A variety of strategies are used by researchers to elucidate the basis of thermophilic proteins’ thermostability. There are three common approaches. Firstly, large data sets of solved structures or amino acid sequences of thermophilic proteins and homologous mesophilic proteins are compared. Many of the differences between these groups are likely the result of thermophilic adaptions to high temperature. Secondly, the effects of specific amino acid substitutions on thermostability are studied using site-directed mutagenesis. This allows the role of specific interactions to be probed. Lastly, in silico simulations of thermophilic proteins allow the free energy resulting from different types of interactions to be calculated and compared. The basis of high thermostability in thermophilic proteins is complex and not yet fully elucidated. However, it is clear that hydrophobic interactions, ion pair networks, disulfide bonds, and amino acid composition are important factors.
Thermodynamics of Protein Stability at High Temperature
Protein folding as a function of temperature can be understood through stability curves, plots of the free energy of unfolding against temperature (Fig. 1). The most important parameter in quantifying protein stability as a function of temperature is Tm, the melting temperature. At this temperature the probabilities of the protein being folded or unfolded are equal and the Gibbs energy change associated with folding is zero (Fig. 2).
The free energy of stabilization of a protein (∆G) as a function of temperature (T) is given by a modified form of the Gibbs-Helmholtz equation (Razvi and Scholtz, 2006):
Razvi and Scholtz (2006) compared thermodynamic data for 26 sets of thermophilic and mesophilic homolog protein pairs. They found that increased intrinsic stability (higher ∆G at all temperatures) was the most common strategy by which the Tm of thermophilic proteins were increased. Decreasing ∆Cp was the second most common strategy. The least common strategy was decreasing the entropy of folding. Interestingly, thermophilic proteins employ different combinations of these strategies to attain high Tm values. For instance, some used all three strategies, some only the first, the second, the first and third, and so forth. However, in no cases studied was decreasing the entropy of folding used alone as the sole mechanism. Thus, thermodynamically, diverse combinations of strategies are used by thermophilic proteins to attain stability at high temperature.
It is reasonable to suspect that hydrophobic interactions play an important role in protein thermostability since the hydrophobic effect increases with temperature (Reed et al., 2013). Indeed, hydrophobic interactions have been demonstrated to be immensely important. This has been observed in a variety of site-directed mutagenesis studies. For instance, Imanaka, Shibazaki, and Takagi (1986) found that amino acid substitutions that increased internal hydrophobicity enhanced the thermostability of proteases. Furthermore, thermophilic proteins generally contain more interactions between hydrophobic residues than mesophilic proteins. Kannan and Vishveshwara (2000) compared thermophilic and mesophilic homolog crystal structures of 24 protein families using a graph spectral method. They found that the majority of thermophilic proteins contained an increased number of aromatic clusters or expanded aromatic networks.
Furthermore, the stabilizing free energy contribution of hydrophobic interactions is greater in thermophilic proteins. Saraboji, Gromiha, and Ponnuswamy (2005) calculated the free energy contributions of a variety of interactions using the structures of thermophilic and mesophilic proteins from 23 different families. In every case they found that the main-chain hydrophobic Gibbs energy was lower in thermophiles than the corresponding mesophilic homologs.
In terms of molecular interactions, increased hydrophobic interactions appear to be one of the most consistent differences between thermophilic and mesophilic proteins. Gromiha et al. (2013) computed and compared parameters relating to hydrophobicity, inter-residue interactions and hydrogen bonds for a dataset of 373 protein families. They found that 80% of thermophilic proteins had greater hydrophobicity than their corresponding mesophilic homologs, making it the strongest predictor of thermophilicity (Fig. 3). Interestingly, the 20% of thermophilic proteins that had decreased hydrophobic interactions all had increased hydrogen bonding or ion pairing. Thus, although the most common, increased hydrophobic interactions is only one of several possible strategies used by thermophilic proteins to achieve stability at high temperature.
