Physical techniques for characterisation of proteins

1. Physical techniques for characterisation of proteins Chiroptical methods

2. Historical Background • Chemists used the rotation of plane polarised light as a method for characterising saccharides • still employed in processing of sugar beet • Sucrose rotates polarised light clockwise, but a mixture of glucose and fructose rotates it anti-clockwise • So hydrolysis of sucrose (dilute acid) appears to invert the rotation of polarised light • Such a mixture of glucose and fructose is known as “invert sugar”

3. Chiroptical techniques • The “chiroptical” techniques are • OR, optical rotation • CD, circular dichroism • ORD, optical rotatory dichroism • All involve measuring the differential interaction of molecules with polarised light • Chiral molecules show optical rotation

4. Mirror plane Chiral molecules show optical rotation If a structure cannot be superimposed on its mirror image it is said to be chiral

5. Polarised light Plane polarised as per Polaroid glasses Circularly polarised light (left and right) can be produced with specialised filters From Matthews, van Holde, Ahern “Biochemistry”

6. Proteins are chiral • In a protein, all peptide residues have “handedness” even glycine, because of it is connected to other residues • All residues in nature are in the L-form • Therefore all proteins have intrinsic chirality and will interact with polarised light

7. OR, CD and ORD • OR optical rotation • measure amount of rotation at fixed wavelength • CD circular dichroism • measure difference in absorption between left and right hand polarised light at different wavelengths • ORD optical rotatory dispersion • measure OR at different wavelengths • mathematically related to CD

8. Circular dichroism • Circular Dichroism is the difference between the absorption of left and right handed circularly-polarised light and is measured as a function of wavelength • Subtract left and right to get circular dichroism http://www.cryst.bbk.ac.uk/BBS/whatis/cd_website.html

9. CD instrument • Circular Dichroism (CD) is observed when optically active matter absorbs left and right hand circular polarised light slightly differently. It is measured with a CD spectropolarimeter (~£50k) • The instrument needs to be able to measure accurately in the far UV at wavelengths down to 190-170 nm. The CD is a function of wavelength

10. Significance of CD • CD spectra for distinct types of secondary structure present in peptides, proteins and nucleic acids are different • The analysis of CD spectra can therefore yield valuable information about secondary structure of biological macromolecules

11. CD sensitivity requirements • The difference in left and right handed absorbance L-R is very small (usually in the range of 0.0001 absorbance units) • Need very specialised equipment (hence price) • Results usually expressed as ellipticity, r

12. Advantages of CD • Sensitive to peptide secondary structure • Very different spectra for • -helix • -sheet • “random coil” • Contributions are additive • can determine the amount of -helix, -sheet and “random coil” by solution measurement

13. Interpretation • Standard spectra for • -helix, -sheet, “random coil” • Model data sets • Greenfield and Fasman, 1969 • Chen, Yang and Chau, 1974 • Computer fits and gives percentage of -helix, -sheet, “random coil”

14. CD spectra • Differential absorption (left and right polarised light absorption L-R)of secondary structure elements versus wave-length  • The CD spectrum of a protein in solution can be resolved into the three elements -helix, -sheet and “random coil” From Matthews, van Holde, Ahern “Biochemistry”

15. Collagen • molar ellipticityversus  for • disordered collagen • native collagen M.L.Tiffany and S. Krimm, Biopolymers (1972)

16. CD apparatus Electro-optical polariser and detector Sample Output device, chart, PC Scanning monochromator

17. Experimental factors Typical Initial Parameters: • Protein Concentration: 0.5 mg/mL • Cell Path Length: 0.5 mm • Stabilizers (Metal ions, etc.): minimum • Buffer Concentration : 5 mM or as low as possible while maintaining protein stability

18. Obtaining secondary structure • Use a linear combination of the different shaped model spectra to reproduce the measured spectrum • New spectrum = 33% (helix) + 33%(sheet) + 34% (coil)

19. Fitting data • New spectrum is calculated for: • 33% (helix) + 33%(sheet) + 34% (coil) • If we do not know %ages but we have measured the CD spectrum • use the computer to “iterate” these values with model data sets until we match the observed spectrum

20. Subtilisin • A potent proteolytic enzyme • serine protease • derived from bacteria • subtilisin A, B variants • very undiscriminating • will hydrolyse almost all enzymes • The most common “enzyme” in biological washing powders • subtilisin gains stability by binding Ca2+ • In commercial detergents Ca2+ is normally chelated to soften water, so engineered versions replace the calcium site with a disulfide bond for better control

21. Subtilisin secondary structure 58% helix 26% sheet 16%coil Obtained by computer fitting CD spectra

22. Subtilisin - 3D structure 58% helix 26% sheet 16%coil Obtained by computer fitting of CD spectra; agrees well with X-ray diffraction

23. CD techniques - the future • Use more extended model data-sets and more sophisticated computer methods • 5-component model can also distinguish • parallel -sheet • antiparallel -sheet • -turn • VU-CD • vacuum ultraviolet CD - down to say 140nm - gives improved resolution