35. We have obtained very similar results using Microcal Origin for
ITC, Affinimeter, and Sedphat. Affinimeter or Sedphat offers a
variety of additional possibilities for global data analysis and
modeling. For this chapter, we have used Sedphat in order to
perform error surface projection calculations for direct comparison with the fits for other methods.
36. The SPR experiments were more recently developed and therefore have been subject to relatively little repetition compared to
the other experiments. The protocol has been reproduced in
another lab with essentially identical results, but we have
observed considerable variability in the number of response
units of HEWL immobilized (and hence in signal change in
the final titration). We suspect that this is correlated with the
age and storage of the coupling reagents and would therefore
recommend starting with fresh reagents or reagents that have
been stored in aliquots at À80
C.
References
1. Jolle `s P, Jolle `s J (1984) What’s new in lysozyme
research. Mol Cell Biochem 63:165–189
2. Blake CCF, Koenig DF, Mair GA, North ACT,
Phillips DC, Sarma VR (1965) Structure of hen
egg-white lysozyme: a three-dimensional Fourier synthesis at 2 A ˚ resolution. Nature 206:757
3. Cheetham JC, Artymiuk PJ, Phillips DC
(1992) Refinement of an enzyme complex
with inhibitor bound at partial occupancy:
hen egg-white lysozyme and tri-N-acetylchitotriose at 1.75 A ˚ resolution. J Mol Biol
224:613–628
4. Moorman VR, Valentine KG, Wand AJ (2012)
The dynamical response of hen egg white lysozyme to the binding of a carbohydrate ligand.
Protein Sci 21:1066–1073
5. Lumb KJ, Cheetham JC, Dobson CM (1994)
1H nuclear magnetic resonance studies of hen
lysozyme-n-acetylglucosamine oligosaccharide
complexes in solution: application of chemical
shifts for the comparison of conformational
changes in solution and in the crystal. J Mol
Biol 235:1072–1087
6. Garcı ´a-Herna ´ndez E, Zubillaga RA, ChavelasAdame EA, Va ´zquez-Contreras E, RojoDomı ´nguez A, Costas M (2003) Structural
energetics of protein-carbohydrate interactions: insights derived from the study of lysozyme binding to its natural saccharide
inhibitors. Protein Sci 12:135–142
7. Bjurulf C, Wadso ¨ I (1972) Thermochemistry
of lysozyme-inhibitor binding. Eur J Biochem
31:95–102
8. Banerjee SK, Rupley JA (1973) Temperature
and pH dependence of the binding of oligosaccharides to lysozyme. J Biol Chem
248:2117–2124
9. Brandts JF, Lin LN (1990) Study of strong to
ultratight protein interactions using differential
scanning
calorimetry.
Biochemistry
29:6927–6940
10. Niesen FH, Berglund H, Vedadi M (2007) The
use of differential scanning fluorimetry to
detect ligand interactions that promote protein
stability. Nat Protoc 2:2212–2221
11. Holdgate GA, Ward WHJ (2005) Measurements of binding thermodynamics in drug discovery. Drug Discov Today 10:1543–1550
12. Duhr S, Braun D (2006) Why molecules move
along a temperature gradient. Proc Natl Acad
Sci 103:19678–19682
13. Seidel SAI, Dijkman PM, Lea WA, van den
Bogaart G, Jerabek-Willemsen M, Lazic A,
Joseph JS, Srinivasan P, Baaske P,
Simeonov A, Katritch I, Melo FA, Ladbury
JE, Schreiber G, Watts A, Braun D, Duhr S
(2013) Microscale thermophoresis quantifies
biomolecular interactions under previously
challenging conditions. Methods 59:301–315
14. Wiseman T, Williston S, Brandts JF, Lin LN
(1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179:131–137
15. Freire E, Mayorga OL, Straume M (1990) Isothermal titration calorimetry. Anal Chem
62:950A–959A
78
Xiaochun Li-Blatter et al.
