Abstract
Single‐domain antibodies (sdAbs) function like regular antibodies, however, consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (T m) values and known structures. We applied molecular dynamics (MD) simulations to estimate their relative stability and compared them with the experimental data. High‐temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as a control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the T m values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q values of hydrophilic residues exhibited an even better correlation, suggesting that stabilization is correlated with favorable interactions of hydrophilic residues. Measuring the Q value on a per‐residue level enabled us to identify residues that contribute significantly to the instability and thus demonstrating how our analysis can be used in a mutant case study.
Highlights
High-temperature molecular dynamics (MD) simulations could provide us valuable information for protein design, such as which residues are flexible and whether native-like interactions are preserved or not.[21]. We previously employed such high temperature simulations to qualitatively estimate the stability of predicted binding configurations between an enzyme, cyclin-dependent kinase 2, and one of its inhibitors.[22]. In another recent paper, we showed the effect of Ca2+ concentration differences on the stability of cutinase, a hydrolytic enzyme, by analyzing our MD trajectories using the fraction of native contacts, Q value, which matched well with our observations from experiments.[23]
Continuing our efforts to fine-grain stability measurements, we investigated the contribution of each amino acid residue to the thermal stability by calculating the Q value of each individual residue from the MD simulations at 400 K and identifying the unstable residues
The correlation coefficient of the average Q value over the residues of each system at 400 K with respect to the corresponding experimental Tm values, shows a relatively high correlation (r = 0.85, Q value data not shown), which suggests that the residues identified by our protocol should be representative of those that significantly contribute to the instability of an single-domain antibodies (sdAbs)
Summary
The variable fragment of antibodies (Fv), which consists of heavy (VH) and light chains (VL), binds to Additional Supporting Information may be found in the online vCeorrsreiocntioonfatdhdisedaortnicDlee.cember 17, 2019. HThigishilsigahntso: peTno aeccsetismsaatertictleheunsdtearbtihlietyteormf ssoinf gthlee-dCoremaativine CaonmtibmoodnsieAsttr(isbduAtiobnsL),icwenesec, hwohsiceh spervmeints sudseA,bdsistwribhuictiohn haandvereaprowdiudcertiaonngineanoyf mmeedlituinmg, ptreomvidpeedrathtueroersigiananldwohrak viseprkonpoewrlyncistetrdu.ctures. Good correlation between the fraction of native contacts (Q). Highlights: To estimate the stability of single-domain antibodies a(snddAbst)h,ewemcheoltsinegsevteenmspdeArabtsurweh.ichFuhartvheeramwoirdee, raonugre opf rmoteoltcinogl etenmapbelreadtureuss atnod ihdaevnetikfynowunnsstatrubcletureres.siAdnuaelyss, isfroofmourwhmioclhecuwlaer ddyensaigmnicesdsismeuvlaetrioanl svsirhtuowaledmautgaonotdsctrourrcetluartieosn baestwaeecnatshee fsratuctdioyn, wofhnicahtivsehcoownetadctism(pQr)ovaendd tshteabmileitlytining steilmicpoe.rature. Our protocol enabled us to identify unstable residues, from which we Gdersaingnt esdposenvseorra:l Jvairptuaanl mSuotacniet tystrfuocrtutrhees aPsroamocatisoen sotuf dSy,ciwenhicceh JshPo1w6eKd0i7m3p3ro1v.ed stability in silico. Grant sponsor: Japan Society for the Promotion of Science
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