Disulfide bonds can have significant contributions to the thermostability of thermophilic proteins. Cacciapuoti et al. (2012) demonstrated this for the hyperthermophilic protein 5′-deoxy-5′-methylthioadenosine phosphorylase II. This protein contains a CXC motif (C259/C261) and two intrasubunit disulfide bonds (C138-C205 and C200-C262). The Tm of the wild-type protein was greater than 108 °C, while the C262S and C259S/C261S mutants had Tm values of 102 and 99 °C respectively. Hence, the contribution of the C200-C262 disulfide bond to the protein’s thermostability is large. Furthermore, the CXC motif has been shown to form a strained 11-atom disulfide ring which can act as a reducing agent (Woycechowsky and Raines, 2003). The CXC motif is therefore important in maintaining the native disulfide linkages. This accounts for the even lower Tm of the C259S/C261S double mutant. The importance of disulfide bonds is clear: their contribution is considerable and this protein has even evolved an elaborate mechanism to maintain these linkages.
Disulfide bonds may be particularly important for the stability of quaternary structure. Boutz et al. (2007) performed a proteomic analysis on disulfide linkages in Pyrobaculum aerophilum, a hyperthermophilic archaeon. Disulfide bonds were found to be important for the stability of a variety of protein-protein complexes. They obtained a crystal structure for one of these complexes, a citrate synthase homdimer. Each subunit contained a single intramolecular disulfide bond, cyclizing the protein chain. Remarkably, this cyclization occurred such that the chains of each subunit were topologically interlinked, holding the dimer together (Fig. 4).
Electrostatic Interactions and Ion Pair Networks
Electrostatic interactions contribute significantly to the thermostability of thermophilic proteins. Although they do often have more salt bridges than mesophilic proteins, the stabilization comes about principally through large ion pair networks (Fig. 5). Yip et al. (1998) studied the relationship between thermal stability and ion pair networks in glutamate dehydrogenases. They used a homology-based modelling approach with known sequences from different species. Lower thermostability correlated with more fragmented ion pair networks.
Indeed, there is considerable evidence that the positioning of the ion pairs, rather than the number, is the most energetically important consideration. Xiao and Honig (1999) calculated electrostatic contributions to the folding free energy of hyperthermophilic proteins and their mesophilic homologs. In all pairs studied, they found that the free energy contribution of electrostatic interactions was greater in the hyperthermophilic proteins. Interestingly, the free energy did not correlate well with the number of ionisable residues or ion pairs. Instead, it originated from the optimum placement of charged residues. Bakker, Hunenberger, and McCammon (1999) provided additional evidence that the optimum organization of ion pairs in networks is the principle electrostatic contribution to thermostability. They performed molecular dynamics simulations on the hyperthermophilic protein Sac7d from 300 to 500 K. The majority of electrostatic stabilization came from three large ion pair networks. Despite this, when considered in isolation, the stabilization from individual salt bridges was calculated to be small, some even were even destabilizing. Thus, the energetic stabilization resulting from ion pair networks show a high degree of cooperativity. This accounts for why it is the organization and positioning, rather than the number of ion pairs, which determines the free energy contribution.
Another important finding of Bakker et al. (1999) was that upon raising the simulation temperature from 300 K to 360 K, the protein-solvent interaction energy became less favourable, while the intraprotein interaction energy became more favourable. Both of these effects were principally a consequence of electrostatic contributions. This may be due to decreased desolvation energy, the energetic cost associated with the loss of solvent interactions when a residue becomes buried in a protein. The desolvation energy (∆W) of an ion is given by (Karshikoff and Ladenstein, 2001):
The thermodynamic mechanism by which ion pairs stabilize thermophilic proteins at high temperature is, at least in part, by lowering ∆Cp. Chan, Yu, and Wong (2011) demonstrated this by individually mutating ion pairs with site-directed mutagenesis. Measurement of the ∆Cp of the mutants and comparison to the wild-type protein allowed the contribution of each ion pair to ∆Cp to be calculated. Two ion pairs were identified whose interaction reduced ∆Cp by 0.8 to 1.0 kJ mol-1 K-1.
