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The protein)-5.4( co)]TJ 9.8216 0 TD -0.0003 Tc 0.1881 Tw [(ntainin)-6.1(g)-1.2( fractions )]TJ -29.9933 -1.847 TD 0.0005 Tc 0.1583 Tw [(were )-5.8(pooled )-5.8(and )-5.8(dial)-8.4(y)8.1(z)1.3(e)-5.9(d)0.2( int)-9.1(o)-1( th)-5.3(e s)5.3(t)-3.3(ora)9.9(g)-6.2(e buff)6.6(er o)-6.8(v)-2.9(er night)-9.1( )5.9(with)-5.3( two buffer)-6.5( )]TJ 0 -1.8412 TD -0.0005 Tc 0 Tw [(exch)-6.3(anges)-7.3(.)3.4( )]TJ T* 0 Tc ( )Tj /TT6 1 Tf T* -0.0005 Tc -0.0005 Tw [(No s)-7.8(a)3.3(l)-4.7(t)-2.7( Buff)-5.3(er)5.7( )]TJ /TT4 1 Tf T* -0.0019 Tc 0.0096 Tw [(10 mM )5.8(T)-5.9(r)-3.1(is pH 8 )]TJ T* 0.0006 Tc 0.0013 Tw [(0.2)-2.8(5)3( m)-2.3(M)1.9( C)-5.4(a)4.2(Cl)]TJ 6.6044 0 0 6.6044 181.5 325.38 Tm 0 Tc 0 Tw (2)Tj 10.3303 0 0 10.3303 185.16 325.86 Tm ( )Tj -7.2253 -1.847 TD -0.0011 Tc 0.0088 Tw [(0.1 mM )5.8(E)-5.1(G)-6.1(T)-5.1(A)6.8( )]TJ 0 -1.8412 TD -0.0016 Tc 0.0093 Tw (0.2 mM )Tj /TT9 1 Tf 3.8043 0 TD 0 Tc 0 Tw <0045>Tj /TT4 1 Tf 0.546 0 TD 0.0045 Tc [(ME)6.3( )]TJ -4.3503 -1.847 TD 0 Tc ( )Tj /TT6 1 Tf 0 -1.8412 TD 0.0001 Tc -0.0011 Tw [(Hig)-7.7(h)2( salt Buffe)-4.5(r )]TJ /TT4 1 Tf T* -0.0019 Tc 0.0096 Tw [(10 mM )5.8(T)-5.9(r)-3.1(is pH 8 )]TJ T* 0.0006 Tc 0.0013 Tw [(0.2)-2.8(5)3( m)-2.3(M)1.9( C)-5.4(a)4.2(Cl)]TJ 6.6044 0 0 6.6044 181.5 211.2001 Tm 0 Tc 0 Tw (2)Tj 10.3303 0 0 10.3303 185.16 211.6201 Tm ( )Tj -7.2253 -1.847 TD -0.0011 Tc 0.0088 Tw [(0.1 mM )5.8(E)-5.1(G)-6.1(T)-5.1(A)6.8( )]TJ 0 -1.8412 TD -0.0016 Tc 0.0093 Tw (0.2 mM )Tj /TT9 1 Tf 3.8043 0 TD 0 Tc 0 Tw <0045>Tj /TT4 1 Tf 0.546 0 TD 0.0045 Tc [(ME)6.3( )]TJ -4.3503 -1.8412 TD -0.0009 Tc 0.0028 Tw [(0.5 M )-5.8(N)-4.6(a)2.7(Cl)-9.8( )]TJ T* 0 Tc 0 Tw ( )Tj /TT6 1 Tf T* -0.0006 Tc -0.0004 Tw [(Storag)-8.4(e Bu)-4.6(f)-5.4(f)0.4(er )]TJ /TT4 1 Tf T* -0.0008 Tc 0.0085 Tw [(25 mM He)-7.2(pes )5.8(p)-4(H)8.6( 7.4 )]TJ 0 -1.847 TD -0.0016 Tc 0.0093 Tw [(25 mM N)-5.3(a)-3.9(Cl)-4.7( )]TJ ET endstream endobj 44 0 obj << /ProcSet [/PDF /Text ] /Font << /TT2 4 0 R /TT4 5 0 R /TT6 6 0 R /TT9 18 0 R >> /ExtGState << /GS1 8 0 R >> /ColorSpace << /Cs6 9 0 R >> >> endobj 48 0 obj << /Length 9624 >> stream BT /TT2 1 Tf 11.2898 0 0 11.2898 110.52 36.54 Tm /Cs6 cs 0 0 0 scn /GS1 gs 0 Tc 0 Tw ( )Tj /TT4 1 Tf 10.3303 0 0 10.3303 489.96 36.48 Tm -0.0024 Tc (78)Tj -36.7307 64.7144 TD -0.0015 Tc 0.0092 Tw [(10 mM Mg)-8.2(Cl)]TJ 6.6044 0 0 6.6044 173.94 704.58 Tm 0 Tc 0 Tw (2)Tj 10.3303 0 0 10.3303 177.6 705 Tm ( )Tj -6.4935 -1.8412 TD -0.0011 Tc 0.0088 Tw [(0.1 mM )5.8(T)-5.1(C)-1.3(E)-5.1(P)4.1( )]TJ T* 0 Tc 0 Tw ( )Tj /TT8 1 Tf 13.1527 0 0 13.1527 110.52 633.84 Tm 0.0008 Tc -0.0001 Tw [(RLC Phosph)7.9(orylation via MLCK-A Kinase )]TJ /TT6 1 Tf 12.2496 0 0 12.2496 110.52 599.34 Tm 0.0001 Tc 0.0017 Tw [(MLCK-A)10( Protein Expr)6.4(essi)]TJ 11.5645 0 TD 0.0002 Tc -0.0032 Tw [(on and Purifi)4.8(cation )]TJ /TT4 1 Tf 10.3303 0 0 10.3303 110.52 575.82 Tm -0.0004 Tc 0.1708 Tw [(T)-4.4(h)-0.4(e my)7.2(os)-7.2(in)-6.2( l)-3.5(i)2.3(ght)-4.2( ch)-6.2(ai)-3.5(n)-6.2( k)7.9(i)2.3(n)-12(a)9(s)-7.2(e)-1( )-5.8(A )-5.8(\()14.4(M)-5(LCK-)-5.2(A\) )-5.8(mu)-8.2(t)-4.2(a)3.2(nt)-4.2( T)-4.4(1)2(6)-3.8(6)2(E w)-5.7(a)3.2(s expre)-6.8(s)-1.4(sed usi)-3.5(n)-0.4(g )]TJ 0 -1.8412 TD 0 Tc 0.1065 Tw [(t)-9.6(h)0(e p)8.4(E)-4(T15b )5.8(ex)-7.9(pr)4.6(ess)-6.8(i)2.7(on)-5.8( plasmid to N-t)-9.6(e)-0.6(r)4.6(m)-2.9(in)-5.8(ally )-5.8(\(His)-6.8(\))]TJ 6.6044 0 0 6.6044 367.62 556.32 Tm 0 Tw (6)Tj 10.3303 0 0 10.3303 371.28 556.8 Tm 0.0023 Tc 0.1042 Tw [( tag the p)10.7(r)1.1(otein.)6.2( )-5.8(A)4.4(f)2.6(ter the )]TJ -25.2422 -1.847 TD 0.0036 Tc 0.3004 Tw [(p)6.2(u)1.7(rifi)6.3(ca)7.2(ti)6.3(on,)13.3( the )-5.8(\()12.6(H)1.3(is\))]TJ 6.6044 0 0 6.6044 219 537.3 Tm 0 Tc 0 Tw (6)Tj 10.3303 0 0 10.3303 222.72 537.72 Tm 0.0047 Tc 0.2993 Tw [( )5.8(ta)14.1(g wa)14.1(s )-5.8(re)9.9(m)7.6(o)3.2(ved by)6.5( c)7(l)1.6(ea)14.1(vage )5.8(wi)7.4(th )5.8(th)10.5(romb)7.3(i)7.4(n)]TJ 6.6044 0 0 6.6044 474.78 540.54 Tm 0.0091 Tc 0 Tw (72)Tj 10.3303 0 0 10.3303 482.16 537.72 Tm -0.0039 Tc 0.3079 Tw [(. 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The c)5.9(e)-2.8(lls )5.8(were grown )]TJ T* 0.0009 Tc 0.1579 Tw [(a)16.1(t)8.7( 2)9.1(8)9.1()11.7(C)12.4( b)9.3(e)6.1(c)9(a)16.1(us)11.6(e)11.9( h)6.7(i)9.4(g)11.6(h)6.7(e)11.9(r)11.3( t)8.7(e)0.3(mp)9.3(er)-6.2(atures led )-5.8(to insoluble pr)5.5(ot)-8.7(e)6.1(i)3.6(n)-4.9(.)4.8( )-5.8(Th)-4.9(e cells)5.7( )-5.8(wer)5.5(e)-5.5( )]TJ T* -0.002 Tc 0.1782 Tw [(in)-7.8(du)-9.8(ced )5.8(w)-7.3(i)-5.1(t)-5.8(h)-7.8( )5.8(0.4 )168.5(mM IPT)-6(G)-1.2( at)-5.8( an )5.8(OD)]TJ 6.6044 0 0 6.6044 300 442.14 Tm 0 Tc 0 Tw (600)Tj 10.3303 0 0 10.3303 310.98 442.56 Tm 0.0025 Tc 0.1679 Tw [( of 0.)6.4(45.)6.4( )-5.8(A)4.6(f)2.8(ter 4 hour)7.1(s t)-7.1(h)2.5(e )5.8(c)4.8(u)0.6(lture was )]TJ -19.405 -1.8412 TD 0.0002 Tc 0.1992 Tw [(cen)-5.6(t)-3.6(rif)6.3(u)-7.6(ge)-6.2(d )-5.8(a)9.6(t)-3.6( 7000 )197.5(r)-6.8(p)-3(m)8.9( )-5.8(for 10 )-5.8(minu)-7.6(tes )5.8(in)-5.6( a)9.6( )-5.8(Beckma)-7.9(n J-6M sw)-5.1(in)-5.6(gi)8.7(n)-5.6(g)-0.7( bucket )]TJ T* 0.0889 Tw [(cen)-5.6(t)-3.6(rif)6.3(u)-7.6(g)5.1(e)-6.2(. )5.8(The pellet was )5.8(re)-6.2(s)5(u)-7.6(spen)-5.6(d)5.7(e)-0.4(d)-5.9( )5.9(in)-5.6( )5.8(20 )87.2(ml )5.9(l)-8.7(y)2(si)8.7(s)-6.6( buff)6.3(er an)-5.6(d f)6.3(r)-6.8(ozen)-5.6( )5.8(in )5.8(l)-8.7(i)-2.9(q)8.6(u)-7.6(id )]TJ 0 -1.847 TD 0.0078 Tc 0.1684 Tw [(ni)10.5(t)4(r)6.6(o)12.1(g)6.9(e)7.2(n)7.8( )-5.8(d)13.3(r)0.8(o)6.3(p)16.2(s.)11.7( T)3.8(h)7.8(e c)4.3(e)7.2(l)4.7(l)4.7(s)12.6( l)4.7(y)9.6(s)12.6(e)1.4(d)7.5( up)10.4(o)6.3(n)7.8( th)7.8(a)5.5(w)8.3(i)10.5(n)2(g)12.7(.)11.7( )-11.6(I)13.2(m)4.9(m)4.9(e)7.2(d)7.5(i)4.7(a)11.4(t)4(e)7.2(l)4.7(y)9.6( )-5.8(a)11.4(f)13.9(te)7.2(r)6.6( th)7.8(a)11.4(w)2.5(i)16.3(n)2(g)6.9( t)4(h)7.8(e )]TJ 0 -1.8412 TD 0.0024 Tc 0.2028 Tw [(c)4.7(e)1.8(lls,)12.1( 10)4.8( )197.5(mM )-5.8(MgCl)]TJ 6.6044 0 0 6.6044 205.5 365.94 Tm 0 Tc 0 Tw (2)Tj 10.3303 0 0 10.3303 209.22 366.42 Tm 0.0004 Tc 0.0015 Tw [(,)10.1( )-214.9(1 mM )-203.3(P)-6(M)1.7(SF)-8.4(,)15.9( )-214.9(an)-5.4(d )-203.3(5 )-11.2(g)5.3(/m)9.1(l )-209.1(DNAs)-6.4(e I)11.6( )-214.9(were )-209.1(ad)5.9(de)-6(d )-209.1(and)-5.7( )-203.3(t)-9.2(h)0.4(e)5.6( )]TJ -9.5544 -1.8412 TD 0.0024 Tc 0.075 Tw [(mi)5.1(xture was sti)5.1(r)1.2(r)7(e)1.8(d ab)5(out 1)4.8(5)4.8( m)5.3(i)5.1(nutes)7.2( a)6(t)-1.4( roo)6.7(m)11.1( )-5.8(temp)10.8(erature )5.8(unti)5.1(l )5.8(the )5.8(vi)5.1(sc)4.7(o)6.7(s)-4.4(i)5.1(t)-1.4(y of )]TJ T* 0.0079 Tc 0.0695 Tw [(th)7.9(e)7.3( s)6.9(o)6.4(l)10.6(u)11.8(t)4.1(i)10.6(o)6.4(n)7.9( )-5.8(d)13.4(e)7.3(c)10.2(r)6.7(ea)11.5(se)7.3(d)7.6(.)11.8( T)9.7(h)2.1(e)7.3( ly)15.5(sa)17.3(te)13.1( )-5.9(wa)17.3(s)6.9( c)10.2(e)1.5(n)7.9(t)-1.7(r)6.7(i)16.4(f)8.2(u)6(g)7(e)7.3(d)7.6( 1)4.5(5)10.3( m)5(i)10.6(n)7.9(u)6(te)7.3(s)12.7( a)17.3(t)-1.7( 1)10.3(7)4.5(0)10.3(0)4.5(0)10.3( )75.5(r)6.7(p)4.7(m)10.8( i)4.8(n)2.1( )]TJ T* -0.001 Tc 0.2527 Tw [(a )-5.8(Bec)-4.5(k)1.5(m)-3.9(a)-9.1(n J)-7(A)1.1(20 )-5.8(ro)-2.5(t)-4.8(o)-2.5(r )-5.8(at)-4.8( 4C)-7(.)-2.9( T)-5(h)-1(e su)-8.8(per)-8.1(n)-1(at)-4.8(ant)-10.6( w)-6.3(a)2.6(s l)-4.1(o)-2.5(a)-3.3(d)4.5(e)-7.4(d)-1.3( )-5.8(o)-2.5(n)-1(t)-10.6(o)3.3( )-5.8(a)8.4( )-5.8(N)-4.7(i)7.5(-)-11.7(N)1.1(T)-5(A)1.1( )]TJ 0 -1.847 TD 0.0028 Tc 0.1211 Tw [(c)5.1(o)1.3(lum)5.7(n)-3( )5.8(t)-6.8(h)8.6(a)6.4(t)-1( wa)12.2(s )-5.8(p)11.2(r)1.6(evi)5.5(o)1.3(usly)10.4( eq)11.2(u)-10.8(i)11.3(lib)5.4(ra)6.4(ted wi)5.5(t)-6.8(h)2.8( )5.8(the 2)5.2(0)5.2( )122(m)5.7(M)4.1( )-5.8(i)5.5(m)-0.1(i)5.5(d)-3.3(a)12.2(z)-8(ol)5.5(e b)5.4(u)0.9(ffer.)6.7( The )]TJ 0 -1.8412 TD 0.0016 Tc 0.1049 Tw [(c)3.9(o)0.1(lum)4.5(n)-4.2( )5.8(was wa)11(shed )-5.8(wi)4.3(th 3)4( c)3.9(o)0.1(lu)5.5(m)4.5(n)-4.2( )5.8(volum)4.5(e)1(s wi)4.3(th 2)4(0)4( )104.6(mM im)4.5(ida)5.2(z)2.4(ol)-7.3(e b)10(u)-6.2(f)7.7(f)1.9(er.)5.5( )5.8(T)-8.2(h)1.6(e )]TJ T* 0.0005 Tc 0.2047 Tw [(protein )-5.8(was eluted with an imid)-5.6(azole gr)]TJ 20.282 0 TD 0.0021 Tc 0.2031 Tw [(a)5.6(d)-4(i)4.8(e)1.5(nt from)5( )-5.8(20)4.5( )203.3(mM to 50)4.5(0)4.5( )197.5(m)10.8(M)-2.5( i)4.8(n)2.1( )-5.8(20)4.5( )]TJ -20.282 -1.8412 TD 0.0001 Tc 0.269 Tw [(minu)-7.7(tes. The f)6.2(l)-3(ow r)-7(a)3.7(t)-9.5(e)-0.5( wa)9.5(s )-5.8(1 )261.4(ml/min )-5.8(fo)]TJ 20.0033 0 TD 0.0009 Tc 0.2682 Tw [(r )-5.8(a)10.3(ll wash)-4.9( )-5.8(a)10.3(n)-10.7(d)6.4( elut)-8.7(ion)-4.9( steps)5.7(.)4.8( )-5.8(Pr)-6.2(otein )]TJ -20.0033 -1.8412 TD 0.004 Tc 0.1431 Tw [(c)6.3(o)2.5(nta)7.6(i)6.7(ni)6.7(n)4(g)-2.7( )11.6(f)4.3(r)2.8(ac)6.3(ti)6.7(on)4(s)3( w)4.5(e)3.4(r)8.6(e)3.4( i)6.7(d)3.7(e)3.4(n)-1.8(ti)6.7(f)10.1(i)6.7(ed)3.7( )5.8(by)5.8( )11.6(SDS )11.6(PA)6.1(G)4.8(E)5.8( a)13.4(n)-7.6(d)9.5( p)12.4(ooled)3.7(. )5.8(T)5.8(o)2.5( )5.8(c)6.3(l)0.9(ea)13.4(ve)3.4( th)4(e)3.4( )]TJ T* -0.009 Tc 0 Tw [(\(H)-17.1(i)-12.1(s)-10(\))]TJ 6.6044 0 0 6.6044 131.28 194.7001 Tm 0 Tc (6 )Tj 10.3303 0 0 10.3303 137.22 195.1801 Tm 0.0013 Tc 0.0471 Tw [(t)-8.3(a)4.9(g)-5.4(,)11( 5 )46.5(mg Ni-NTA )5.8(pur)-5.8(i)4(fi)9.8(e)-5.1(d)1( )5.8(MLC)-4.7(K)5.5(-)-9.4(A)3.4( pr)5.9(ot)-8.3(ei)9.8(n)-4.5( )5.8(was dialy)8.9(z)-3.7(ed )-5.8(i)9.8(n)-4.5(to )5.8(the cle)-5.1(a)4.9(v)-7.9(a)-1(g)6.2(e)-5.1( )]TJ -2.5846 -1.847 TD 0.0028 Tc 0.1792 Tw [(b)5.4(u)0.9(ffe)8(r)1.6( wi)5.5(th f)8.9(r)1.6(eshly)10.4( )-5.8(p)5.4(r)1.6(ep)5.4(a)6.4(r)-4.3(e)8(d)2.5( 8)5.2( )174.3(U)7.1(/)-1.6(m)5.7(l)-0.3( thr)7.4(o)1.3(m)5.7(b)-0.4(in.)6.7( F)5.6(o)1.3(r)7.4( )-5.8(the c)5.1(l)-0.3(ea)6.4(va)6.4(ge reac)5.1(ti)5.5(on,)6.7( the )]TJ T* -0.0011 Tc 0.0553 Tw [(prot)-4.9(ein w)-6.4(a)-9.2(s incu)-14.7(bat)-4.9(e)-1.7(d )-5.8(at)-4.9( 3)-4.5(7)1.3(C )-5.8(for 50)-4.5( )-11.6(mi)7.4(n)-6.9(u)-3(t)-4.9(e)-1.7(s )-5.8(an)-6.9(d )-5.8(qu)-8.9(en)-6.9(ched )-5.8(wit)-4.9(h)-6.9( 1 )52.3(m)-4(M)0.2( P)-7.5(M)-5.7(SF)-4.1(. )]TJ 0 -1.8412 TD 0.0029 Tc 0.121 Tw [(The p)11.3(r)1.7(otei)5.6(n was)7.7( loa)6.5(d)2.6(ed )5.8(onto )5.8(a)6.5( Ni)5.6(-N)5(TA)5( c)5.2(o)1.4(lum)5.8(n)2.9( tha)12.3(t)-6.7( )5.8(wa)6.5(s )5.8(eq)5.5(ui)5.6(lib)11.3(ra)6.5(ted )5.8(wi)5.6(th )5.8(the )]TJ T* 0.0027 Tc 0.0283 Tw [(c)5(l)-0.4(ea)6.3(v)-6.5(a)12.1(ge )-5.8(b)11.1(u)-5.1(ffer)7.3( to rem)5.6(o)1.2(v)5.1(e)2.1( the )11.6(cl)5.4(ea)6.3(ved)8.2( )-11.6(\()11.7(H)-5.4(i)11.2(s)-4.1(\))]TJ 6.6044 0 0 6.6044 336.6 118.5601 Tm 0 Tc 0 Tw (6)Tj 10.3303 0 0 10.3303 340.26 118.98 Tm 0.0022 Tc 0.023 Tw [( )-5.8(tag.)6.1( The )-5.8(i)4.9(m)-0.7(m)10.9(e)-4.2(dia)11.6(t)-7.4(e flow t)-7.4(h)2.2(ro)6.5(ugh)8( )]TJ -22.2394 -1.8412 TD 0.0005 Tc 0.135 Tw [(con)-5.3(t)-3.3(ain)-5.3(e)-0.1(d)6( )-5.8(the clea)9.9(ve)-5.9(d )5.8(MLC)-5.5(K)4.7(-)-10.2(A)2.6( prot)-9.1(ein)-5.3(.)4.4( 50% gl)-8.4(y)8.1(c)2.8(erol was ad)6(de)-5.9(d)6( to the f)6.6(l)-2.6(ow )]TJ ET endstream endobj 49 0 obj << /ProcSet [/PDF /Text ] /Font << /TT2 4 0 R /TT4 5 0 R /TT6 6 0 R /TT8 7 0 R /TT11 19 0 R >> /ExtGState << /GS1 8 0 R >> /ColorSpace << /Cs6 9 0 R >> >> endobj 51 0 obj << /Length 4396 >> stream BT /TT2 1 Tf 11.2898 0 0 11.2898 110.52 36.54 Tm /Cs6 cs 0 0 0 scn /GS1 gs 0 Tc 0 Tw ( )Tj /TT4 1 Tf 10.3303 0 0 10.3303 489.96 36.48 Tm -0.0024 Tc (79)Tj -36.7307 64.7144 TD 0.0004 Tc 0.2977 Tw [(t)-9.2(h)0.4(rough)6.2( for stora)9.8(g)-0.5(e p)8.8(u)-7.4(r)-6.7(p)8.8(oses an)-5.4(d the ali)8.9(q)3(u)-7.4(o)4.7(t)-9.2(s)5.2( were stor)5(ed at)-9.2( )5.8(-80C. )-5.9(A)8.3(f)0.7(ter )]TJ 0 -1.8412 TD -0.0023 Tc 0.0042 Tw [(t)-11.9(h)-2.3(awin)-8.1(g an)-8.1( al)-5.4(i)-5.4(q)6.1(u)-10.1(o)-3.8(t)-11.9(,)7.4( t)-6.1(h)-2.3(e MLCK-)-13(A)-0.2( )-5.8(pro)-3.8(t)-6.1(ein)-8.1( wa)]TJ 20.9848 0 TD 0.0013 Tc 0.0006 Tw [(s stored)6.8( in -20)6.2(C f)7.4(o)-0.2(r subs)6.1(e)-5.1(q)3.9(uent use. )]TJ /TT6 1 Tf -20.9848 -1.8412 TD 0.0017 Tc -0.0027 Tw [(Lysis Bu)9.4(ffer )]TJ /TT4 1 Tf T* -0.0016 Tc 0.0093 Tw [(20 mM )5.8(T)-5.6(r)-2.8(is pH 7.9 )]TJ 0 -1.847 TD -0.0005 Tc 0.0024 Tw [(5 m)-3.4(M)0.8( i)-3.6(m)-3.4(idazol)-3.6(e )]TJ 0 -1.8412 TD -0.0013 Tc 0.009 Tw [(50)-4.7(0 mM )5.8(N)-5(a)-9.4(Cl)-4.4( )]TJ T* 0 Tc 0 Tw ( )Tj /TT6 1 Tf T* 0.0007 Tc 0.0041 Tw [(Clea)-7.1(vage B)5.8(u)-3.2(ffer)6.9( )]TJ /TT4 1 Tf T* -0.0016 Tc 0.0093 Tw [(20 mM )5.8(T)-5.6(r)-2.8(is pH 8.4 )]TJ T* 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403.5 453.54 Tm -0.0025 Tc 0.002 Tw [( l)-7.8(e)-4.5(adi)-7.8(n)-2(g to)-6.7( a )-7.1(d)-4.8(e)-4.5(cre)-11.6(a)0.4(se )-7.1(of)-7.4( )]TJ -34.6005 -1.8423 TD -0.0041 Tc -0.0035 Tw [(th)-3.6(e)-6.1( )-14.2(abs)-5.6(o)-1.2(r)-8(p)-9.3(ti)-9.4(on)-3.6( a)-8.3(t)-5.4( )-7.1(340)-9.7( n)-10.7(m)-8.5(.)]TJ /TT6 1 Tf 12.4004 0 TD 0 Tc 0 Tw ( )Tj /TT4 1 Tf -12.4004 -2.5084 TD ( )Tj 10.3303 0 0 10.3303 110.52 399.3 Tm 0.0029 Tc 