proteins require optimized stability and 
 flexibility to perform 
 their biological roles , including interacting with binding partners , 
 responding to their environment and resisting aggregation . protein 
 function depends on both structure and dynamics ; making the folded 
 state of a protein more stable by rigidifying it does not necessarily 
 lead to enhanced functionality . comparisons 
 among homologous proteins from thermophilic , mesophilic , and psychrophilic 
 organisms have often shown that these proteins are comparably flexible 
 when each is considered at its physiologically relevant temperature , 
 even though in thermodynamic terms adaptation to higher temperature 
 often correlates with higher stability . the crystallins , the structural proteins of the eye lens , are unusually 
 stable and thus present an attractive model system for studying questions 
 of protein stability . these proteins create the high refractive index 
 necessary for this specialized tissue to focus light on the retina ; 
 the concentration and distribution of the different crystallins determine 
 the refractive index gradient of the lens . unlike in land animals , 
 where the air  water interface at the cornea provides a significant 
 amount of focusing power , in aquatic organisms the crystallins alone 
 produce the refractive capability of the eye . fish lenses are therefore 
 more spherically shaped than those of land animals and have both increased 
 protein concentrations and greater protein refractivity in comparison 
 to their terrestrial counterparts . while 
 the crystallin concentrations in mammalian lenses can reach up to 
 450 mgml , lenses belonging to aquatic organisms 
 reach up to 1000 mgml . in vertebrates there are two common types of lens proteins : the 
 -crystallins , which are primarily structural , and the 
 -crystallins , which have an additional function of binding 
 damaged structural proteins and preventing aggregation . the 
 -crystallins are believed to have resulted from the gene duplication 
 of an ancestral -crystallin domain ; these proteins are closely 
 related to each other and to other chaperone proteins . the structural crystallins , including the taxon - specific crystallins 
 found in many organisms , and the -crystallins that are 
 the focus of this study have been recruited from diverse abundant , 
 soluble proteins via gene sharing or duplication often followed by 
 selection for increased refractivity of the protein itself . the -crystallin superfamily , which is characterized 
 by two double greek key domains , is thought to be derived from a calcium - binding 
 motif that existed before the evolution of eye lenses , as evidenced by similarity to calcium - binding 
 proteins in sequences from archaea , slime 
 mold , and urochordate . the urochordate ( ciona intestinalis ) protein 
 in particular is highly similar to the vertebrate members of the family , 
 but with two notably different features : it has only one domain rather 
 than two , and it contains two calcium binding sites . despite this 
 common evolutionary history , functional mammalian lens proteins lack 
 calcium - binding activity . a study of 
 the structure and dynamics of zebrafish ( danio rerio ) m7-crystallin by solution - state nmr has revealed a potentially 
 general unfolding pathway for all -crystallin domains . although this protein has the same overall fold 
 as the mammalian -crystallins , there are significant 
 primary sequence differences , including enhanced methionine content 
 as well as the absence of some of the tryptophan residues that are 
 strongly conserved in mammals . in teleost fishes , the m - crystallins 
 are the most common structural proteins in the lens , while -crystallins 
 are more common in humans . although s - crystallins 
 are a relatively minor subclass in both cases , they were chosen as 
 the focus of this study because their amino acid sequences are strongly 
 conserved among all vertebrates and because of their ability to resist 
 cold cataract . crystallins , particularly 
 in fish lenses , are enriched in highly 
 polarizable amino acids and exiguous in aliphatic amino acids as a 
