Supplemental Material uman NF-α amin acid sequence: 1 VRSSSRPS PVAVVANP QAEQLQWLN RRANALLAN VELRNQLVV PSELYLIYS 61 QVLFQP SVLLI SRIAVSYQ VNLLSAISP QREPEAE APWYEPIYL 121 VFQLE RLSAEINRP YLFAESQV YFIIAL Extinctin cefficient used fr determinatin f cncentratin: ε NF(278nm) = 60500 M -1 cm -1 Fab f adalimumab amin acid sequence: Light chain: 1 IQMQSPSS LSASVRV IRASQIR NYLAWYQQP APLLIYA ASLQSVPS 61 RFSSS FLISSLQP EVAYYQR YNRAPYFQ VEIRV AAPSVFIFPP 121 SEQLSA SVVLLNNFY PREAVQWV NALQSNSQ ESVEQS SYSLSSL 181 LSAYE VYAEVQ LSSPVSFN RE eavy chain: 1 EVQLVES LVQPRSLRL SAASFF YAMWVRQA PLEWVSA IWNSIY 61 ASVERFI SRNANSLY LQMNSLRAE AVYYAVS YLSASSLY WQLVVS 121 SASPSVF PLAPSSSS AALLV YFPEPVV SWNSALS VFPAVLQS 181 SLYSLSSVV VPSSSLQ YINVNP SNVVE PS Extinctin cefficient used fr determinatin f cncentratin: ε Fab(278nm) = 66400 M -1 cm -1 SS PAE electrphresis: SS PAE electrphresis was emplyed t cnfirm suitability f the mnmlecular denaturatin mdel used fr the interpretatin f urea induced unflding curves. he data suggests that Fab is cmpsed f tw plypeptide chains f similar length (20-0 ka) cnnected with a disulphide bnd. his is als in accrdance with the amin acid sequence data presented abve. SI-Figure 1. SS PAE electrphresis f Fab under reducing nn-reducing cnditins. 1
hermdynamic Analysis f Experimental ata lbal Mdel Analysis f Urea-induced Unflding urves Mnitred by -Urea denaturatin f Fab, NF-α the Fab-NF-α cmplex can be successfully described in terms f a tw-state r three-state mdel. he mdels can be defined as fllws, 1(,u ) I F1(,u ) F I Mdels 1- F(,u ) F F F 2(,u ) F2(,u ) F where F, F represent Fab mnmer, NF-α trimer NF-α-Fab heterhexamer in their native (N) states, respectively, whereas superscript dentes the prteins in their denatured () states. he transitins t the intermediate states I I F accmpanying the denaturatin f F cmplex is assumed t be mnmlecular. he apparent equilibrium cnstants in Mdels 1- are F F, functins f temperature () urea cncentratin ( can be defined as: F( I, I I F F I 1(, 2( F1( F. F2 ( he quantities in the square brackets represent the crrespnding equilibrium mlar cncentratins Θ ) at a that are dependent n u. Accrding t the mdels the measured mlar ellipticity ( ( given wavelength, u can be expressed in terms f the crrespnding cntributins N( Θ I( Θ ( that characterize states N, I, as fllws, Θ Θ N(, Θ I(, Θ (, ) (, N(, I(, (, u F Θ, (SI-Eq. 1) where N(, I( ( represent fractins f prteins in states N, I respectively, at given u. In the case f F denaturatin, the fractins are defined as F F ( F ( t ( 1 t N(, u ) F(, u ) t F F, in the case f denaturatin as N( ( t (, u ) in the case f F denaturatin F t N( F (, whereas ( Θ ( are defined as Θ Θ Θ, respectively ([F] t, [ ] t [ F ] ( (, (, F (, ( F ( represent ttal Fab, NF-α trimer Fab-NF-α cmplex cncentratins, respectively). WhileΘ ( Θ N(, Θ ( can be btained frm the experiment ( Θ ( is the measured signal, while Θ N( Θ ( can be estimated at any measured as linear functins f u frm pre- psttransitinal base lines), I( ( can be calculated frm the mdel, as shwn belw. Fr tw state denaturatin f F the secnd term in SI-Eq. 1 is equal t zer. In the case f r F Θ was included in the glbal mdeling as a linear functin f independent f u. denaturatin I( I( ( can be cnnected t the thermdynamics f unflding thrugh prpsed mdels (Mdels 1-) the general characteristics f urea denaturatin that the stard ibbs free energy f unflding ( ) fr any transitin i (i = F, 1, 2, F1 r F2) appears t be a linear functin f u i (, u ) ) i ( i ( mi u (SI-Eq. 2) 2
