdddvpslw contains aspartic acid residues that form ion pairs during 
 assembly into higher order oligomers .
sequence alignment for human , pig , cow , and sheep b3 shows 
 that each contains this aspartic acid repeat motif ( figure s4 ) .
the results in figure 4 suggest that isomerization of either of 
 these peptides leads to dramatic loss of solubility and , by extension , 
 function .
formatting 
 is identical to figure 2 , except for the linker of the structured domains , shown in green . 

asp110 , asp113 , asp133 , asp134 , and asp135 are all in red bold text 
 in the amino acid sequence and are shown explicitly in the human crystal 
 structure ( pdb 3qk3 ) to highlight important regions of isomerization .
his113 from human 
 b3 was mutated to asp113*. the same analysis was carried out on pig and cow lenses , 
 and the 
 results are summarized with the sheep results in figure 5 .
the percent isomerization 
 per protein was determined by taking the sum of the percent of isomerization 
 per peptide and dividing it by the total number of peptides , including 
 a value of zero for those that were not isomerized .
peptides where 
 the degree of isomerization could not be determined were omitted for 
 all samples of the same protein .
for example , tryptic digestion of 
 173 residue a yielded 12 peptides that cover 92% of the sequence . 

contributions from these same peptides were used to calculate the 
 numbers for each of the ws and wi a digests for each animal . 

the data were then normalized to the a digest that was most 
 isomerized , which was the wi sheep digest . the same approach was applied 
 to b and b3 . although there is some variation between 
 species , in general ,
the degree of isomerization is , on average , significantly greater 
 in the wi fractions for all species .
several of the peptides 
 in figures 24 appear to exhibit an unusual degree of isomerization 
 relative to their peers .
for example , qddhgyisr from a is not only is the most isomerized site in 
 the protein but also is significantly more isomerized than any other 
 peptide in the structured -crystallin domain .
quantitatively , 
 the average amount of isomerization per peptide in the structured 
 domain of a is 17.9% for the ws digest and 27.3% in the wi 
 digest . at 76.7 and 89.3%
isomerization in the ws and wi fractions , qddhgyisr is isomerized 4 the 
 average rate in the ordered crystallin region .
the analogous sequence 
 in b , qdehgfisr , is also the most 
 isomerized peptide extracted from the wi fraction , although full quantitative 
 comparisons can not be made due to lack of separation of some isomers 
 for other peptides in the ordered region ( see figure s5 for details ) .
finally , kmeivdddvpslw is the most isomerized peptide in the wi fraction from 
 b3 .
each of these peptides shares a common feature , sequential 
 repeats of acidic residues . in two cases ,
these observations suggest that sequential 
 acidic residues represent sites of greater propensity for isomerization .
there are at least two possible explanations that could account 
 for increased isomerization at acid residue repeats .
first , if there 
 are multiple aspartic acid residues , then isomerization at multiple 
 sites is possible . structural perturbation engendered by the first 
 modification could easily lead to increased local backbone flexibility , 
 facilitating isomerization at additional sites .
indeed , examination 
 of the elution profiles for these peptides reveals an abundance of 
 isomers , indicating modification at more than one residue .
for example , 
 the elution profiles for the ws and wi fractions from pig for pqddhgysir reveal the presence 
 of seven isomers ; see figure 6 . the lowercase  p  at the n - terminus of the 
 sequence denotes pyroglutamate , which is a common product during trypsin 
 digestion .
comparison with synthetically prepared all l - asp 
 isomer was used to confirm the peak corresponding to the canonical 
 peptide .
risomer values ( 4.8 , 5.5 , 3.7 , 
 4.6 , 4.5 , 1.2 , and 3.4 ) relative to the synthetic version confirm 
 that the sixth peak in the digest is the all l - asp isomer 
 ( see figure s6 for details ) .
the ws / wi 
 degree of isomerization for pqddhgysir from sheep is shown in figure 6c , d .
a similar elution profile is seen between 
 wi pig and sheep ; however , the amount of l - asp in the ws 
 pig lens is nearly twice as great as it is in ws sheep .
( a ) ws chromatogram 
 and ( b ) wi chromatogram for pqddhgysir from pig .
( d ) wi sheep . lower case  p  
 indicates the pyroglutamate that forms during tryptic digestion of 
 n - terminal glutamine residues .
isomers were confirmed by ms / ms analysis 
 and are labeled with percent abundance .
another factor contributing to isomerization at sites with 
 multiple 
 acidic residues , which may be more important than increased backbone 
 flexibility , is inhibited repair by protein isoaspartyl methyltransferase 
 ( pimt ) .
pimt is the only known repair enzyme that targets protein 
 damage caused by aging .
it is present and active in bovine lenses 
 and reverts l - isoasp back to l - asp.d - asp is also repaired , although much less efficiently , 
 but d - isoasp is not a substrate .
importantly , sequence effects for substrate recognition have been 
 identified and revealed that pimt has substantially lower affinity 
 for isoasp if the n+1 , n+2 , or n+3 residues are negatively charged or if the n+1 site is proline .
therefore , the sequence 
 regions with multiple acidic residues that we have observed to be 
 highly modified are unlikely to be repaired by pimt , and our results 
 confirm the importance of this enzyme in the repair of long - lived 
 proteins .
acidic residue repeat sites may therefore be prone 
 to sequential 
 isomerization and difficult to repair , a potent combination with important 
 consequences for protein aging .
indeed , examination of other long - lived 
 proteins associated with age - related diseases reveals the presence 
 of numerous acidic acid repeat sites that would similarly be susceptible 
 to isomerization .
for example , amyloid precursor protein associated 
 with alzheimer s disease contains 27 acidic residue repeat 
 sites that would be poor substrates for pimt .
table 1 lists long - lived proteins that contain aspartic 
 acid repeats , are aggregation - prone , and are associated with diseases . 

