P6: Biophysical chemistry and diffraction

What is the phase problem in x-ray difractors? why is it necessary? How to solve this problem?

Detectors measure intensity of diffracted X-rays (gives amplitude) but looses phase information.

Phase info and intensity both needed to reconstruct e- density map

Without Phases we can’t calculate e- density map therefore we can’t determine where the atoms are

Can be fixed using : Heavy atom method, Patterson maps or Multiple Isomorphous replacement (MIR)

What does neutron diffraction measure and what does it use? talk about F and b

Measures the scattering of neutrons by atomic nuclei in a crystal

Good at locating light atomes such as H, C, N

Uses bragg’s law n𝜆 =2𝑑 𝑠𝑖𝑛𝜃

scattering length b is constant

F decreases with angle due to atomic vibration causing destructive interference

What does Fluorescence spectroscopy measure? What does Local probe measure and what does Flyo spec give

It measures the environement of a specific fluorophore as the protein unfolds

Only measures 1 specific region of the protein

Fluo spec give Tm (ie. the melting temperature), detects early/partial unfolding

What does DSC measure ?

Differential Scanning Calorimetry (DSC) measures the heat absorbed by the whole protein as it unfolds with increasing temperature

Measures the unfolding of the entire protein

Gives the change in enthalpy, entropry, gibbs free energy and Tm directly ( more reliable Tm than Fluo spec as it measures the protein as a whole

At Tm: deltaG=0 and K=1

What does powder diffraction measure?

Measures diffraction pattern from a powdered crystalline sample

No need for single crystal

gives ring instead of spots

Used to identify unknown materials and phase identification

Less structure info than single crystal diffraction

Uses bragg’s law to identify d-spacing

What does Equilibrium dialysis measure? How it works? and its key equations

measure binding affinity (Kd) between protein and a ligand

Works :

Membrane sperates 2 compartements (L not the protein)

At equilibrium free ligand is equal on both sides

Difference in L concentration between the 2 compartments = bound Ligand

Key equations:

What is a Unit cell?

A unit cell is the smallest repeating 3D block of a crystal lattice that, when translated in space, can reproduce the entire crystal structure.

What is an asymmetric unit?

The asymmetric unit is the smallest unique portion of a crystal structure from which the entire contents of the unit cell can be generated by applying the symmetry operations of the crystal.

What is a space group?

A space group describes all the symmetry operations present in a crystal structure. These operations include : i, rotation, translation, mirror planes…..

How are unit cell, asymmetric unit, and space group and describe how they are related?

Can be summed like this