 result of their selection for high refractive index . this selective pressure can potentially work against the 
 selective pressure for cold tolerance , which favors a relatively high 
 proportion of hydrophobic residues on the surface . in comparisons 
 of crystallin proteins from different environments , two notions of 
 protein stability are relevant . the thermodynamic 
 stability , g of unfolding , is the difference 
 in gibbs free energy between the folded and unfolded states , measured 
 by reversible denaturation of the protein . the unfolding temperature tm is measured 
 by ( usually irreversible ) direct thermal denaturation . although they are not directly comparable , the 
 thermal and chemical stabilities of similar proteins are often positively 
 correlated ; in a series of homologs or variants , it is common for 
 the ordering of thermal and chemical denaturation resistance to follow 
 the same ordinal ranking . in the case of the eye lens crystallins 
 from the antarctic toothfish ( dissostichus mawsoni ) , comparing the thermal and chemical stabilities of two closely 
 related proteins can potentially provide insight into the different 
 sequence characteristics related to their two major functions : cold 
 tolerance and high refractive index . the antarctic toothfish 
 lives in the cold waters of the southern 
 ocean , where temperatures can be as cold as 2 c . its 
 lens proteins are therefore resistant to both age - related loss of 
 solubility and the formation of cold cataract.d . mawsoni has two s - crystallin 
 paralogs , s1- and s2-crystallin ( abbreviated ts1 
 and ts2 throughout ) , with a sequence identity of 60% . protein 
 turnover is very low in the eye lens , requiring the crystallins to 
 maintain their stability and solubility over the whole lifespan of 
 the organism . in mesophilic organisms , high stability corresponds 
 to a high thermal denaturation temperature and high g of unfolding . quantitative thermodynamic studies 
 of cold - adapted proteins so far have generally found decreased thermodynamic 
 stability . in general , psychrophilic proteins are characterized by decreased 
 core hydrophobicity , exiguous isoleucine content , increased surface 
 hydrophobicity , fewer total charged residues , increased surface charge , 
 a lower arginine / lysine ratio , weaker interdomain and intersubunit 
 interactions , decreased secondary structure content , more and longer 
 loops , more glycine residues , fewer and weaker metal - binding sites , 
 fewer disulfide bonds , fewer electrostatic interactions , and increased 
 conformational entropy of the unfolded state . some of these adaptations conflict with the primary optical function 
 of the -crystallins , for which highly polarizable amino acids 
 are selected . quantitatively , this is described by the refractive 
 index increment dn / dc , the change 
 in refractive index with concentration . although this can be empirically 
 determined , for proteins it is often assumed to be described by a 
 simple additive model in which only the amino acid content is important 
 for determining dn / dc for the entire 
 protein molecule . here we compare 
 the thermal and chemical stabilities of d. mawsoni s1- and s2-crystallins in light 
 of these competing functions . although these proteins have comparable 
 values for g of unfolding , surprisingly , 
 ts1 is more susceptible to thermal denaturation while ts2 
 is more readily unfolded with urea . for related proteins , these quantities 
 are typically positively correlated with each other and with overall 
 thermodynamic stability . the differential resistance to thermal and 
 chemical unfolding in this system underscores the different mechanisms 
 of unfolding involved and the intramolecular interactions involved 
 in resistance to them . plasmids 
 containing the cdna sequences of the human s - crystallin ( hs ) 
 and d. mawsoni s1 ( genbank , dq143971.1 ) 
 and s2 ( genbank , dq143972.1 ) genes were purchased from blue heron biotech , llc . each 
 gene was flanked by regions containing restriction sites for ncoi 
 and xhoi , an n - terminal 6 his tag , and a tev cleavage sequence 
 ( enlfqg ) with the n - terminal methionine of hs , ts1 , 