it is clear that the results obtained here may be relevant on a much 
 broader scale for any system composed of long - lived proteins .
number of asp residues with an acidic 
 residue in the n+1 , n+2 , or n+3 position .
we have also identified isomerized regions with multiple serine 
 residues , such as eekpssapss . in the ws 
 and wi fraction of the sheep lens this peptide
elutes in six different 
 peaks ( figure s7 ) , and comparison of resulting 
 cid spectra confirms that they represent different isomers .
it is 
 interesting to note that the total amount of isomerization in the 
 wi sheep fraction for eekpssapss is much 
 lower than what was observed for qddhgyisr , 27.3% compared with 90.4% , respectively .
it is possible that isomerization 
 of aspartic acid may have a larger influence on local structure than 
 epimerization of serine , inducing isomerization of nearby residues 
 more efficiently .
alternatively , there is no known repair enzyme for 
 epimerization of serine , which may suggest that the failure of pimt 
 to repair isomerization may be the most important factor influencing 
 isomerization at acidic residue repeats .
however , the number of peptides 
 with unambiguous epimerization at serine , that is , peptides that are 
 isomerized and do not contain aspartic acid , is relatively small . 

therefore , more data will need to be acquired before strong conclusions 
 about serine epimerization can be drawn .
peptide 
 epimerization and isomerization are difficult to detect 
 and remain among the least studied ptms despite their potentially 
 important role in numerous diseases .
we have evaluated these modifications 
 in crystallin proteins from the eye lenses of several organisms , and 
 our results suggest that isomerization and epimerization lead to reduced 
 protein solubility in many cases .
although there is variation between 
 species , several common themes emerge from analysis of the data . for 
 example
this finding suggests that many other long - lived 
 proteins with known disordered regions , such as -synuclein , 
 tau , and -amyloid , may also exhibit pathology related to isomerization 
 or epimerization .
although less prone to modification , isomerization 
 within well - structured regions of proteins leads to more drastic changes 
 in behavior , including unchecked aggregation .
it was previously known 
 that serine and aspartic acid are the most easily isomerized among 
 the natural amino acids , but we have further demonstrated that when 
 multiple acidic residues are close in sequence , the propensity for 
 modification is enhanced further .
we postulate that increased flexibility 
 following an initial modification and failure of pimt to repair damaged 
 residues contribute to the dramatic isomerization of sites with multiple 
 acidic residues .
the degree of isomerization at these sites also confirms 
 the importance of pimt for long - term maintenance of protein structure . 

proteins associated with parkinson s disease , alzheimer s 
 disease , amyotrophic lateral sclerosis , and huntington s disease 
 all contain regions with multiple aspartic acid or serine residues 
 in close proximity , suggesting that these proteins may also exhibit 
 hotspots of isomerization .
both a and b exhibit 
 similar structural and functional 
 behavior , yet a is found only in the eye lens .
the isomerization 
 behavior of a and b are quite distinct , with a 
 being more prone to isomerization , but with b suffering greater 
 loss of solubility following isomerization .
the ability of a 
 to sustain isomerization without loss of solubility may make it uniquely 
 suited to the zero - turnover environment of the eye lens .
it is clear 
 that further , detailed examination of isomerization and epimerization 
 in other tissues is needed and will expand our understanding of the 
 role of long - lived proteins in aging - related diseases .
 the eye lens crystallins represent 
 an ideal target for studying 
 the effects of aging on protein structure . 
 herein we examine separately 
 the water - soluble ( ws ) and water - insoluble ( wi ) crystallin fractions 
 and identify sites of isomerization and epimerization . both collision - induced 
 dissociation and radical - directed dissociation 
 are needed for detection 
 of these non - mass - shifting post - translational modifications . 
 isomerization 
 levels differ significantly between the ws and the wi fractions from 
 sheep , pig , and cow eye lenses . 
 residues that are most susceptible 
 to isomerization are identified site - specifically and are found to 
 reside in structurally disordered regions . 
 however , isomerization 
 in structured domains , although less common , often yields more dramatic 
 effects on solubility . 
 numerous isomerization hotspots were also identified 
 and occur in regions with aspartic acid and serine repeats . 
 for example , 128kmeivdddvpslw140 in b3 
 crystallin contains three sequential aspartic acid residues and is 
 isomerized heavily in the wi fractions , while it is not modified at 
 all in the ws fractions . 
 potential causes for enhanced isomerization 
 at sites with acidic residue repeats are presented . 
 the importance 
 of acidic residue repeats extends beyond the lens , as they are found 
 in many other long - lived proteins associated with disease . 