where m i is an empirical parameter crrelated strngly t the amunt f prtein surface area-expsed t the slvent upn denaturatin (1) assumed t be temperature-independent. is the stard ibbs free energy f unflding in the absence f urea (u = 0) that may be expressed in terms f crrespnding stard ibbs free energy ( ) stard enthalpy f unflding ( ) at a reference temperature 0 = 7 º stard heat capacity f unflding ( temperature-independent, thrugh the ibbs-elmhltz relatin (integrated frm). 1 ln i( ) i ( ) 0 i( ) 0 P, i 0 0 0 0 ), assumed t be (SI-Eq. ) It fllws frm SI-Eq. 2 SI-Eq. that the mdel (adjustable) parameters,,, m i define i (, u ) thus the crrespnding i( [ R i ( exp i( ]. nsequently, they specify the ppulatins f species in slutin ( i(,u ) = f ( j( ; j N,I, ), 1 j( ) the mdel functin (SI-Eq. 1) at any u. he best glbal fit values f j j (, (able 1) btained using the nnlinear Levenberg-Marquardt regressin prcedure (2) were used t estimate (integrated frm), i( ) i( ) P, i 0 (frm SI-Eq. ), frm the irchhff s law (SI-Eq. 4) the crrespnding entrpy cntributin, S frm the general relatin (SI-Eq. 5) i( ) i( ) Si ( ) lbal Mdel Analysis f I Binding urves-mdel functin describing binding f Fab t three equivalent independent binding sites n NF-α was fitted t the sets f I curves measured at varius temperatures as described belw. he mdel functin at a given can be defined as fllws (,4) rp,, 1 (SI-Eq. 6) where i( ) n is the stard enthalpy f Fab binding t the binding site f NF-α, is an average n i F i i1 number f Fab bund n NF-α ( t ), r is a mlar rati between ttal cncentratin f Fab NF-α in the measuring cell. he derivative in SI-Eq. 6 can be expressed as r = (12)(1+[-r-c][r 2-2r(-c)+(1+c) 2 ] 12 ), where c = 1( () [ ] t ), () is an apparent P,, n1 cnstant f Fab binding t any f the three binding sits n NF-α [ ] t is the ttal cncentratin f NF-α in the measuring cell. he crrespnding stard ibbs free energy stard enthalpy at 0 = 7 º stard heat capacity f binding temperature independent) define (assumed t be at any by SI-Eqs. 4. hus the values f adjustable parameters, cmpletely define the temperature dependence f the binding cnstant [ i() = exp(- R)], the mdel functin (SI-Eq. 6) at any
cnsequently the crrespnding thermdynamic prfiles (Fig. 6 in the main text). he best fit values f the adjustable parameters (able 1 in the main text) were btained using the nnlinear Levenberg- Marquardt regressin prcedure (2). It shuld be mentined that a significant curvature f the I curve at FabNF-α mlar rati r (Fig 2) frm which a safe binding cnstant can be estimated is bserved nly at the highest measured temperature. herefre, ne may be a bit skeptical abut the accuracy f the btained binding cnstant. wever, since the glbal mdel gives gd descriptin f all I istherms, the ne with the curvature the thers with nticeable breaks at r, we believe that the crrespnding estimate f the binding cnstant is reasnable. Structural interpretatin f NF-α-Fab assciatin- Numerus recent studies f prtein unflding prtein-prtein binding prcesses have shwn that the crrespnding