Protein FT-IR Spectrum -Two standards EPAC +D exchar ge(with 30) uM CAMF No absorbance at 2000-1750cm-1 H/D exchange Amide I/ Amide‖ Wavenumber(cm)
• No absorbance at 2000-1750 cm-1 • H/D exchange: Amide I / Amide II Protein FT-IR Spectrum —Two standards EPAC H-D exchange (with 300 uM cAMP) Wavenumber (cm-1) 130014001500160017001800 Absorbance 0.00 .05 .10 .15 .20 H2O 1' 3' 5' 10' 30' 60' 120' 180' D2O
FT-IR spectra of PK Subtraction of h o will bring big error in absorbance spectrum 75017016501600155015001450140013501300 Wavenumber(cm Small error in second derivative spectrum
FT-IR spectra of PK • Subtraction of H2O will bring big error in absorbance spectrum • Small error in second derivative spectrum Wavenumber (cm-1) 1300135014001450150015501600165017001750 Absorbance Wavenumber (cm-1) 15801600162016401660168017001720 Absorbance/Wavenumber2 PK PK - negative error PK - positive error PK spectra
H/D exchange 2000190018001700160015001400 T 1700 1680 1660 1640 162016 Wavenumber(cm")
1400150016001700180019002000 Absorbance Wavenumber (cm-1) 160016201640166016801700 d 2A/d n 2 H/D exchange
H/D exchange 25 20 15 10 0.00 020406080100120140160180200 EXchanged Time(min)
Exchanged Time (min) 0 20 40 60 80 100 120 140 160 180 200 Ratio of unexchanged proton 0.00 .05 .10 .15 .20 .25 .30 H/D exchange
FT-IR spectra -in the presence of denaturant Have to get good absorbance spectrum first CRP+0.8 M GdnHCI Be careful, when sample in 0. 8 M GdnHCl B the presence of GdnHCl CRP(10×) C 2000190018001700160015001400130 Wavenumber(cm)
Wavenumber (cm-1) 13001400150016001700180019002000 Absorbance 1674 CRP+0.8 M GdnHCl 0.8 M GdnHCl CRP (10x) A B C FT-IR spectra —in the presence of denaturant • Have to get good absorbance spectrum first • Be careful, when sample in the presence of GdnHCl !