Amino Acid Composition
A number of changes in the amino acid composition have been noted between thermophilic proteins and their mesophilic homologs. For instance, Kumar, Tasi, and Nussinov (2000) compared 18 non-redundant families of thermophilic and mesophilic proteins. They found that Arg and Tyr frequencies were increased by a significant extent in thermophilic proteins. They argued that these results could be consequences of the increased salt bridges and hydrophobicity commonly associated with thermophilic proteins.
Furthermore, there are statistically significant differences between the amino acid composition of helices in thermophilic and mesophilic proteins. The frequency of Cys and His are decreased, while the frequency of Arg is increased (Kumar, Tasi, and Nussinov, 2000). Interestingly, Arg is a helix favouring residue while Cys, and His are helix disfavouring. In addition, thermophilic proteins have considerably higher helical content in the majority of protein families (Kumar, Tasi, and Nussinov, 2000). It follows that increased helical content and propensity may be an important mechanism of increasing stability at high temperature.
Lastly, Kumar et al. (2000) also found a statistically significant decrease in the proportion of Cys and Ser in thermophilic proteins. This is not surprising: the polar amino acids Asn, Cys, Gln, and Met are considered thermolabile because at high temperature they may undergo deamidation or oxidation reactions (Russel et al. 1997). Thus, decreased usage of these amino acids could be adaption to high temperature, reducing the risk of these undesirable reactions. Although not statistically significant, the proportion of Asn also decreased by a sizeable amount. However, in apparent opposition to this hypothesis the amount of Gln and Met were similar in mesophilic and thermophilic proteins. This is likely because solvent exposure and the local environment are important determinants of an amino acid’s susceptibility to deamidation and oxidation reactions. The biased amino acid usage therefore occurs almost exclusively among the surface residues. A visually striking example of this difference in surface composition is lumazine synthase (Fig. 6). Thus, differences in amino acid composition between thermophilic and mesophilic proteins can be more clearly elucidating by considering the location of each residue. Fukuchi and Nishikawa (2001) compared the interior and exterior amino acid composition of a variety of mesophilic and thermophilic proteins available on the Protein Data Bank. As expected, the frequency of polar uncharged residues (Asn, Ser, and Thr) on the surface of thermophilic proteins was much lower than for mesophilic proteins. Conversely, the proportion of polar charged residues (Asp, Glu, Arg, and Lys) was higher. Although the surface composition is different, Fukuchi and Nishikawa did not find any significant differences in amino acid composition internally.
A diverse set of strategies are used by thermophilic proteins to achieve their remarkable thermostability. Thermodynamically, the most common mechanism is to increase the overall stabilizing free energy, ∆G. However, alternative mechanisms, including decreasing ∆Cp and/or entropy of folding are also used by many thermophilic proteins.
A variety of molecular interactions are associated with the high thermostability of thermophilic proteins. The contribution of individual disulfide bonds can be significant, greatly increasing the Tm value. They have been found to be especially important in maintaining quaternary structure at high temperature. In addition, increased hydrophobic interactions are a ubiquitous feature of thermophilic proteins. They have more aromatic clusters and a higher stabilizing free energy contribution from hydrophobic interactions. Large, optimally organized ion pair networks also enhance the thermostability of thermophilic proteins.
Lastly, differences in amino acid composition, particularly on the protein’s surface, contribute to their thermostability. Replacement of uncharged polar surface residues with charged residues protects the protein’s amino acids from potentially detrimental amidation or oxidation reactions. Overall, thermostability is a complex property with contributions from a variety of interactions. Thermophilic proteins use different strategies to variable extents to achieve stability at high temperature.