0.2604 Tw [(The )5.8(out)-6.7(p)11.3(ut)4.9( )5.8(va)6.5(lue )5.8(of the)8.1( a)6.5(b)5.5(sorpti)5.6(on )-5.8(\()11.9(A)-0.8(\))6.1( wa)12.3(s i)5.6(n)2.9( [mO)6.6(D)3.6(/)4.3(m)0(i)5.6(n)-2.9(] )5.8(whic)5.2(h wa)6.5(s )5.8(then )]TJ 0 -1.9341 TD -0.001 Tc 0.2004 Tw [(con)-6.8(v)-4.4(ert)-10.6(e)4.2(d t)-4.8(o)-2.5( [OD/s])-10(. Th)-6.8(e )5.8(ext)-4.8(i)1.7(n)-6.8(c)1.3(tion coe)-7.4(f)5.1(f)-6.5(i)-4.1(ci)-4.1(ent)-4.8( of the N)-4.7(A)1.1(D)-6.1(H)2.6( )-5.8(\()]TJ /TT3 1 Tf 11.2898 0 0 11.2898 433.74 379.32 Tm 0 Tc 0 Tw <01c6>Tj /TT4 1 Tf 6.6044 0 0 6.6044 438.54 378.9 Tm 0.0037 Tc (NADH)Tj 10.3303 0 0 10.3303 457.8 379.32 Tm -0.009 Tc 0.2142 Tw [(\) a)-5.4(n)-9(d)-9.3( )5.8(t)-12.8(h)-14.8(e)-15.4( )]TJ -33.6175 -1.9225 TD 0.0012 Tc 0.0298 Tw [(diameter )-11.6(\()10.2(d)0.9(\))10.2( o)-6.1(f)1.5( )5.8(th)-4.6(e )5.8(we)6.4(ll a)10.6(llowed th)-4.6(e )5.8(determin)-4.6(ation of)7.3( th)-4.6(e )5.8(NADH con)-4.6(c)3.5(entration )]TJ 0 -1.8412 TD 0.0032 Tc -0.0013 Tw [(i)5.9(n)-2.6( the a)6.8(c)5.5(ti)5.9(n ac)5.5(ti)5.9(va)6.8(ted)8.7( )-5.8(A)5.3(T)-0.8(Pase )-5.8(a)12.6(s)2.2(sa)6.8(y:)7.1( )]TJ T* 0 Tc 0 Tw ( )Tj /TT3 1 Tf 11.2898 0 0 11.2898 110.52 301.44 Tm <01c6>Tj /TT4 1 Tf 6.6044 0 0 6.6044 115.26 301.02 Tm -0.0016 Tc [(NA)-15.9(DH)]TJ 10.3303 0 0 10.3303 134.52 301.44 Tm -0.0028 Tc 0.0047 Tw [(=)-6.7( 6.22)]TJ 6.6044 0 0 6.6044 163.62 304.26 Tm 0.0091 Tc -0.0043 Tw (3 *)Tj 10.3303 0 0 10.3303 171.9 301.44 Tm 0 Tc 0 Tw (M)Tj 6.6044 0 0 6.6044 181.38 305.22 Tm 0.0041 Tc (-1)Tj 10.3303 0 0 10.3303 187.26 301.44 Tm 0.0035 Tc (cm)Tj 6.6044 0 0 6.6044 203.64 305.22 Tm -0.005 Tc (-1)Tj 10.3303 0 0 10.3303 209.52 301.44 Tm 0 Tc ( )Tj -9.5834 -1.9167 TD 0.0026 Tc -0.0007 Tw [(d=)4.5( 0.)6.5(312)5(5)5( )-5.8(cm)5.5( )]TJ 0 -1.9341 TD 0 Tc 0 Tw (c)Tj 6.6044 0 0 6.6044 117.18 261.1801 Tm -0.0016 Tc [(NA)-15.9(DH)]TJ 10.3303 0 0 10.3303 136.44 261.66 Tm 0.0039 Tc -0.002 Tw [(= A)11.8(/)]TJ 11.2898 0 0 11.2898 157.68 261.66 Tm 0 Tc 0 Tw ( )Tj /TT3 1 Tf 0.2764 0 TD <01c6>Tj /TT4 1 Tf 6.6044 0 0 6.6044 165.6 261.1801 Tm -0.0021 Tc (NADH )Tj 10.3303 0 0 10.3303 186.6 261.66 Tm -0.0003 Tc 0.0022 Tw (* d )Tj -7.3647 -1.9167 TD 0 Tc 0 Tw ( )Tj 0 -1.847 TD 0.0007 Tc 0.1406 Tw [(The value of)6.8( c)]TJ 6.6044 0 0 6.6044 185.46 222.3 Tm -0.0061 Tc 0 Tw [(NA)-11.3(DH)]TJ 10.3303 0 0 10.3303 204.66 222.78 Tm 0.0018 Tc 0.1395 Tw [( )5.8(c)4.1(o)0.3(rresp)4.4(onds )5.8(to t)3.8(h)1.8(e vel)4.5(o)0.3(c)4.1(i)10.3(t)-13.6(y)9.4( )-5.8(\()16.6(v)-7.4(\))5( of the a)5.4(c)4.1(ti)4.5(n )-5.8(a)5.4(c)4.1(ti)4.5(va)5.4(ted A)3.9(T)-8(P )]TJ -9.113 -1.8412 TD 0.0005 Tc 0.0014 Tw [(h)-5.3(y)2.3(d)6(r)-0.7(ol)-8.4(y)8.1(s)-6.3(is by)8.1( )-5.8(a g)-6.2(i)9(ven)-5.3( concent)-9.1(r)5.1(ation of act)-9.1(i)3.2(n \()9.5(A)-3.2(\). )]TJ 0 -1.847 TD 0 Tc 0 Tw ( )Tj /TT6 1 Tf 0 -1.8412 TD -0.0001 Tc -0.0009 Tw [(Final assay )-5.8(co)-4.7(nce)-4.7(n)7.6(t)-8.1(r)0.3(ati)-4.3(o)-4.7(n)7.6(s)-1.6( )-5.8(of t)-8.1(h)1.8(e)-4.7( AT)-4.2(P)-3.4( rege)-4.7(ne)-4.7(rati)-4.3(ng syste)-4.7(m)4.7( )]TJ /TT4 1 Tf T* 0.0035 Tc -0.0016 Tw [([)6.1(P)2.9(K] 0.)7.4(12)5.9( )-5.8(mg)8.4(/m)12.2(l )]TJ T* -0.0025 Tc 0.0044 Tw [([L)-5.2(D])-11.5( 0)-5.9(.)-4.4(02 )-5.8(mg/ml)-5.6( )]TJ 0 -1.847 TD -0.001 Tc 0.0029 Tw [([PE)-5(P)-1.6(])-4.2( 3 m)-3.9(M)-5.5( )]TJ 0 -1.8412 TD -0.0018 Tc 0.0037 Tw [([N)-5.5(AD)-6.9(H])-5( 0)-5.2(.)2.1(3 )-5.8(mM )]TJ T* 0 Tc 0 Tw ( )Tj ET endstream endobj 61 0 obj << /ProcSet [/PDF /Text ] /Font << /TT2 4 0 R /TT3 62 0 R /TT4 5 0 R /TT6 6 0 R >> /ExtGState << /GS1 8 0 R >> /ColorSpace << /Cs6 9 0 R >> >> endobj 64 0 obj << /Length 7316 >> stream BT /TT2 1 Tf 11.2898 0 0 11.2898 110.52 36.54 Tm /Cs6 cs 0 0 0 scn /GS1 gs 0 Tc 0 Tw ( )Tj /TT4 1 Tf 10.3303 0 0 10.3303 489.96 36.48 Tm -0.0024 Tc (83)Tj /TT6 1 Tf -36.7307 64.7144 TD 0.0031 Tc -0.0041 Tw [(A)5.2(T)-1(P)5.6(a)1.1(se Bu)5(ffe)4.3(r)9.3( )]TJ /TT4 1 Tf 0 -1.8412 TD -0.002 Tc 0.0097 Tw [(35 mM M)-6.6(O)-4.1(PS pH )5.8(7)-5.4(.)-3.9(4 )]TJ T* -0.0008 Tc 0.0085 Tw [(0.1 mM )5.8(M)-5.3(g)-7.5(Cl)]TJ 6.6044 0 0 6.6044 176.82 666.48 Tm 0 Tc 0 Tw (2)Tj 10.3303 0 0 10.3303 180.48 666.9601 Tm ( )Tj -6.7723 -1.8412 TD -0.001 Tc 0.0087 Tw [(25 mM )5.8(KCl)-9.9( )]TJ 0 -1.847 TD 0.003 Tw [(2 m)-4(M)0.2( DTT)-5.1( )]TJ /TT6 1 Tf 0 -1.8412 TD 0 Tc 0 Tw ( )Tj /TT8 1 Tf 13.1527 0 0 13.1527 110.52 576.72 Tm 0.0011 Tc 0.0007 Tw [(Pr)6.2(obi)-5.5(n)3.6(g RLC and HC interactio)]TJ 14.5841 0 TD 0.0005 Tc -0.002 Tw [(n vi)-6.1(a Cysteine Footpri)-6.1(n)7.6(ting )]TJ /TT6 1 Tf 12.2496 0 0 12.2496 110.52 542.22 Tm -0.0003 Tc -0.0003 Tw [(Cysteine F)4.6(ootprinting )]TJ 10.4477 0 TD -0.0019 Tc 0.0029 Tw [(via NT)-3.2(CB Labe)-3.4(ling )]TJ /TT4 1 Tf 10.3303 0 0 10.3303 110.52 518.7 Tm 0.0018 Tc 0.0176 Tw [(The f)7.9(o)0.3(llowing p)10.2(r)0.6(otoco)-5.5(l)-1.3( ca)11.2(n be sca)5.4(l)-1.3(ed)7.3( pr)]TJ 19.4631 0 TD 0.0055 Tc 0.0139 Tw [(o)4(p)8.1(or)4.3(ti)8.2(o)4(n)5.5(a)9.1(lly)7.3( )5.8(d)5.2(e)-0.9(p)8.1(e)4.9(nd)11(i)8.2(n)-0.3(g)10.4( on)5.5( the)10.7( a)9.1(m)8.4(ou)3.6(n)5.5(t)7.5( )]TJ -19.4631 -1.847 TD 0.0008 Tc 0.0883 Tw [(of the protein. To )-5.8(a)10.2(l)-8.1(kyla)10.2(te t)-8.8(h)0.8(e solvent expos)5.6(e)0.2(d sulfur r)5.4(e)-5.6(sidues )-5.8(in th)-5(e myos)5.6(in)-5( )-5.8(S1)-8.4(, )]TJ 0 -1.8412 TD 0.0009 Tc 0.4308 Tw [(2 )429.8(l of)7( )-5.8(s)5.7(o)-0.6(lu)-6.9(tion )429.8(I)6.3( )5.8(was )-5.8(a)10.3(d)-5.2(d)6.4(e)-5.5(d )5.8(t)-8.7(o)5.2( 18 )429.8(l )-5.8(\()15.7(1)-8.3(8 )429.8(M\) pr)5.5(ot)-8.7(ei)9.4(n a)4.5(n)-4.9(d incu)-6.9(ba)4.5(ted )5.8(fo)5.2(r)-6.1( )]TJ T* 0 Tc 0.1123 Tw [(5 )110.4(minu)-7.8(tes a)9.4(t)-9.6( )5.8(roo)-7.3(m)2.9( )5.8(t)-9.6(e)-6.4(mper)-7(a)9.4(t)-9.6(ure. The re)-6.4(act)-9.6(i)2.7(on)-5.8( was )-5.8(q)8.4(u)-13.6(enched)-6.1( )-5.8(by )-5.8(pip)8.4(e)-0.6(t)-9.6(t)-3.8(ing the )]TJ T* 0.0009 Tc 0.0243 Tw [(rea)4.5(c)3.2(tion )-5.8(mix into 20 )23.3(l of )5.8(solut)-8.7(i)9.4(on )23.3(I)6.3(I)0.5(.)4.8( 40 )]TJ 18.5454 0 TD -0.0015 Tc 0.0267 Tw [(l)-4.6( )-5.8(\()13.3(e)-7.9(q)-4.7(u)-3.4(al)-4.6( v)-4.9(o)2.8(l)-4.6(u)-9.3(me o)-3(f)-1.2( t)-5.3(h)-1.5(e react)-5.3(i)1.2(o)-3(n)-7.3( )5.8(m)-4.4(i)1.2(x)-3.6(t)-5.3(u)-3.4(r)3.1(e)-13.7(\))1.7( )]TJ -18.5454 -1.8412 TD 0.0022 Tc 0.1798 Tw [(of sol)4.9(u)-5.6(ti)4.9(on )5.8(II)7.6(I )-5.8(wa)5.8(s)7( )-5.8(added to la)5.8(b)4.8(e)-4.2(l the dena)5.8(tur)6.8(e)1.6(d,)17.7( ther)6.8(efore s)7(o)0.7(lvent)-7.4( ex)5.9(p)4.8(o)0.7(sed,)6.1( )]TJ T* 0.0003 Tc 0.3908 Tw [(sulfur re)5.5(sid)-5.8(u)-1.6(es with )5.8(NTCB. The mi)8.8(xt)]TJ 18.0866 0 TD 0.0005 Tc 0.3906 Tw [(u)-7.3(r)-0.7(e )5.8(was )5.8(incu)-7.3(ba)9.9(t)-9.1(e)-0.1(d)6( 5 )-5.8(minutes at r)5.1(o)4.8(om )]TJ -18.0866 -1.847 TD -0.001 Tc 0.1075 Tw [(t)-10.6(e)-1.6(m)7.7(p)1.6(e)-7.4(r)-8.1(a)8.4(t)-10.6(ure. 4)-4.4(00 )104.6(l)-4.1( of)-6.5( 0.05)-4.4(% N)-4.7(a)-3.3(D)-6.1(O)8.5(C)-7( )-5.8(\()8(5)1.4(X v)-10.2(o)-2.5(lu)-8.8(me )5.8(of th)-6.8(e )5.8(react)-4.8(i)1.7(o)-8.3(n)-1( mixt)-10.6(ure)-7.4(\))8( w)-6.3(a)8.4(s )]TJ 0 -1.8412 TD 0.276 Tw [(add)-7.2(e)-1.7(d )5.8(t)-4.9(o)-2.6( t)-4.9(h)-6.9(e m)7.6(i)1.6(xt)-4.9(u)-8.9(r)-2.3(e, fol)-4.2(l)-4.2(o)-2.6(wed b)-4.3(y)6.5( 48 )273(l)-10( )5.8(of 50)-4.5(%)7.1( T)-5.1(C)-7.1(A)6.8( )-5.8(\()13.7(1)-4.5(/)-5.5(1)1.3(0)]TJ 6.6044 0 0 6.6044 422.46 369.24 Tm -0.0027 Tc 0 Tw [(th)-10.6( )]TJ 10.3303 0 0 10.3303 433.38 366.42 Tm 0.0021 Tc 0.267 Tw [(volum)5(e)1.5( of)8.2( )-5.8(the)7.3( )]TJ -31.2536 -1.8412 TD -0.0013 Tc 0.0149 Tw [(re)-7.7(act)-5.1(i)1.4(on m)-4.2(i)-4.4(xt)-10.9(ure\))7.7(. 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