Methodology in protein science

Yun-Ru (Ruby) Chen 陳韻如 Ph.D.

The Genomics Research Center

(office at 7th floor)

yrchen@gate.sinica.edu.tw

2789-9930 ext 355

Protein synthesis in cell

Incorporate non-natural amino acid in cell

Protein Expression and Purification Volume 38, Issue 1, November 2004, Pages 37-44

Preparing protein samples

What is the point?

Endogenous proteins are not enough!

Amplification

Way to go

Hetero expression

Bacteria: E. Coli

Insect cells: baculo virus infection

Mammalian cells: human suspension cells

Molecular cloning Heterogeneous expression Harvest and purification

Bioorganism factory!

Expression level, solubility, purification procedures, yield

Detection/Quantification of proteins

Quantification of pure protein

UV absorbance: Tyr, Trp,

Beer’s law A=bc extinction coefficient

Edhock equation. Abs280=1280*(# of Tyr)+2560*(# of Trp)

• Specific Assays (functional based)1. Catalytic activity2. Ligand binding3. Antibody binding (western blot)

• Nonspecific assays 1. Biuret ( rx peptide backbone)2. Lowry( rx peptide backbone)3. Ninhydrin (rx free amino group)4. Fluorescamine (rx free amino group)5. Coomassie stain, noncovalent complex, ~10-7g detection6. Silver stain, <10-9g detection7. Direct blue8. Flamingo (fluorescence staining)9. SyproRuby (fluorescence staining)

electrophoresis

SDS-PAGEsodium dodecyl sulfate-polyacrylamide gel el

ectrophoresis (1 SDS molecule H-bonding with 2 residues)

Agarose gelDNA

Native-PAGE

chromatography

Size-exclusion Ion-exchange Affinity hydrophobicity

Size-exclusion chromatography

Ion-Exchange

Anion exchange Cation exchange

Affinity Chromatography

Common Fusion Tags and Purification Conditions

Fusion Tag

Immobilized Ligand

Binding Conditions Elution Conditions Available Formats

Glutathione S-transferase(GST)

Reduced glutathione

Neutral (physiologic) pH, and non-denaturing; glutathione must be reduced and GST must be active

Free reduced glutathione at neutral pH (competitor)

Prepacked column kits, spin cup column kits, SwellGel Discs, coated microplates

Histidine-tagged

Chelated Nickel or Cobalt

Neutral (physiologic) pH without reducing or oxidizing agents; small tag must be accessible in fusion protein structure; high ionic strength and denaturants (chaotropes such as 8 M urea) compatible.

>200 mM Imidazole, low pH, or strong chelators

Prepacked column kits, spin cup column kits, SwellGel Discs, Swell- Gel Discs in 96-well filter plates, coated microplates

Maltose Binding Protein (MBP)

Dextrin

Neutral (physiologic) pH and non-denaturing; NaCl added to reduce nonspecific binding

Maltose at neutral pH (competitor)

Gel slurry, coated microplates

Green Fluorescent Protein (GFP)

Anti-GFP antibody

Neutral (physiologic) pH and non-denaturing

Usual antibody/antigen elution buffers (e.g., low pH or chaotropic salts)

Coated microplates

Peptide synthesis

The longer the more expensive Limitation at ~100 residues Relatively clean

Methods to detect protein primary structure

Protein sequencingBy Edman degradationPhenylisothiocyanate (PITC)React with N- free amino acidApply to chromatographyEach amino acid eluted by Abs254nm

(Error are cumulative)

Instrument setup of Mass spectrometry

•MALDI•ESI•Ion bombardment•Chemical ionization•Electron impact ionization

•Magnetic•Quadrupole•Ion trap•TOF(Time of flight)•FT-MS

(no detergent, desalt)

Mass Spectrometry

Combine with Edman degradation Combine with limited proteolysis (ex:Trypsin digestion) De novo sequencing is sometimes difficult (tandom mass) (LC/

MS/MS)

AdvantageProtein doesn’t need high purityPicomoles of sample are requiredAlso detect post-translational modification

Methods to detect protein secondary structure

Circular Dichroism Infrared spectrometry (FTIR)