 and ts2 replaced by the final glycine in the cleavage sequence . the biophysical experiments described below were performed without 
 removing the n - terminal 6 his tag . the toothfish crystallin 
 genes were amplified using oligonucleotide primers purchased from 
 sigma - aldrich ( st . louis , mo ) , and the resulting gene products were 
 individually cloned into pet28a(+ ) vectors ( novagen , darmstadt , germany ) . hs , ts1 , and ts2 were overexpressed in rosetta 
 ( de3 ) escherichia coli using standard iptg - induced 
 overexpression protocols at 25 c in standard luria broth ( lb ) the 
 cells were lysed by sonication , and cell debris was removed by centrifugation . his - tev - hs , his - tev - ts1 , and his - tev - ts2 were 
 purified on a ni - nta column ( applied biosystems , foster city , ca ) . the pure protein was collected from the column elution fraction and 
 then dialyzed extensively against 10 mm phosphate buffer , ph 6.9 , 
 for all experiments . purified ts1 
 and ts2 
 were diluted to 0.125 mgml with 10 mm phosphate 
 buffer at ph 6.9 for the collection of full circular dichroism ( cd ) 
 spectra and to 0.25 mgml with 10 mm phosphate 
 buffer at ph 6.9 , 150 mm nacl , and 1 mm dtt for unfolding experiments . measurements were taken on a j-810 spectropolarimeter ( jasco , easton , 
 md ) equipped with a thermal controller . for unfolding measurments , the samples were heated at a rate of 2 cmin . for thermal denaturation curves , the cd at 218 nm was monitored 
 and the curves were fit to a two - state equilibrium unfolding model 
 to determine the thermal denaturation temperature ( tm ) . uv fluorescence 
 measurements 
 were made on ts1 and ts2 at a concentration of 0.075 
 mgml in 10 mm phosphate buffer , ph 6.9 . samples for chemical unfolding curves were prepared with increasing 
 concentrations of 10 m urea ( fisher scientific , waltham , ma ) . samples were allowed to equilibrate for at least 
 24 h before absorption  emission fluorescence spectra were obtained 
 using a f4500 fluorescence spectrophotometer ( hitachi , tokyo , japan ) 
 with a ex of 280 nm . the ratio of baseline - corrected 
 emission intensities at 360 and 320 nm was used for analysis . to determine 
 the thermodynamic parameters ( gw and m values ) , g[urea ] was 
 calculated from the normalized equilibrium unfolding data and a linear 
 least - squares fit was performed in mathematica to the line1where gw is the value 
 of g at 25 c , extrapolated to zero concentration 
 of denaturant , and m is a measure of the dependence 
 of g on denaturant concentration . dynamic light scattering ( dls ) 
 measurements were obtained with a zetasizer nano zs ( malvern instruments , 
 malvern , u.k . ) on s1 and s2 at a concentration of 1.0 
 mgml in 10 mm phosphate buffer , ph 6.9 . 
 at each temperature , the sample was allowed to equilibrate for 2 min 
 before measurements were obtained , after which scattering measurements 
 were performed in triplicate , resulting in a heating rate of 0.5 
 cmin . transmittance was obtained using a cary 
 4000 uv  vis ( agilent technologies , santa clara , u.s . ) on hs , 
 ts1 , and ts2 at a concentration of 10.0 mgml in 10 mm phosphate buffer , ph 6.9 , from 25 to 5 c . the structural -crystallins 
 share a common fold consisting 
 of paired homologous double greek key domains , each with two sets 
 of four adjacent antiparallel -strands linked by short loops . this protein architecture has been identified as contributing to the 
 very high stability of the -crystallins , particularly the tight interdomain interface 
 that contains several critical hydrophobic interactions . homology models for t1 and ts2 
 based on the solution structure of hs ( pdb code 2m3 t ) were constructed using swissmodel . and are shown in figure 1 . homology models of d. mawsoni s1- ( gray ) 
 and s2-crystallin ( blue ) based upon the solution nmr structure 
 of human s - crystallin . the primary sequence of s - crystallin is highly conserved 
 across diverse species including fish , mammals , and birds . a sequence alignment for hs , ts1 , ts2 , and 
 a selection of their orthologs from other fish species is shown in 
 figure 2 . both ts1 and ts2 have 
 moderate sequence identity to hs ( 57% and 53% , respectively ) . 
