Tutorial 6 (Protein Structure) SS10 v1 0

Drug Design PHCM 81

Outline

-Protein Structure

-X Ray Crystallography• steps• getting the crystal• getting the structure• problems / assessment

- NMR Spectroscopy• basics• multi-dimensional NMR• NOE• Comparison X-Ray vs. NMR

- Electron Cryomicroscopy

Protein Structure Determination

Protein structureX-ray crystallographyNMR spectroscopyElectron cryomicroscopy

Drug Design PHCM 81

Outline

-Protein Structure




We differentiate between:

Which of those is the "structure" we want to determine?

Protein Structure

Drug Design PHCM 81

Outline

-Protein Structure




Protein Backbone / Torsion AnglesDifferent Angles:

Which of those are important for the 3D structure?

Drug Design PHCM 81

Outline

-Protein Structure




…gives information about the structure of a protein:

What do the four quadrants tell us?What do the dark regions tell us?

Ramachandran Plot

Drug Design PHCM 81

Outline

-Protein Structure




X-Ray Crystallography -1 The Steps:1. generating and growing a suitable crystal (>100 µm in each direction, pure,

regular)2. expose the crystal to X-rays to generate the diffraction pattern (gradually

rotating > 180 deg.) intensity of every spot is recorded at every orientation of the crystal

3. generation of the computational model of atoms arrangement in the crystal

How is the diffraction pattern generated?• incident beam causes each scatterer to re-radiate a small portion of its

energy as a spherical wave • the diffracted waves from different layers interfere more or less constructively

and give a pattern of dots (diffraction pattern) • if scatterers are arranged symmetrically

with a separation d, these spherical waves will add 100% constructively where Bragg's law is fulfilled

nd sin 2Bragg's law:

Drug Design PHCM 81

Outline

-Protein Structure




X-Ray Crystallography -2How the crystals are generated:

"Hanging Drop Method"

A few microliters of protein solution are mixed with an about equal amount of reservoir solution containing the precipitants.A drop of this mixture is put on a glass slide which covers the reservoir.As the protein/precipitant mixture in the drop is less concentrated than the reservoir solution (remember: we mixed the protein solution with the reservior solution about 1:1), water evaporates from the drop into the reservoir.As a result the concentration of both protein and precipitant in the drop slowly increases, and crystals may form.

Drug Design PHCM 81

Outline

-Protein Structure




X-Ray Crystallography -3From the crystal to the structure:•diffraction pattern can be interpreted mathematically by a computer to give an electron density map•then, we build-up the protein, residue by residue, fitting it inside to the electron density

Drug Design PHCM 81

Outline

-Protein Structure




X-Ray Crystallography -4 What is the Problem?• in order to obtain an interpretable electron density map (using Fourier

transform) both amplitude (intensity) and phase of the interfereing scattered wave beams must be known

• "phase" means here: how big is the shift of two (or more) interfering waves (beams)?

• however, in crystallography we canNOT directly record the phase of the scattered beams, only their intensities (amplitudes):"phase problem"

• there are several approaches to overcome this problem

Descriptors for the Quality of an X-Ray Structure:•B-Factors or Debye-Weller-Factors (DWF):differences between the actual shape of the protein in the flexible regions and the picture we get from the "frozen" shape of the protein in the crystal•R-Factor (or residual factor or reliability factor):agreement between the crystallographic model and the experimental data low B factor: blue

high B factor: red

Drug Design PHCM 81

Outline

-Protein Structure




NMR Spectroscopy -1

• absorbance of certain (“quantized”) amounts of electro-magnetic radiation causes different effects in an atom or molecule, depending on the energy (resp. frequency) of radiation

• moving charge causes a magnetic field

• magnetic field induces current in a coil

• when a nuclear spin is under the influence of an external magnetic field, “precession” (tumbling motion) occurs

• precession evokes new magnetic field

• magnetic field causes voltage in a receiving coil

Basics

Principle of Measurement

Drug Design PHCM 81

Outline

-Protein Structure




NMR Spectroscopy -2Principle:

•an atomic nucleus absorbs electromagnetic radiation that has got exactly the energy that is used for the nucleus to oscillate between different spin states•this absorption is recorded and gives information about the close environment of each nucleus in a molecule, in this way the method can be used for structure determination•the electromagnetic radiation is radio waves (MHz frequencies)•the different spin states are only experienced when a magnetic field is present•only nuclei with odd number of protons or odd number of neutrons (or both) are NMR active (H-1, H-2 (=D), C-13, N-14, N-15, F-19)

not a smiley!

