studying interactions between protein, small molecules and...

Studying Interactions between Protein, Small Molecules and Water by DSC

Recent DSC work – interaction of small molecules with proteins

Differential scanning calorimetry scans of lysozyme with varying

sodium phytate concentrations in 10mM NaCl, pH 7.0.

2

The Effect of the Anions Phosphate, Sulphate and Chloride on 1 mg/ml Lysozyme

pH 7

Binding of anions to proteins effect on stability does not follow the

Hofmeister series

(mM)

At higher anion concentrations there are two distinct phases of stabilisation

4

Sodium Chloride

5

At higher anion concentrations there are two distinct phases of stabilisation

Disodium Sulphate

6

At higher anion concentrations there are two distinct phases of stabilisation

Disodium Hydrogen Phosphate

7

Sodium Iodide

Sodium Perchlorate

Low charge density ions behave like detergents

Current theory of stabilisation

+ve

Anionic

molecule -

High charge density ions bind to the

protein’s basic side chains by

electrostatic interaction. Alteration of

the charge results in a change in force

applied to the peptide backbone.

Mechanism 1. Strong direct interaction of anions with opposite

charged side chains

8

Current theory of stabilisation

Anionic

molecule -

Anions interact with the protein’s

backbone by electrostatic

interaction – has a weak effect on

stability

Mechanism 2. Weak direct interaction of anions with partially

charged peptide backbone

9

10

Current theory of stabilisation

Mechanism 3. Competition for water

between the solvation layers of the

protein and the ions

Mechanism 4. Wetting agent or detergent-like mechanism

Low charge density ions reduce the

free energy required to wet the

newly exposed hydrophobic interior

of the protein.

ΔΔGsolv layer

+ve

Chaotropic anions or

charged organic molecule

11

Current theory of stabilisation

Sodium Iodide

Sodium Perchlorate

Reality – multiple mechanisms may occur concurrently

• Direct strong interaction

• Competition for water

+ve

Phosphate

12

Current theory of stabilisation

13

Arginine – a widely used excipient in

bio-pharmaceutical formulations

14

Glycine

Guanidinium HCl

Arginine – a widely used excipient in

bio-pharmaceutical formulations

15

Arginine – a widely used excipient in

bio-pharmaceutical formulations

Arginine

16

Arginine – a widely used excipient in

bio-pharmaceutical formulations

Glycine

17

Arginine – a widely used excipient in

bio-pharmaceutical formulations

Guanidinium HCl

18

Arginine – a widely used excipient in

bio-pharmaceutical formulations

Glycine

Guanidinium HCl

Arginine

19

Glycine

Guanidinium HCl

Arginine – an excipient that could have

been reverse engineered

Mechanism 2 & 3

Mechanism 4

Goals

• Better understand the role of small molecules in protein stability

• Challenge existing theories on the interaction between water and

cosolutes (small inorganic, organic and macromolecules)