 conserved residues include the four tryptophans , several glycines 
 in loops , and other structurally important residues . a blast search reveals many other homologues from mesophilic 
 fish species ; here we examine some examples from classes chondrichthyes 
 ( cartilaginous fish , such as sharks and rays ) and osteichthyes ( teleost 
 fish , which includes d. mawsoni , and the well - known 
 model organism danio rerio ) . although other putative 
 sequences identified from dna open reading frames were more closely 
 related in some cases , our analysis is limited to a selection of sequences 
 confirmed from mrna transcripts . the sequences whose properties are 
 summarized in table 1 include the zebrafish 
 ( danio rerio ) b - crystallin ( db ) and s3-crystallin ( ds3 ) , the chinese catfish ( clarias fuscus ) s1- and s2-crystallins ( cls1 and cls2 ) , and the slender bamboo shark ( chiloscyllium 
 indicum ) s1- and s2-crystallin ( chs1 
 and chs2 ) . sequence alignment of 
 human s - crystallin ( hs ) , d. mawsoni s1- and s2-crystallin ( ts1 
 and ts2 ) , d. rerio sb- ( db ) 
 and s3-crystallin ( ds3 ) , c. fuscus s1- 
 and s2-crystallin ( cls1 and cls2 ) , and c. indicum s1- and s2-crystallin ( chs1 
 and chs2 ) . d. mawsoni s1 and s2 
 have overall sequence identities of 57% and 53% with s - crystallin , 
 respectively , and 60% sequence identity to one another . the residues 
 are colored according to their chemical properties as follows : green , 
 hydrophobic residues ( avfpmilw ) ; blue , acidic residues ( de ) ; magenta , 
 basic residues ( rk ) ; black , all other residues ( styhcngq ) . in general , the amino acid composition of proteins 
 is relatively 
 constant , as it primarily depends on factors such as the codon redundancy 
 and mutation tolerance , such that deviation 
 from the average amino acid frequency is often indicative of selection 
 for a particular function or environmental adaptation . for example , 
 thermophilic proteins are often enriched in arginine because of its 
 importance in forming stabilizing salt bridges . many psychrophilic proteins are exiguous in proline content . eye lens proteins , which have been selected for their high refractive 
 index increments , are enriched in highly polarizable amino acids such 
 as trp , tyr , phe , arg , met , and cys and exiguous in aliphatic residues . the amino acid frequencies for hs , ts1 , 
 ts2 , and the other fish -crystallins described above 
 are given in table 1 along with the average 
 values for vertebrate proteins and the contribution of each amino 
 acid type to dn / dc . the grand 
 average of hydropathicity index ( gravy ) predicts the 
 hydrophobic character of the proteins where more positive values indicate 
 higher hydrophobicity . table 2 summarizes the sequence characteristics of hs , 
 ts1 , ts2 , and several homologous proteins from mesophilic 
 fish , as calculated from the expasy protparam tool . the aliphatic index measures the relative volume occupied 
 by aliphatic side chains ( nonpolar , hydrophobic ) including alanine , 
 valine , isoleucine , and leucine . all 
 of the -crystallins investigated here are moderately hydrophilic , 
 with small negative gravy values . ts2 is the most hydrophobic 
 of the proteins studied , yet it has a lower aliphatic index than hs , 
 reflecting its enhanced content of aromatic residues . hs has 
 the next most positive gravy value and the highest aliphatic character , 
 along with both zebrafish proteins . thus , in terms 
 of gravy index and aliphatic character , the toothfish crystallins 
 are within the range of variance established by the comparison group 
 of mesophilic fish sequences . selection for cold tolerance often leads 
 to decreased numbers of hydrophobic residues in the protein core , 
 and more on the surface , reflecting the 
 reduced entropy cost of exposing hydrophobic groups to solvent at 
 low temperatures . a large proteomic analysis of the amino acid composition 
 of psychrophilic and mesophilic proteins found that psychrophilic 
 proteins have a larger number of hydrophobic residues in loops and 
 a smaller number in helices , relative to their mesophilic counterparts . this was consistent with the well - known idea 
 that aliphatic amino acids in the core of the protein contribute to 
 stability via the hydrophobic effect . some sequence commonalities can be observed among all the 
 fish 
 s - crystallins investigated here , due to their both their common 
 ancestry and the selective pressures on lens proteins . in general , 
 alanine , valine , isoleucine , and leucine are expected to be selected 