Drug Design PHCM 81

Outline

-Protein Structure




NMR Spectroscopy -3 Nuclei under investigation:• ideally C-13 and N-15: mainly for proteins from the lab• protons (H-1): only for proteins from natural sources

Sample handling:• purified protein is dissolved in a buffer solution and adjusted to

the desired conditions (concentration, pH...) • sample volume: approx. 500 µL, conc. range approx.

0.1 – 3 mM• due to the presence

of 1000s of nuclei, a simple "1D" NMR experiment would not be good enough (overlap and disturbance of many signals); this is why MULTIDIMENSIONAL experiments are carried out instead

1 D 1H-NMR spectrum =>

Drug Design PHCM 81

Outline

-Protein Structure




NMR Spectroscopy -42D NMR Spectroscopy:correlating two ("normal") 1D spectra

for protein structure elucidation we use (among others):

HSQC - Heteronuclear Single Quantum Correlation: 2D heteronuclear spectroscopy gives a peak for each H-1 that is bound to a heteronucleus

Drug Design PHCM 81

Outline

-Protein Structure




NMR Spectroscopy -53D NMR Spectroscopy:correlating a 2D spectrum with a 1D spectrum:

Using N-15: TOCSY-N HSCQ // NOESY-N HSCQ

• like “normal” HSQC with additional proton dimension (for the correlation!)

• each peak of the HSQC has the TOCSY or NOESY peaks “attached”

Using N-15 and C-13: HNCO, HNCACO, HNCA, HNCOCA, HNCACB and CBCACONH

• like “normal” HSQC with additional carbon dimension (for the correlation!)

Schematic of an HNCA and HNCOCA.Each box displays peaks that “belong” to one nitrogen nucleus.

In the HNCA each nitrogen senses the alpha carbon of the same AA and the preceding residue.

In the HNCOCA each nitrogen senses only the alpha carbon of the preceding residue.

Drug Design PHCM 81

Outline

-Protein Structure




NMR Spectroscopy -6Nuclear Overhauser Effect:

•used to investigate which nuclei (of one sort) are spatially close

•sample is irradiated with exact resonance frequency of one of the nuclei

•the peak of this nucleus will disappear (or even appear as a negative peak)

•peaks of nuclei that are close to this one will be enhanced

Drug Design PHCM 81

Outline

-Protein Structure




NMR vs. X-Ray Crystallography

technique >

V criterionNMR X-Ray

Crystallography

sample (usually) solution single crystal

molecular size < 20 kDa (one domain) any size, domain,

complexanalyte's nature

functional active site domains any domain or complex

analytical feature atomic nuclei electron density

resolution 2 – 3.5 Å 2 – 3.5 Å

requistites pure protein perfect crystal of a pure protein

Drug Design PHCM 81

Outline

-Protein Structure




Electron CryomicroscopyRationale and Development of the Method:

•decreasing radiation damage occurring in electron microscopy (due to high energy of electrons and heating of the specimen)•first trials with vitreous (amorphous) ice (temperatures around – 140 °C) allowed imaging of adenovirus (Jacques Dubochet, 1982)

Conducting Cryomicroscopy: Thin Film Method•biological material is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid freezing, usually in liquid ethane near liquid nitrogen temperature•thin film method is limited to thin specimens (< 500 nm) because electrons cannot cross thicker samples without multiple scattering events•thicker specimens can be vitrified e.g. by high pressure freezing (up to hundreds of μm); they can then be cut in thin sections (40 to 200 nm thick)

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Tutorial 6 (Protein Structure) SS10 v1 0