SAXS CryoEM

MS

NMR

CD EPR

X-RAY FRET

Hybrid protein structure determination

Mark Berjanskii, Edmonton, July 2015

Definition

Hybrid protein structure determination is the 3D modelling of a protein structure using experimental data from different experimental methods

How is it relevant?

Prion protein Mad Cow disease

-synuclein Parkinson's disease

Amyloid beta (Aβ or Abeta) Alzheimer's Disease

Proteins investigated in Wishart’s group can not be studied by traditional high-resolution methods of structure determination

Outline

1) Traditional methods:

X-ray crystallography

Solution NMR

2) Low-resolution methods

Mass-spectrometry

EPR spin-labeling

FRET

SAXS

3) GAMDy -> Hybrid-GAMDy

Why do we need to know protein structure?

Traditional methods of protein structure determination

SAXS CryoEM

MS

NMR

CD EPR

X-RAY FRET

X-ray crystallography

X-ray crystallography

Data analysis: indexing, merging , scaling, phasing

Resolution of X-ray structures

Pros and cons of X-ray crystallography

Advantages: Disadvantages

Provides high-resolution information Unlike solution NMR, does not require a protein be soluble in a high-concentrated solution Unlike solution NMR, can be applied to proteins with MW > 200kD

Requires a protein crystal Can not be used with amyloid fibrils Crystal contacts can distort protein structure Can not be used with very flexible molecules

SAXS CryoEM

MS

NMR

CD EPR

X-RAY FRET

Solution NMR spectroscopy

Summary of solution NMR spectroscopy

Experiment Spectra processing Spectra assignment

NOE assignment

Distance restraints

Model generation

Resolution of NMR structures

Macromolecular NMR spectroscopy for the non-spectroscopist. Kwan AH, Mobli M, Gooley PR, King GF, Mackay JP. FEBS J. 2011 Mar;278(5):687-703

Pros and cons of solution NMR spectroscopy

Advantages: Disadvantages

Provides high-resolution information Unlike XRAY, does not require a protein crystal and is not affected by crystal contacts Can be used to study flexible proteins Reflects conformational averaging

Requires high concentrations of soluble protein Can not be applied to large proteins (800kD max so far) Can not be used with amyloid fibrils

Solid-state NMR

Solid-state NMR with magic angle

spinning for insoluble proteins

HET-S prion 2008

Pros and cons of solid-state NMR spectroscopy

Advantages: Disadvantages

Can be used to study poorly soluble proteins

Can not be applied to large proteins Requires highly homogeneous sample Has troubles with flexible protein regions Limited resolution and sensitivity

SAXS CryoEM

MS

NMR

CD EPR

X-RAY FRET

Mass spectrometry

Distance restraints from MS cross-linking experiments

Distance restraints

Model generation

Pros and cons of MS cross-linking

Advantages: Disadvantages

Unlike X-ray, it does not require protein crystal Unlike solution NMR, it does not require highly concentrated soluble protein Can work with large proteins above the NMR limit

Requires protein modification that can alter protein properties. Provides only sparse restraints Can’t cross-link buried residues

MS cross-linking and prions

HET-S prion

25-hour H-D exchange of PrP

Large cross-linking agents will not penetrate tight fibril core

β-Sheet core of human prion protein amyloid fibrils as determined by hydrogen/deuterium exchange Xiaojun Lu, Patrick L. Wintrode, and Witold K. Surewicz

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) ?????

Are there other methods to study fibril core?

SAXS CryoEM

MS

NMR

CD EPR

X-RAY FRET

Electron paramagnetic resonance

Electron paramagnetic resonance Principle

- the Bohr magneton

- Landé g-factor 2.0023 for free electron

Electron paramagnetic resonance Spectra

Electron paramagnetic resonance Spectrometer

Site-directed spin labeling for EPR

EPR dipolar broadening and exchange narrowing

5-6 Å

Electron-electron dipolar interactions

Spin exchange

8–25 Å

EPR spectra of spin-labeled PrPSC

50 nitroxide-derivatized Cys mutants of huPrP90–231

Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):18946-51. Molecular architecture of human prion protein amyloid: a parallel, in-register beta-structure. Cobb NJ1, Sönnichsen FD, McHaourab H, Surewicz WK.

PrPSC is in-register parallel β-sheet

Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):18946-51. Molecular architecture of human prion protein amyloid: a parallel, in-register beta-structure. Cobb NJ1, Sönnichsen FD, McHaourab H, Surewicz WK.