Light Waves

farUV CDCircular dichroism (CD) is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light. It can be used to determine the structure of macromolecules (must be asymmetric)

n -> pi* centered around 222 nmPart of pi -> pi* centered around 208 nmpi -> pi* centered around 190 nm

n -> pi* involves non-bonding electrons of O of the carbonylpi -> pi* involves the p-electrons of the carbonyl

Far UV CD spectraDetect peptide backbone through pi bond formed by overlapping of 2 p orbitalAlpha helix: min @222 and 208nmBeta sheet: min @216nm, max @195nmRandom coil: decreasing signal below 200nm, slight increase @218nm

Signal to noise ratio:Protein concentration, Path length, salt in buffer, response time

Units: ellipticity (θ), 32.98 θ = 33.98 ΔAbs Ellipticity: millidegreeMolar ellipticity ([θ]) is CD corrected for concentration. molar elliplicity are historical (deg cm2/dmol)the sample concentration (g/L), cell pathlength (cm), and the molecular weight (g/mol) must be known

% alpha-helix = (-[θ]222nm +3000)/39000Biochemistry. 39, 11657-11666, 2000Secondary Structure Prediction needs spectra down to at least 200nm (some need 178nm)

Infrared spectra

The frequencies with which bonded atoms vibrate relative to each other determine the vibrational spectrum of a molecule.

High background of water. Often use D2O

Amide I is the most sensitive Spectra need to be deconvoluted

Methods to detect protein tertiary/quaternary structural changes

Size exclusion chromatography Fluorescence spectroscopy Near UV Circular dichroism Analytical Ultracentrifugation (AUC) NMR X-ray crystallography

Fluorescence

Fluorescence Wavelength scan Fluorescence Anisotropy Fluorescence correlation spectrum Fluorescence Life Time Fluorescence energy transfer

Highly sensitive Small amount (ug) Give total conformational information

FluorescenceJablonski diagram

quantum yield=(# of (3))/(# of (1))

0

50E+05

10E+06

15E+06

20E+06

25E+06

300 350 400 450 500 550 600 650 700 750

cps/uA / Wavelength (nm)

File # 2 = Y-M2A emission wavelength, nm

fluo

resc

en

ce in

ten

sity

(cp

s/u

A)

λem ＞ λex

Stern-Volmer equation, Ksv=t0kq

Fluorescence instrumentation

Fluorescence Anisotropy

Polarization techniques can provide average size and shape of rotating fluorophores and macromolecules

Polarization (P) = (Iv - Ih) / (Iv+ Ih)Anisotropy (r) = (Iv - Ih) / (Iv+ 2Ih) where Iv is the intensity parallel to the excitation plane and Ih is the emission perpendicular to the excitation plane. They are interchangeable quantities and only differ in their normalization. Polarization P ranges from –0.33 to +0.5 while the range for anisotropy r is –0.25 to +0.4.

Fluorescence correlation spectrumUsing confocal or two photon microscopy, light is focused on a sample and the measured fluorescence intensity fluctuations (due to diffusion, chemical reactions, aggregation, etc.). FCS is the fluorescent counterpart to dynamic light scattering, FCS obtains quantitative information such asdiffusion coefficients, hydrodynamic radii, average concentrations, kinetic chemical reaction rates.

Fluorescence Life TimeThe fluorescence lifetime refers to the average time the molecule stays in its excited state before emitting a photon. Fluorescence typically follows first-order kinetics:where [S]t is the concentration of excited state molecules at time t, [S]0 is the initial concentration and Γ is the decay rate or the inverse of the fluorescence lifetime. This is an instance of exponential decay. [S]t = [S]0 exp (Γ* t)

Fluorescence energy transferA donor chromophore in its excited state can transfer energy by a nonradiative, long-range dipole-dipole coupling mechanism to an acceptor chromophore in close proximity (typically <10nm).

What Information Can AUC Provide?

Molecular weight Stoichiometry Oligomerization

(Kd from 10-3-10-8 M)

Shape and size Number of species Diffusion constant

Sedimentation equilibrium

Sedimentation velocity

The first model was built in 1924 by Theodore (The) Svedberg.