enthalpy ( ) heat capacity ( ) changes can be parameterized in terms f changes in slvent accessible plar (ΔA P ) nnplar (ΔA N ) surface areas accmpanying these prcesses (1,5-9). Such a parameterizatin is based n the estimatin f the nnplar (A N ) plar (A P ) slvent-accessible areas f prteins in the initial (unbund) final (bund) states. A N A P f NF-α-Fab cmplex were calculated frm structural mdel btained by mlecular mdeling (described in the main text) by applying the prgram NAESS versin 2.1 using the prbe size f 1.4 Å (10). A N A P f unbund NF-α Fab were btained frm the structural mdel f the cmplex by deleting the crdinates f either f the binding partners by applying the same prgram as fr the cmplex. ΔA N, BIN ΔA P, BIN were estimated as a difference between A N (A P ) f the NF-α-Fab cmplex the sum A N (A P ) f the unbund NFα Fab. he heat capacity ( ) enthalpy changes ( P, BIN BIN ( ) BIN ( ) ( )) accmpanying binding can be expressed as the sum f nnplar (subscript N) P, BIN plar (subscript P) cntributins (5-9). P, BIN P, BIN, N P, BIN, P aan, BIN bap, BIN (SI-Eq. 7) BIN ( ) BIN ( ), N BIN ( ), P c a( ) A N, BIN d b( ) A P, BIN (SI-Eq. 8) Parameters a = 0.45 calml -1-1 Å -2, b = -0.26 calml -1-1 Å -2, c = -8.44 calml -1 Å -2, d = 1.4 calml -1 Å -2 are btained frm Murphy Freire (5) Xie Freire (6), whereas is parameterized as i( ) can dap represents the enthalpy f unflding bserved with mst glbal prteins at their median transitin temperature f = 60 º. he entrpy change ( ) accmpanying binding prcesses can be expressed as fllws, S BIN, S ln( ) S BIN, P, BIN S R (SI-Eq. 9) where first term in SI-Eq. 9 is an estimate f change in slvatin entrpy upn binding S 112 º is the reference temperature at which slvatin entrpy is assumed t be zer (7). he secnd term SR = -50 cal ml -1-1 is an estimate f the translatinal rtatinal entrpy lss accmpanying a rigid-bdy assciatin (8). Empirical relatins between structural thermdynamic parameters in cmbinatin with experimentally btained quantities enable us t parse the thermdynamics f binding int a cntributin f rigid-bdy assciatin ( ) cntributin f cnfrmatinal change ( F ONF ; see Eq. 1 in the main text). F F F, (SI-Eq. 10) BIN ONF F BIN 4
where F,, S,. mentined abve while P F was btained frm the I experiments. was estimated as F F ONF ONF F F BIN F BIN was calculated as. he calculated P, values enable us t estimate changes in slvent accessible plar nnplar surface ONF( ) areas accmpanying cnfrmatin change ( A P, ONF, A N, ONF ) by emplying SI-Eqs. 7 8. References: 1. Mayers, J.., Pace,. N., Schltz, M. J., (1995) Prtein Sci. 4, 218-2148 2. Press, W.., Flannery, B. P., euklvski, S. A., Vetterling, W.., (1992) Numerical Recipes, Oxfrd, U: ambridge University Press. Lah, J., rbnak, I., linar, M., Vesnaver,., (2008) Nucleic Acid Res. 6, 897-904 4. Bnčina, M., Lah, J., Reščič, J., Vlachy, V. (2010) J.Phys. hem. B. 114, 41-419. 5. Murphy,. P., Freire, E. (1992) Adv. Prtein hem. 4, 1-61 6. Xie,., Freire, E. (1994) Prteins Struct. Funct. enet. 19, 291-01 7. Baldwin, R. L., (1986) Prc. Natl. Acad. Sci. U.S.A 8, 8069-8072 8. Splar, R. S., Recrd, M.., Jr. (1994) Science 26, 777-784 9. Makhatadze,. I., Privalv, P. L. (1995) Adv. Prtein hem. 47, 07-425. 10. ubbard, S. J., hrntn, J. M. (199) NAES, University llege, Lndn ONF 5