Determination of kinetic and thermodynamic protein stability

1

• Determination of kinetic and thermodynamic
  protein stability
• Direct measurement
• labeling
• crosslinking
• Spectra
• Competition binding

2

• Qualitative detection of binding labelling
• J.S. Holcenberg, Biochemistry, 7 Feb 1978, 17(3),
  411-417

1. reaction
OH


2. Reduction with NaBH4
3. Proteolytic digest
6-diazo-5-oxo-norleucine
A conserved threonine is modified (Thr12 in
Acinetobacter glutaminase-asparaginase, Thr20 in
Pseudomonas 7A glutaminase-asparaginase)
3

• Qualitative detection of binding labelling
• K. Aghaiypour, Biochim. Biophys. Acta, 17 Dec
  2001, 1550(2), 117-128
• Crystal structure of the modified enzyme (without
  reduction).

A threonine and a tyrosine react with the
diazonorleucine.
4

• Qualitative detection of binding crosslinking
• Protein chains can be connected (crosslinked)
  with various bifunctional reagents, most common
  glutar(di)aldehyde.

With increasing reaction time with glutaraldehyde
(ca. 0.1), the dimer of XS1S2 gets covalently
linked
A. Kuusinen, J. Biol. Chem., 8 Oct 1999, 274(41),
2893728943
5

• Direct measurement of the equilibrium in a
  dialysis experiment.
• In the equilibrium
• Protein Ligand ProteinLigand
• all three concentrations must be known to
  calculate the
• Equilibrium constant K ProteinLigand/(Protei
  nLigand)

6

• Equilibrium dialysis

P free protein PL protein-ligand complex L
free ligand
P
L
PL
L
L
L
P
PL
L
L
L
L
dialysis membrane

• P PL is usually known (the amount of added
  protein in the beginning).
• L and L PL are measured
• Radioactivity of labeled compounds
• Metal content by AAS
• HPLC (denature PL and set L free before the
  measurement)
• UV/Vis or fluorescence spectra of L and PL

7

• Measurement of free and complexed protein
• Some measurable parameter must be altered when
  the ligand binds so that free and complexed
  protein can be distinguished.
• Example binding of iodide to GFP (especially
  Thr203Tyr mutants).
• Measurable parameter fluorescence
• Model

GFP (fluorescent) I- GFP-I-
(nonfluorescent)
8

• Binding of iodide to GFP
• R.M. Wachter, J. Mol. Biol., 2000, 301, 157-171

Apparent dissociation constants Kapp(pH 8.0)
431 mM Kapp(pH 6.0) 22 mM At K, 50 of the
GFP molecules seem to be complexed with I- (and
nonfluorescent).
9

• Binding of iodide to GFP
• Iodide binds close to the chromophore. This
  encourages protonation of the chromophore,
  because two close negative charges (I- and
  deprotonated chromophore) are unfavorable. The
  protonated chromophore does not fluoresce.

4.4
10

• Binding of iodide to GFP
• The close interaction with the chromophore is
  also the reason that binding seems pH dependent

Binding of iodide depends on the protonation of
the chromophore there are two different binding
constants for iodide (K1 and K2).
11

• Binding of calcium to a GFP-calmodulin-GFP fusion
  protein
• R. Heim, Current Biol., 1996, 6(2), 178-182
• FRET (fluorescence resonance energy transfer) If
  two fluorescent molecules are close (lt10 nm)
  emission from one molecule can directly excite
  the second molecule.

BFP
Excitation 381 nm, Emission 445 nm (blue)
Excitation 470 nm, Emission 508 nm (green)
GFP
Excitation 381 nm, Emission 508 nm (green)
BFP
GFP
12

• Binding of calcium to a GFP-calmodulin-GFP fusion
  protein
• A. Myawaki, Nature, 1997, 388(6645), 882-887

13

• Binding of calcium to a GFP-calmodulin-GFP fusion
  protein

different mutants
14

• Binding of calcium to a GFP-calmodulin-GFP fusion
  protein

15

• Design of metal binding sites
• T.A. Richmond, Biochem. Biophys. Res. Commun.,
  2000, 268(2), 462-465
• Metal ions have characteristic coordination
  spheres. Mutations in in silico allow to design
  proteins with binding sites for specific metals.
• Transition metal binding sites have been
  engineered into GFP by inserting several
  mutations. They lead to metal ion quenching at
  lower concentration than with wild type.