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Mbinu mbalimbali hutumiwa na watafiti kueleza msingi wa thermophilic protini <unk> thermostability. Kuna mbinu tatu za kawaida. Kwanza, seti kubwa za data za miundo iliyofumuliwa au mfululizo wa asidi ya amino ya protini za thermophilic na protini za mesophilic za homologous zinalinganishwa. Tofauti nyingi kati ya vikundi hivyo huenda zikawa matokeo ya mabadiliko ya thermophilic kwa joto la juu. Pili, athari za substitutions maalum amino asidi juu ya thermostability ni alisoma kwa kutumia tovuti-kuelekezwa mutagenesis. Hii inaruhusu jukumu la mwingiliano maalum kuchunguzwa. Mwishowe, katika silico simulations ya protini thermophilic kuruhusu nishati huru inayotokana na aina tofauti ya mwingiliano kuwa mahesabu na kulinganishwa. Msingi wa thermostability ya juu katika protini thermophilic ni tata na bado si kikamilifu elucidated. Hata hivyo, ni wazi kwamba mwingiliano hydrophobic, ion jozi mitandao, disulfide vifungo, na amino asidi utungaji ni mambo muhimu. Thermodynamics ya protini utulivu katika joto la juu: protini folding kama kazi ya joto inaweza kueleweka kwa njia ya mviringo utulivu, plots ya nishati ya bure ya unfolding dhidi ya joto. 1) Kiwango muhimu zaidi katika quantifying protini utulivu kama kazi ya joto ni Tm, joto kuyeyuka. Katika joto hili, uwezekano wa protini kuwa folded au unfolded ni sawa na mabadiliko ya nishati ya Gibbs kuhusishwa na folding ni sifuri. 2) Nishati ya bure ya utulivu ya protini (G) kama kazi ya joto (T) hutolewa na fomu iliyobadilishwa ya usawa wa Gibbs-Helmholtz (Razvi na Scholz, 2006) na kulinganisha data ya thermodynamic kwa seti 26 za jozi za protini za thermophilic na mesophilic. Waligundua kuwa kuongezeka kwa utulivu wa ndani (ng'ambo ya juu ya G katika joto lote) ilikuwa mkakati wa kawaida ambao Tm ya protini za thermophilic ziliongezwa. Kupunguza <unk>Cp ilikuwa mkakati wa pili wa kawaida. Mkakati wa kawaida zaidi ulikuwa kupunguza entropy ya folding. Kwa kupendeza, protini thermophilic kutumia mchanganyiko tofauti ya mikakati hii kufikia thamani ya juu ya Tm. Kwa mfano, wengine walitumia mbinu zote tatu, wengine mbinu ya kwanza, ya pili, ya kwanza na ya tatu, na kadhalika. Hata hivyo, katika kesi yoyote alisoma ilikuwa kupungua entropy ya folding kutumika peke yake kama utaratibu pekee. Hivyo, thermodynamically, mchanganyiko mbalimbali wa mikakati ni kutumika na protini thermophilic kufikia utulivu katika joto la juu. Ni jambo la busara kuamini kwamba athari za hydrophobic zina jukumu muhimu katika thermostability ya protini, kwa kuwa athari ya hydrophobic huongezeka na joto (Reed et al., 2013). Kwa kweli, mwingiliano wa hydrophobic umeonyeshwa kuwa muhimu sana. Hii imeonekana katika aina mbalimbali ya utafiti wa mutagenesis iliyoelekezwa kwenye tovuti. Kwa mfano, Imanaka, Shibazaki, na Takagi (1986) waligundua kwamba ubadilishaji wa asidi ya amino ambao uliongeza hydrophobicity ya ndani uliongeza thermostability ya proteases. Zaidi ya hayo, protini thermophilic kwa ujumla kuwa na maingiliano zaidi kati ya mabaki hydrophobic kuliko protini mesophilic. Kannan na Vishveshwara (2000) walilinganisha thermophilic na mesophilic homogeneous crystal miundo ya familia 24 protini kutumia graph spectral mbinu. Walipata kwamba wengi wa protini thermophilic