ITC, Affinimeter, and Sedphat. Affinimeter or Sedphat offers a
variety of additional possibilities for global data analysis and
modeling. For this chapter, we have used Sedphat in order to
perform error surface projection calculations for direct comparison with the fits for other methods.
36. The SPR experiments were more recently developed and therefore have been subject to relatively little repetition compared to
the other experiments. The protocol has been reproduced in
another lab with essentially identical results, but we have
observed considerable variability in the number of response
units of HEWL immobilized (and hence in signal change in
the final titration). We suspect that this is correlated with the
age and storage of the coupling reagents and would therefore
recommend starting with fresh reagents or reagents that have
been stored in aliquots at À80
C.
References
1. Jolle `s P, Jolle `s J (1984) What’s new in lysozyme
research. Mol Cell Biochem 63:165–189
2. Blake CCF, Koenig DF, Mair GA, North ACT,
Phillips DC, Sarma VR (1965) Structure of hen
egg-white lysozyme: a three-dimensional Fourier synthesis at 2 A ˚ resolution. Nature 206:757
3. Cheetham JC, Artymiuk PJ, Phillips DC
(1992) Refinement of an enzyme complex
with inhibitor bound at partial occupancy:
hen egg-white lysozyme and tri-N-acetylchitotriose at 1.75 A ˚ resolution. J Mol Biol
224:613–628
4. Moorman VR, Valentine KG, Wand AJ (2012)
The dynamical response of hen egg white lysozyme to the binding of a carbohydrate ligand.
Protein Sci 21:1066–1073
5. Lumb KJ, Cheetham JC, Dobson CM (1994)
1H nuclear magnetic resonance studies of hen
lysozyme-n-acetylglucosamine oligosaccharide
complexes in solution: application of chemical
shifts for the comparison of conformational
changes in solution and in the crystal. J Mol
Biol 235:1072–1087
6. Garcı ´a-Herna ´ndez E, Zubillaga RA, ChavelasAdame EA, Va ´zquez-Contreras E, RojoDomı ´nguez A, Costas M (2003) Structural
energetics of protein-carbohydrate interactions: insights derived from the study of lysozyme binding to its natural saccharide
inhibitors. Protein Sci 12:135–142
7. Bjurulf C, Wadso ¨ I (1972) Thermochemistry
of lysozyme-inhibitor binding. Eur J Biochem
31:95–102
8. Banerjee SK, Rupley JA (1973) Temperature
and pH dependence of the binding of oligosaccharides to lysozyme. J Biol Chem
248:2117–2124
9. Brandts JF, Lin LN (1990) Study of strong to
ultratight protein interactions using differential
scanning
calorimetry.
Biochemistry
29:6927–6940
10. Niesen FH, Berglund H, Vedadi M (2007) The
use of differential scanning fluorimetry to
detect ligand interactions that promote protein
stability. Nat Protoc 2:2212–2221
11. Holdgate GA, Ward WHJ (2005) Measurements of binding thermodynamics in drug discovery. Drug Discov Today 10:1543–1550
12. Duhr S, Braun D (2006) Why molecules move
along a temperature gradient. Proc Natl Acad
Sci 103:19678–19682
13. Seidel SAI, Dijkman PM, Lea WA, van den
Bogaart G, Jerabek-Willemsen M, Lazic A,
Joseph JS, Srinivasan P, Baaske P,
Simeonov A, Katritch I, Melo FA, Ladbury
JE, Schreiber G, Watts A, Braun D, Duhr S
(2013) Microscale thermophoresis quantifies
biomolecular interactions under previously
challenging conditions. Methods 59:301–315
14. Wiseman T, Williston S, Brandts JF, Lin LN
(1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179:131–137
15. Freire E, Mayorga OL, Straume M (1990) Isothermal titration calorimetry. Anal Chem
62:950A–959A
78
Xiaochun Li-Blatter et al.