Diagram of a Sedimentation Experiment

Fc: centrifugal forceFb: buoyancyFd: frictional force

Sedimentation EquilibriumChange in Concentration Over Time

Basic Theory of Sedimentation Equilibrium

C

rm rbC: concentrationr: radius: angular velocityJS: Flux of sedimentationJD: Flux of diffusion (Fick’s Law)s: sedimentation coefficient

ecrc 0)( )22

(2

02 rr

Curve fitting with assumed model or plotting method

Different Cases for Sedimentation Equilibrium

1. Single species (monomer)2. self-association (homooligomer)3. Heterogeneous mixtures (heterooligomer)4. Multiple component versus multiple species5. Heterogeneous associations6. Non ideally

ideal

Plotting the residual as a function of radial distance

Determining the rate of movement of a solute under a centrifugal field

The centrifugal force on the particle (solute) is equal to the friction of the particle.

dt

drffvrwMrwMb 22 )1(

Sedimentation Velocity

Comparison to Other Useful Techniques

AUC Mass Spectrometry

Micro-Calorimetry

Fluorescence spectrometry,CD, Light scattering

Solution MW MW Thermo-

dynamics

(G, H, C)

Enzyme kinetics, 2nd structures,

solution mass

Stoichiometry, Assembly Model

Non covalent interactions

Molar ratio Stoichiometry

Thermodynamics( G)

Stoichiometry Folding stability Folding stability

Conformational changes, shape

Epitope mapping Conformational changes

Conformational changes

Kd 10-3~10-8 Identify unknowns Kd 10-3~10-11;

10-6~10-20 M

Kd 10-6~10-11 M

Methods to detect atomic level protein structure

X-ray Crystallography

NMR

EM

Amount

10mg/ml

pro con

Atomic levelLarge protein(50-100kD)

Magic requiredRigid structureMostly native state

Atomic levelFlexible proteinsDynamicsOver large time scale

Size limit(<50kD)

10mg/ml

Data derived from physical techniques for probing structure, the interpretation is not unambiguous and entails assumptions and approximations often depending upon knowledge of the proteins from other sources (biology)

X-ray Crystallography

Bragg’s Law

The interference is constructive when the phase shift is a multiple to 2π; this condition can be expressed by Bragg's law:

Both entail a droplet containing purified protein, buffer, and precipitant being allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. Initially, the droplet of protein solution contains an insufficient concentration of precipitant for crystallization, but as water vaporizes from the drop and transfers to the reservoir, the precipitant concentration increases to a level optimal for crystallization.

Crystallizing proteins

NMR spectroscopy(nuclear magnetic resonance)

Determine internuclear distances by measuring perturbations between assigned resonances from atoms in the protein in solution

1D NMRProton: 1H, Isotope labeled carbon 13C and nitrogen 15N, 19FDifferent nuclei in a protein absorb electromagnetic energy (resonance) at different frequencies because their local electromagnetic environment differ

Useful parameters

Chemical shift (freq) Chemical structure

Spin-spin coupling (through bond)

3JNHtorsion angle for structural constraint

Signal intensity Concentration

Nuclear overhauser effect (NOE)

(through space)

Intermolecular distance

Relaxation time (T1, T2) Motional dynamics

Signal linewidth dynamics

2D NMR

Electron Microscope

Electron beam is stronger than X-ray. No need for 3D crystal

Achieve atomic level resolution

electrons interact more strongly with atoms than X-rays

Phase problem less severe

But could destroy sample

a) Wireb) Ribbonc) Ball and Stickd) space filling as a sphere of va

n der Waals radiuse) surface representation GRAS

P image topology of protein surface (red negative, blue positive)

Protein structure determination methodsHigh resolution X-ray crystallography | NMR | Electron crystallography

Medium resolution

Cryo-electron microscopy | Fiber diffraction | Mass spectrometry | SAXS

Spectroscopic NMR | Circular dichroism | Absorbance | Fluorescence | Fluorescence anisotropy

Translational Diffusion

Analytical ultracentrifugation | Size exclusion chromatography | Light scattering | NMR

Rotational Diffusion

Fluorescence anisotropy | Flow birefringence | Dielectric relaxation | NMR

Chemical Hydrogen-deuterium exchange | Site-directed mutagenesis | Chemical modification

Thermodynamic Equilibrium unfolding

Computational Protein structure prediction | Molecular docking