zilizomo idadi kubwa ya makundi ya harufu au kupanuliwa mitandao ya harufu. Zaidi ya hayo, kuimarisha mchango wa nishati huru ya mwingiliano hydrophobic ni kubwa katika protini thermophilic. Sarabogi, Gromihal na Ponnuswamy walichunguza mchango wa nishati ya bure ya maingiliano mbalimbali kwa kutumia muundo wa protini za thermophilic na mesophilic kutoka familia 23 tofauti. Katika kila kesi waligundua kwamba nishati ya Gibbs ya hydrophobic ya mlolongo kuu ilikuwa chini katika thermophiles kuliko homologs zinazolingana za mesophilic. Kwa upande wa mwingiliano wa molekuli, kuongezeka kwa mwingiliano wa hydrophobic inaonekana kuwa moja ya tofauti thabiti kati ya protini za thermophilic na mesophilic. Gromiha et al. Kwa mfano, katika utafiti wa 2013 na 2013 wa hydrophobicity, interresidual interactions na vifungo vya hidrojeni, familia 373 za protini zilikuwa na athari kubwa kwa viwango vya hydrofobicity. Kwa mfano, asilimia 80 ya protini zenye thermophilic zina hydrophobicity kubwa kuliko zile zinazofanana na homophilic, na hivyo kuwa ni kiashiria cha nguvu zaidi cha thermophilicity. 3) Kwa kushangaza, asilimia 20 ya protini zenye joto la joto ambazo zilikuwa na mwingiliano wa hydrofobic uliopungua, zote zilikuwa na uhusiano wa hidrojeni ulioongezeka au kuunganishwa kwa ioni. Hivyo, ingawa kawaida zaidi, kuongezeka kwa mwingiliano hydrophobic ni moja tu ya mikakati kadhaa iwezekanavyo kutumika na protini thermophilic kufikia utulivu katika joto la juu. Viunganishi vya disulfide vinaweza kuwa na michango muhimu kwa thermostability ya protini thermophilic. Cacciapuoti et al. Kwa mfano, katika utafiti wa 2012 wa hyperthermophilic protein 5′-deoxy-5′-methylthioadenosine phosphorylase II, Protini hii ina CXC motif (C259C261) na vifungo viwili vya disulfide vya intrasubunit (C138C205 na C200C262). Tm ya protini ya aina ya mwitu ilikuwa zaidi ya 108 ° C, wakati mutants ya C262S na C259S na C261S zilikuwa na Tm ya 102 ° C na 99 ° C. Kwa hiyo, mchango wa C200-C262 disulfide dhamana ya protini ya thermostability ni kubwa. Kwa kuongezea, CXC imethibitishwa kuunda pete ya disulfide ya atomu ya 11 ambayo inaweza kutenda kama wakala wa kupunguza (Wojciechowski na Raines, 2003). Kwa hiyo CXC motif ni muhimu katika kudumisha asili disulfide viungo. Hii inaelezea Tm ya chini zaidi ya mutant mbili ya C259S / C261S. Umuhimu wa vifungo vya disulfide ni wazi: mchango wao ni mkubwa na protini hii hata imebadilika utaratibu wa kina ili kudumisha vifungo hivi. Viunganishi vya disulfide vinaweza kuwa muhimu hasa kwa utulivu wa muundo wa quaternary. Boutz et al. Utafiti wa proteomic wa viungo vya disulfide katika Pyrococcus aerophilus, archaeon ya hyperthermophilic (2007) Viunganishi vya disulfide viligunduliwa kuwa muhimu kwa utulivu wa aina mbalimbali za vipengele vya protini-protein. Walipata muundo wa kioo kwa moja ya misombo hii, homdimer ya citrate synthase. Kila subunit ilikuwa na kiunganishi kimoja cha disulfide ndani ya molekuli, kinachozunguka mlolongo wa protini. Kwa kushangaza, hii cyclization ilitokea hivyo kwamba minyororo ya kila subunit walikuwa topologically interlinked, kushikilia dimer pamoja (Fig. 4) Electrostatic Maingiliano na Ion Pair Mitandao Electrostatic maingiliano kuchangia kwa kiasi kikubwa kwa thermostability ya protini thermophilic. Ingawa