 against in lens proteins because these amino acids have low refractive 
 index increments . as expected based on the requirement for higher 
 refractivity in the lenses of aquatic organisms , all of the fish crystallins 
 have higher predicted dn / dc values 
 than hs . consistent with this idea , ts1 has the lowest 
 aliphatic index , as well as a higher predicted dn / dc than hs . selection for increased refractive 
 index leads to reduced fraction of aliphatic residues in favor of 
 those with more polarizable side chains . this would lead to the expectation that the s - crystallins 
 would have an unusually high proportion of the aromatic amino acids 
 phe , tyr , and trp to compensate , which is the case for all of the 
 proteins listed in table 1 . for example , toothfish 
 s1 , which has the highest predicted dn / dc , has 9.1% tyr , almost 3 times the average value , and is 
 also enriched in phe ( 6.8% vs the average of 4.0% ) . other 
 highly polarizable residues that would be expected to be 
 enriched in lens proteins include arg , met , cys , and his . methionine 
 in particular has been cited as being particularly important in providing 
 the high refractivity required for fish crystallins , as it is greatly 
 enriched in the abundant m - crystallin of the zebrafish lens . although all the proteins have a met content 
 greater than its average value of 1.8% , the level of enrichment varies 
 greatly : the catfish proteins cls1 and cls2 have the 
 highest met content ( 12.7% and 9.3% , respectively ) , while the other 
 crystallins have more moderate levels of enrichment ranging from 2.3% 
 to 5.8% , with ts1 and t2 falling within this range . 
 the situation for his and cys is comparable ; all of the crystallins 
 studied have his contents close to the average value , while cys levels 
 are slightly elevated for the toothfish crystallins but also for the 
 catfish proteins . the major difference between the toothfish crystallins 
 and their mesophilic homologues is in their arg content . arg is often 
 exiguous in cold - adapted proteins because of its role in the formation 
 of stabilizing salt bridges all the -crystallins 
 investigated are significantly enriched in arg except for ts2 , 
 which has approximately the average value . the relative amounts 
 of arg in the two d. mawsoni crystallins may provide 
 insight into their denaturation behavior , which is described in the 
 next section . taken together , these observations suggest that rather 
 than any one residue being critical for determining the high dn / dc values required for function in fish 
 lens proteins , this function can be acquired by different combinations 
 of high - refractivity amino acids , within the constraints imposed by 
 the additional selection for cold tolerance in the d. mawsoni proteins . the toothfish crystallins have lower isoelectric 
 point ( pi ) values 
 with respect to hs , meaning that they have much more negative 
 charge at neutral ph . this is consistent with the previous observation 
 that psychrophilic proteins often have more acidic pi values than 
 homologous mesophilic or thermophilic proteins , possibly because negatively 
 charged residues are important for mediating interactions with the 
 solvent and hence maintaining flexibility in cold environments . alternatively , in this case it may simply reflect 
 the reduction in positive charge due to the decreased arginine content 
 of ts1 and ts2 relative to the s - crystallins 
 from mesophilic fish . by use of psipred , a secondary structure 
 prediction software , the sequences for both ts1 
 and ts2 were predicted to have primarily -sheet 
 secondary structures . circular dichroism ( cd ) spectra were collected 
 for both ts1 and ts2 to assess the overall general secondary 
 structures of the proteins ( figure 4 ) . a comparison 
 of the circular dichroism spectra of ts1 and ts2 at 
 25 c indicates that both proteins have primarily -sheet 
 secondary structures . the negative ellipticities of ts1 and 
 ts2 occur at 216 and 217 nm , respectively . these values are 
 in the range that is typical of -sheet proteins and is consistent 
 with the predicted secondary structure results and with the experimental 
 results for hs . predicted secondary structures 
 of d. mawsoni ( a ) 
 s1 and ( b ) s2 . the intrinsic fluorescence of crystallin proteins is an important 
 indication of the degree of folded structure in the double greek key 
 domains , as the fluorescence is primarily due to four highly conserved 
 tryptophan residues in the protein core . both ts1 and ts2 
 share these conserved tryptophans ; ts2 also has a fifth tryptophan . 
