119.0 1:0 -0.05 ppm 119.5 1:0.2 120.0 1:0.5 1:1.0 120.5 121.0 12 10.2 10.0 9.8 9.6 94 Ko-250 nM Mapping Binding Contacts
10.2 10.0 9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 119.0 119.5 120.0 120.5 121.0 121.5 1:1.0 1:0.5 1:0.2 1:0 ~0.05 ppm KD ~ 250 nM Mapping Binding Contacts
Chemical Shift Perturbation Mapping Slow or fast exchange A=X6NHP+(615N2 x=4,10....or some other number Mott et al (2003)J.Biol Chem.27817053-17 Binding a(larger)protein ligand to a labelled protein gCD2(10 kDa)titrated with CD48(540 kDa-glycosylated A 1:0 B 1:02 11 s52 0s52 S52 0 . o. 117- 90 o moar ratio of D4 McAlister et al(1996)Biochemistry 355982-5991
1:0 1:0.5 1:1.0 Chemical Shift Perturbation Mapping Slow or fast exchange # = [x($NH)2 + ($15N)2] 1/2 x = 4, 10 ….or some other number 40 50 60 70 80 90 100 0 1 2 3 4 5 0 10 20 30 Mott et al (2003) J. Biol Chem. 278 17053-17059 Binding a (larger) protein ligand to a labelled protein Overall broadening due to increase in %c. Selective broadening of some residues e.g. CD2 (10 kDa) titrated with CD48 (~35-40 kDa - glycosylated) McAlister et al (1996) Biochemistry 35 5982-5991 Fractional reduction in peak height #h = (hi -hx)/(hi -hf ) plotted against molar ratio of CD48 1:0 1:0.2 1:1
Comparison between intensity loss and chemical shift changes A Loss of intensity in strands C.C'correlates with shift changes 86epes
Comparison between intensity loss and chemical shift changes Loss of intensity in strands C, C’ correlates with shift changes Free Complex
Hydrogen Exchange (high affinity binding) Patterson et al(1990)Science 249755-759 ncubate inD hr to days HN ChetehgRanneastyangeaerncaiaionntepmesenek小adahenceK all residues-there should be a sharp distinction between protected and Assumes that there are no co-operative unfolding events as a result of binding the other molecule L64 …free form bound form Protection factors (k/ 8湖 598 02 Protection factors3 30 nge time (h ours)
Hydrogen Exchange (high affinity binding) Patterson et al (1990) Science 249 755-759 Incubate in D2O 1hr to days HN HN HN HN O DN HN DN/HN HN O Dissociate at low pH (H/D exchange is slow) and isolate labelled protein DN HN DN/HN HN O HSQC to identify remaining NHs Calculate the rate of intensity change after incubation in the presence (kbound) and absence (kfree) of the binding partner Calculate kfree/kbound for all residues - there should be a sharp distinction between protected and unprotected residues Assumes that there are no co-operative unfolding events as a result of binding the other molecule Exchange time (hours) free form bound form Protection factors (kfree/kbound): K60 >60 L64 >50 E66 8 D93 ~0 Residues divided into 2 groups: Protection factor ! 7 Protection factor " 3
Cross-saturation Takahashi et al(2000)Nat.Struc.Biol.7220-223 Protein Proteir Strong binding case 1 NH -CH cross-saturation etoe2laosmcean子ame23taionsapdyansendbyspndmsonoe Cross-saturation to theNHin the Measure peak vstime of saturation tofind residues in more quickly. 言06 N9 04 Chemical shift Cross-saturatior H0215AN 0.2 ppm
Cross-saturation Takahashi et al (2000) Nat. Struc. Biol. 7 220-223 -NH -CH -15NH -C2H Protein 2 Protein 1 R.F. Band-selective proton saturation, followed by TROSYHSQC Saturate the aliphatics of protein 2 - magnetization is rapidly transferred by spin diffusion to the rest of protein 2 i.e. aromatics and amides. Cross-saturation to the 15NH in the interface on protein 1 Measure peak intensity vs time of saturation to find residues in interface Strong binding case cross-saturation Plot intensity ratio vs saturation time. Residues in the interface get saturated more quickly. X-ray structure - buried residues Chemical shift perturbation (#HN2 + 0.15#N2)1/2 > 0.2 ppm H/D exchange protection factor > 10 Cross-saturation (red> light blue > yellow > green > dark blue)