mara nyingi huwa na madaraja ya chumvi zaidi kuliko protini za mesophilic, utulivu hutokea hasa kupitia mitandao mikubwa ya jozi ya ioni (Fig. 5) Yip et al. Utafiti wa 2008 ulionyesha uhusiano kati ya utulivu wa joto na mitandao ya jozi ya ioni katika glutamate dehydrogenases. Walitumia mbinu ya kuiga inayotegemea homolojia na mfululizo unaojulikana kutoka kwa spishi tofauti. Thermostability ya chini inahusiana na mitandao zaidi ya jozi ya ioni iliyovunjika. Kwa kweli, kuna uthibitisho mkubwa kwamba nafasi ya jozi za ioni, badala ya idadi, ni kuzingatia muhimu zaidi ya nishati. Xiao na Honig (1999) mahesabu mchango electrostatic kwa folding bure nishati ya protini hyperthermophilic na homologues yao mesophilic. Katika jozi zote zilizochunguzwa, waligundua kwamba mchango wa nishati ya bure ya mwingiliano wa umeme ulikuwa mkubwa katika protini za hyperthermophilic. Kwa kupendeza, nishati ya bure haikuhusiana vizuri na idadi ya mabaki ya ionizable au jozi za ioni. Badala yake, ilitokana na mahali panapofaa pa kuweka mabaki yenye nishati. Bakker, Hunenberger, na McCammon (1999) walitoa ushahidi wa ziada kwamba shirika bora la jozi za ioni katika mitandao ni mchango wa msingi wa umeme kwa thermostability. Kwa mfano, katika utafiti wa molekuli ya protini ya hyperthermophilic Sac7D, ambayo ilifanya kazi kwa joto la 300K, imara ya umeme ilitokana na mitandao mitatu ya jozi ya ioni. Hata hivyo, wakati kuchukuliwa peke yake, utulivu kutoka madaraja ya chumvi ya mtu binafsi ilikuwa mahesabu kuwa ndogo, baadhi hata walikuwa destabilizing. Hivyo, utulivu wa nishati unaotokana na mitandao ya jozi ya ioni huonyesha kiwango cha juu cha ushirikiano. Hii inaelezea kwa nini ni shirika na nafasi, badala ya idadi ya jozi ya ioni, ambayo huamua mchango wa nishati ya bure. Ugunduzi mwingine muhimu wa Bakker et al. Katika utafiti wa 1999 iligunduliwa kwamba baada ya kuongeza joto la simulation kutoka 300K hadi 360K, nishati ya mwingiliano wa protini-solvent ilizidi kuwa nzuri, wakati nishati ya mwingiliano wa intraprotein ilizidi kuwa nzuri. Athari zote mbili hizi zilikuwa hasa matokeo ya michango ya electrostatic. Hii inaweza kuwa kutokana na kupungua kwa nishati ya desolvation, gharama ya nishati inayohusiana na hasara ya mwingiliano wa mchanganyiko wakati mabaki yanapozikwa katika protini. Nishati ya desolvation (W) ya ion ni inayotolewa na (Karshikoff na Ladenstein, 2001) na utaratibu thermodynamic ambayo jozi ya ioni kuimarisha protini thermophilic katika joto la juu ni, angalau kwa sehemu, kwa kupunguza Cp. Chan, Yu, na Wong (2011) walionyesha hili kwa kubadilisha jozi za ioni kwa mtu mmoja-mmoja na mutagenesis iliyoelekezwa kwenye tovuti. Upimaji wa <unk>Cp ya mutants na kulinganisha na protini ya aina ya mwitu iliruhusu mchango wa kila jozi ya ioni kwa <unk>Cp kuhesabiwa. Kwa mfano, kuna jozi mbili za ioni ambazo zinashughulikia uharibifu wa 0.8 - 1.0 kJ mol-1 K. Amino Acid Composition Miongoni mwa mabadiliko katika amino asidi muundo wamekuwa alibainisha kati ya protini thermophilic na mesophilic homologs yao. Kwa mfano, Kumar, Tasi, na Nussinov (2000) walilinganisha familia 18 zisizo za ziada za protini za thermophilic na mesophilic. Walipata kwamba Arg na Tyr masafa walikuwa kuongezeka kwa kiasi kikubwa katika protini thermophilic. Walidai kwamba matokeo haya