 in mammalian eye lenses , the positioning of the conserved tryptophan 
 side chains is essential for the rapid quenching of uv fluorescence 
 hypothesized to protect crystallins from photochemical degradation 
 in species that are subject to strong uv light exposure . their conservation in this aquatic species may 
 be due to their contributions to the dn / dc , hydrophobic packing , or both . uv fluorescence spectra 
 for d. mawsoni s1 and s2 are shown 
 in figure 4 . the emission maxima in the fluorescence 
 spectra for excitation at 280 nm ts2 has increased fluorescence 
 intensity in comparison to ts1 due to the presence of the additional 
 tryptophan in its sequence . tryptophans that are exposed to 
 water have emission maxima between 340 and 350 nm , whereas completely 
 buried tryptophans have maxima around 330 nm . the slight red shifts 
 of both ts1 and ts2 with respect to hs indicate 
 that the tryptophans in both toothfish proteins are more exposed to 
 water , suggesting that ts1 and ts2 are less compactly 
 folded and more structurally flexible than hs , as expected . s1 displays a negative ellipticity 
 at 216 nm , while s2 displays a negative ellipticity at 217 
 nm . ts1 has an emission maximum at 
 331 nm , and the emission maximum for ts2 is 336 nm . cold denaturation , protein unfolding due to the 
 decreased energetic 
 cost of exposing hydrophobic resides to solvent , does not occur for 
 most globular proteins until well below the freezing point of water . except in special cases , experimental studies of cold denaturation 
 have required the use of chemical denaturants , high pressure , encapsulation in reverse micelles , or limiting the sample volume to small capillaries to study the unfolding intermediates . thus , 
 cold cataract , the low - temperature opacity of many protein solutions 
 such as mammalian lenses , results from liquid  liquid phase 
 separation rather than protein unfolding . the toothfish eye lens does 
 not undergo cold cataract above its freezing point of 12 c , 
 in contrast to mammalian lenses , which form them at much higher temperatures 
 ( 20 c ) . s - crystallins 
 in general are resistant to cold cataract and are thought to play 
 an important role in maintaining solubility in multicomponent crystallin 
 mixtures ; e.g. , bovine s - crystallin has a theoretical liquid liquid 
 phase separation temperature of 28 c and its presence in concentrated solutions of other -crystallins 
 results in a lowered phase separation temperature . transmission measurements at 600 nm were taken as a function 
 of temperature in order to establish that solutions of our in vitro 
 generated protein constructs are transparent over the experimentally 
 relevant temperature range ( i.e. , cold cataract does not occur ) . these 
 results , summarized in table 3 , indicate that 
 hs , s1 , and s2 all remain transparent at 5 c , 
 consistent with previous measurements of s - crystallins . although 
 solutions of each of the -crystallins studied here remain transparent 
 at 5 c , further investigations will be needed to assess their 
 ability to stabilize mixtures of other crystallins as a function of 
 temperature . enhanced low - temperature stability has been previously 
 observed in cold - adapted teleost a - crystallins , which have 
 greater hydrophobic character and are better able to maintain their 
 chaperone activity at lower temperatures than their mesophilic orthologs 
 at the cost of high thermal stability . the overall thermodynamic 
 stability of the s - crystallin fold is highly relevant to the 
 biological function of the eye lens because of the lack of protein 
 turnover in the lens ; the crystallins must remain stable and soluble 
 for decades . in general , cold - stable proteins are generally more susceptible 
 to chemical denaturation than their higher - temperature counterparts . factors affecting overall protein stability 