EPR pros and cons

Advantages: Disadvantages

Unlike X-ray crystallography, EPR does not require a protein crystal Unlike NMR, does not require a protein be soluble in a high-concentrated solution EPR has better sensitivity than NMR Proteins are labeled before fibrillization, no need to penetrate fibril core

Requires multiple site-directed mutations Properties of spin-labeled proteins may be different from the original Can provide only sparse distance restraints

SAXS CryoEM

MS

NMR

CD EPR

X-RAY FRET

Förster resonance energy transfer

Förster resonance energy transfer

R0 - Förster distance of the pair of donor and acceptor, i.e. the distance at which the energy transfer efficiency is 50%

Q0 is the fluorescence quantum yield of the donor, κ2 is the dipole orientation factor, n is the refractive index of the medium, NA is Avogadro's number, and J is the spectral overlap integral

FRET applications Protein-protein interactions Protein conformation change

Unfolding of apolipoprotein fibril on lipid bilayer

Soft Matter. 2015 Jul 29;11(31):6223-34. FRET evidence for untwisting of amyloid fibrils on the surface of model membranes. Gorbenko G1, Trusova V, Girych M, Adachi E, Mizuguchi C, Akaji K, Saito H.

Donor: Fluorescent probe Laurdan Acceptor: Thioflavin T (ThT)

FRET pros and cons

Advantages: Disadvantages

Unlike X-ray crystallography, FRET does not require a protein crystal Unlike NMR, does not require a protein be soluble in a high-concentrated solution Proteins are labeled before fibrillization, no need to penetrate fibril core

Requires multiple site-directed mutations Properties of labeled proteins may be different from the original Can provide only sparse distance restraints

SAXS CryoEM

MS

NMR

CD EPR

X-RAY FRET

Small angle X-ray scattering

SAXS equipment

Bruker NANOSTAR X-RAY scattering system

Small-angle X-ray scattering

Analysis of SAXS curves

SAXS and amyloid proteins

Metallomics. 2015 Mar;7(3):536-43 Small angle X-ray scattering analysis of Cu(2+)-induced oligomers of the Alzheimer's amyloid β peptide. Ryan TM1, Kirby N, Mertens HD, Roberts B, Barnham KJ, Cappai R, Pham Cle L, Masters CL, Curtain CC.

Effect of Cu(2+) concentration on Abeta oligomers

SAXS pros and cons

Advantages: Disadvantages

Unlike X-ray crystallography, SAXS does not require a protein crystal Does not require a protein be soluble in a high-concentrated solution Does not require protein modification

No high-resolution information

CryoEM SAXS

MS

NMR

CD EPR

X-RAY FRET

Cryo-electron microscopy (CryoEM)

Cryo-electron microscopy

Image formation in the electron microscope. (a) Electrons, emitted by a source that is housed under a high vacuum, are accelerated down the microscope column . After passing through the specimen, scattered electrons are focused by the electromagnetic lenses of the microscope (b) Schematic illustrating the principle of data collection for electron tomography. As the specimen is tilted relative to the electron beam, a series of images is taken of the same field of view. (c) Rendering of selected projection views generated during cryo-electron tomography

3D image from Cryo-EM

Examples of Cryo-EM images

(a,b) Illustration of spiral architecture of the nucleoid in Bdellovibrio bacteriovorus showing (a) a 210 Å thick tomographic slice through

the 3D volume of a cell (b) a 3D surface rendering of the same cell,

with the spiral nucleoid highlighted (c) Higher magnification view of a

tomographic slice through the cell, showing well-separated nucleoid spirals and ribosomes (dark dots) distributed at the edge of the nucleoid.

(d) Expanded views of 210 Å thick tomographic slices, showing top-views of polar chemoreceptor arrays.

Cryo-EM revolution in structural biology

Cryo-EM can now achieve a resolution necessary for de novo structure determination

Cryo-EM structures <5Å

Examples of “high-resolution” de novo structures from Cryo-EM

A) transient receptor potential cation channel subfamily V member 1 (TRPV1) ion channel

B) F420-reducing [NiFe] hydrogenase

C) large subunit of the yeast mitochondrial ribosome

D) γ-secretase.

CryoEM pros and cons Advantages: Disadvantages

CryoEM can achieve high enough resolution Unlike X-ray crystallography, CryoEM does not require a protein crystal Unlike solution NMR, CryoEM does not require a protein be soluble in a high-concentrated solution Unlike mass-spectrometry, CryoEM does not require protein modification

Complex measurements and data analysis Difficult to use for proteins with MW below < 300KDa CryoEM application for high-resolution structure determination is still new and has not been thoroughly tested by the scientific community Shortage of labs with proper equipment and expertise

How can we contribute to hybrid structure determination?

GAMDy

Genetic Algorithm for biased Molecular Dynamics

CONTRA MD biasing

CS-GAMDy performance Distorted model of ubiquitin

(reference PDB ID: 1UBQ) Distorted model of Q5E7H1

(reference PDB ID: 2JVW)

Comparative model of NFU1 homolog (reference PDB ID: 2M5O,

template PDB ID: 1TH5, sequence ID: 20%)

Comparative model of ubiquitin (reference PDB ID: 1UBQ,

template PDB ID: 1IYF, sequence ID: 30%)

Hybrid-GAMDy

Torsions Distances

XPLOR 3.81

SAXS Distances

XPLOR NIH

CryoEM NAMD

NMR, FRET, MS, EPR distances, X-ray data, NMR torsion angles

MD biasing

Genetic algorithm

In-house scoring functions for SAXS, CryoEM, MS