yanaweza kuwa matokeo ya kuongezeka kwa madaraja ya chumvi na hydrophobicity kawaida kuhusishwa na protini thermophilic. Zaidi ya hayo, kuna tofauti za takwimu muhimu kati ya amino asidi muundo wa helices katika protini thermophilic na mesophilic. Mzunguko wa Cys na His hupungua, wakati mzunguko wa Arg unaongezeka (Kumar, Tasi, na Nussinov, 2000). Kwa kuvutia, Arg ni helix kupendelea mabaki wakati Cys, na His ni helix disfavouring. Kwa kuongezea, protini za thermophilic zina maudhui ya juu zaidi ya helical katika familia nyingi za protini (Kumar, Tasi, na Nussinov, 2000). Inafuata kwamba kuongezeka kwa maudhui ya helical na propensity inaweza kuwa utaratibu muhimu wa kuongeza utulivu katika joto la juu. Mwishowe, Kumar et al. Pia iligundua kupungua kwa kiasi muhimu cha Cys na Ser katika protini za thermophilic (2000). Hii si ya kushangaza: amino asidi za polar Asn, Cys, Gln, na Met zinachukuliwa kuwa thermolabile kwa sababu kwa joto la juu zinaweza kupitia athari za deamidation au oxidation (Russel et al. 1997) na Kwa hivyo, kupungua kwa matumizi ya asidi hizi za amino inaweza kuwa marekebisho ya joto la juu, kupunguza hatari ya athari hizi zisizohitajika. Ingawa si muhimu kwa takwimu, uwiano wa Asn pia ilipungua kwa kiasi kikubwa. Hata hivyo, kinyume na nadharia hii kiasi cha Gln na Met walikuwa sawa katika protini mesophilic na thermophilic. Hii ni uwezekano kwa sababu solvent mfiduo na mazingira ya ndani ni muhimu determinants ya amino asidi ya unyeti kwa deamidation na athari oxidation. Kwa hiyo matumizi ya asidi ya amino bias hutokea karibu peke kati ya mabaki ya uso. Mfano wa kuvutia wa tofauti hii katika muundo wa uso ni lumazine synthase (Fig. 6) Hivyo, tofauti katika amino asidi muundo kati ya thermophilic na mesophilic protini inaweza kuwa wazi zaidi kueleza kwa kuzingatia eneo la kila mabaki. Fukuchi na Nishikawa (2001) walilinganisha muundo wa ndani na nje wa asidi ya amino ya aina mbalimbali ya protini za mesophilic na thermophilic zinazopatikana kwenye Benki ya Takwimu za Protini. Kama ilivyotarajiwa, mzunguko wa mabaki ya polar uncharged (Asn, Ser, na Thr) juu ya uso wa protini thermophilic ilikuwa chini sana kuliko kwa protini mesophilic. Kinyume chake, uwiano wa mabaki ya polar charged (Asp, Glu, Arg, na Lys) ilikuwa ya juu. Ingawa muundo wa uso ni tofauti, Fukuchi na Nishikawa hawakupata tofauti yoyote muhimu katika muundo wa asidi ya amino ndani. Mfumo mbalimbali wa mikakati hutumiwa na protini thermophilic kufikia thermostability yao ya ajabu. Thermodynamically, utaratibu wa kawaida ni kuongeza jumla ya utulivu bure nishati, <unk>G. Hata hivyo, utaratibu mbadala, ikiwa ni pamoja na kupungua <unk>Cp na / au entropy ya folding pia hutumiwa na protini nyingi thermophilic. Tofauti ya maingiliano ya molekuli ni kuhusishwa na thermostability ya juu ya protini thermophilic. Mchango wa vifungo vya disulfide vya mtu binafsi unaweza kuwa muhimu, kuongeza sana thamani ya Tm. Zimeonekana kuwa muhimu hasa katika kudumisha muundo wa quaternary katika joto la juu. Kwa kuongezea, kuongezeka kwa mwingiliano wa hydrophobic ni sifa ya kila mahali ya protini za thermophilic. Wana makundi zaidi ya harufu na mchango mkubwa wa nishati ya bure ya utulivu kutoka kwa mwingiliano wa hydrophobic. 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