 include hydrophobic interactions , hydrogen bonds , and conformational 
 entropy . ts1 and ts2 were subjected to chemical 
 denaturation 
 with increasing concentrations of urea , while fluorescence spectroscopy 
 was used to monitor unfolding . each sample was allowed to equilibrate 
 for at least 24 h before fluorescence measurements were collected . the excitation wavelength was 280 nm , and emission spectra were collected 
 between 300 and 500 nm . fluorescence maximum intensities were normalized 
 by taking the f360/320 ratio at each concentration of denaturant and 
 plotted as fraction unfolded vs denaturant concentration ( figure 5a ) . the data points indicated with open circles 
 in figure 5a represent dilution of the samples 
 to 2 m urea after full unfolding at 7 m urea , demonstrating the reversibility 
 of this transition . figure 5b is a plot of 
 g vs denaturant concentration for the transition 
 regions of the unfolding curves used to extrapolate values for gw , the g at 25 c in the absence of any 
 denaturant . ts1 is more susceptible 
 to unfolding by urea ; the [ urea]1/2 of s1 is equal 
 to 3.8 and 5.6 m for ts2 . gw is 13.35 
 and 18.74 kj mol for ts1 and ts2 , 
 respectively . urea unfolding 
 is thought to be driven by a combination of indirect and direct mechanisms . it weakens the hydrophobic effect by disrupting 
 the hydrogen bonding network of the solvent , as well as stabilizing 
 unfolded states via direct electrostatic and hydrogen bonding interactions 
 with side chain and backbone groups . more urea may be needed 
 to unfold ts2 because it has nearly twice as many aliphatic 
 residues as ts1 and fewer hydrophilic amino acid residues.the 
 slope , m , describing the dependence of g on denaturant concentration , is very similar for both 
 ts1 and ts2 . ( a ) chemical denaturation curves of hs , d. mawsoni s1 and s2 with varying amounts 
 of urea , measured by 
 fluorescence spectroscopy and plotted as fraction unfolded . all three 
 proteins exhibit two - state equilibrium unfolding behavior by urea 
 denaturation . data points designated with open circles represent samples 
 of hs , ts1 , and ts2 that were first unfolded 
 with 7 m urea and then diluted to 2 m urea to indicate that urea denaturation 
 is reversible . ( b ) g vs denaturant concentration 
 for the transition regions of the chemical unfolding curves used to 
 extrapolate values for g(h2o ) . s1 is more susceptible to unfolding by urea at lower concentrations 
 where g(h2o ) is 13.35 and 18.74 
 kjmol for s1 and s2 , respectively . thermal denaturation provides complementary 
 information regarding 
 protein stability and aggregation propensity . for some proteins , e.g. , 
 lysozyme , the thermally denatured state 
 has been shown to differ form that induced by chemical denaturation . 
 furthermore , this process is often irreversible , making the calculation 
 of thermodynamic quantities problematic ; however , the midpoint of 
 the unfolding transition ( tm ) is itself 
 a useful measure of protein stability . the thermal denaturation of 
 ts1 and ts2 was monitored by circular dichroism at 218 
 nm ( figure 6 ) and fit to a two - state equilibrium 
 unfolding model . the cd melting curves obtained provide information 
 about the overall stability of the protein folds . the difference in 
 thermal stabilities between the two proteins is quite different ; ts1 
 has a tm of 68.5  0.1 c , while 
 ts2 had a tm of 58.0  0.1 
 c . for comparison , human s - crystallin has a tm of 72.0  0.1 c under the same conditions . the relationship between thermal stability , 
 hydrophobicity , and aliphatic index is not immediately clear for these 
 proteins ; ts2 , the most hydrophobic crystallin studied , has 
 the lowest tm while s1 , the least 
 hydrophobic , does not have the highest tm value . the highest tm belongs to hs , 
 which has a hydrophobic content between those of ts1 and ts2 . 
 the explanation for this surprising observation may be a result of 
 selection for high dn / dc . ts1 , 
 but not ts2 , is enriched in arg , which is known for increasing 
 stability in thermophilic proteins , due to the ability of the guanidinium 
 group to form stabilizing salt bridges . although increased arginine 
 content should stabilize the protein with respect to thermal denaturation , 
 as seen for hs and ts1 , chemical denaturation by urea 
 should affect these salt bridges the same way as any other polar interaction , 
 meaning that other factors such as hydrophobicity come into play in 
 ts2 . thermal unfolding curves of ts1 and ts2 measured 
 by monitoring the circular dichroism signal at 218 nm , with best - fit 
 unfolding curves . aggregation under thermal stress was also measured as a function 
 of temperature using dynamic light scattering ( dls ) for both toothfish 
 s - crystallins to ascertain their aggregation propensity . aggregation 
 propensity is not always directly correlated with thermal stability . at even moderately high concentrations , protein 
 aggregates can form well below the thermal denaturation temperature 
 as a result of interactions between transiently exposed groups in 
 conformationally mobile protein monomers . the measurements were made 
 in 10 mm phosphate buffer with no additional reducing agents to avoid 
 interfering with any attractive intermolecular forces that may be 
 responsible for aggregation . for each data point , taken in triplicate , 
 the sample was allowed to equilibrate for 2 min prior to measurement 
 at a given temperature . three scans of % abundance by number were 
 averaged at each temperature and then fit to a gaussian function using 
 nonlinear regression . the average apparent particle size is plotted 
 as a function of temperature in figure 7 . ts1 
 remains monomeric until 35.0 c where intermediate aggregates 
 in the range 30100 nm begin to form before quickly transitioning 
 into larger aggregates up to 1200 nm in size at 47.5 c . ts2 
 follows a similar trend , but intermediate sized aggregates begin forming 
 at 34.0 c and large aggregates at 42.0 c . in comparison , intermediate aggregates of hs do not begin forming until around 
 49.0 c while larger aggregates appear at 58.5 c . both ts1 and ts2 are less thermally 
 stable and more aggregation prone than hs . ts1 is monomeric until 35.0 c where it 
 begins to form intermediate aggregates and then finally to form large 
 aggregates around 48.0 c . ts2 behaves similarly , but 
 because it is not as thermally stable , it forms intermediate sized 
 aggregates at 34.0 c and large aggregates 42.0 c . in summary , the biophysical characterization 
 of ts1 and 
 ts2 showed that while both proteins have the primarily -sheet 
 secondary structures characteristic of -crystallins , they appear 
 to have slightly less overall -sheet character than their human 
 homologue . ts1 and ts2 also appear to have greater structural 
 flexibility as observed in the red - shifted tryptophan fluorescence 
 spectra , indicating that the structurally conserved tryptophans in 
 the core of both proteins are more accessible to water . of the two 
 crystallins , the less structurally rigid ts2 has the lowest 
 thermal stability as determined by thermal denaturation despite having 
 a higher gw . nevertheless , both 
 toothfish crystallins have lower thermal stabilities than hs 
 and begin forming high molecular weight aggregates at lower temperatures . the biophysical characterization of the d. mawsoni s1- and s2-crystallins demonstrates an unusual set 
 of protein homologues in which thermal stability does not directly 
 correlate with g , and provides a useful 
 model system for future structural and mutagenesis studies pinpointing 
 the molecular determinants of protein solubility , thermal stability , 
 and denaturation resistance .
the s1- and s2-crystallins , 
 structural eye lens proteins 
 from the antarctic toothfish ( dissostichus mawsoni ) , are homologues of the human lens protein s - crystallin . 
 although s1 has the higher thermal stability of the two , it 
 is more susceptible to chemical denaturation by urea . 
 the lower thermodynamic 
 stability of both toothfish crystallins relative to human s - crystallin 
 is consistent with the current picture of how proteins from organisms 
 endemic to perennially cold environments have achieved low - temperature 
 functionality via greater structural flexibility . in some respects , 
 the sequences of s1- and s2-crystallin are typical of 
 psychrophilic proteins ; however , their amino acid compositions also 
 reflect their selection for a high refractive index increment . like 
 their counterparts in the human lens and those of mesophilic fish , 
 both toothfish crystallins are relatively enriched in aromatic residues 
 and methionine and exiguous in aliphatic residues 
 . the sometimes contradictory 
 requirements of selection for cold tolerance and high refractive index 
 make the toothfish crystallins an excellent model system for further 
 investigation of the biophysical properties of structural proteins .