Proteomics 2013 蛋白質體學 Protein Analysis (II) 陳威戎

1. Circular Dichroism (CD) 2. X-ray Crystallography 3. Nuclear Magnetic Resonance (NMR) 4. Cryo-Electronic Microscopy (Cryo-EM) 5. Protein Data Bank (PDB) Methods for determining protein structure

Circular Dichrosim (CD) Spectroscopy - Introduction 1. When plane polarized light passes through a solution containing an optically active substance the left and right circularly polarized components of the plane polarized light are absorbed by different amounts. 2. When these components are recombined they appear as elliptically polarized light. The ellipticity is defined as . 3. CD is the ellipticity (difference) in absorption between left and right handed circularly polarized light that measured with spectropolarimeter. 4. Proteins and nucleic acids contain elements of asymmetry and thus exhibit distinct CD signals.

The principle behind CD spectroscopy The light from UV1 is passed into a Photo Elastic Modulator (PEM) which converts the linear polarized light into alternating left and right handed polarized light. The two polarizations are differently absorbed, and the difference in absorption is detected with a Photo Multiplier Tube (PMT)

The principle behind CD spectroscopy

Polarization Vertically Polarised Light Horizontally Polarised Light Left Circularly Polarised (LCP) Light Right Circularly Polarised (RCP) Light

Spectropolarimeter

Cuvette for Circular Dichroism

Far-UV ( nm) CD for determining protein secondary structure  The far-UV CD spectrum of proteins can reveal important characteristics of their secondary structure.secondary structure  CD cannot say where the alpha helices that are detected are located within the molecule or even completely predict how many there are.  It can be used to study how the secondary structure of a molecule changes as a function of temperature or of the concentration of denaturing agents, e.g. Guanidinium chlorideor urea, thus revealing important thermodynamic information about the molecule.Guanidinium chlorideurea  Anyone attempting to study a protein will find CD a valuable tool for verifying that the protein is in its native conformation before undertaking extensive and/or expensive experiments with it.

Far-UV ( nm) CD for determining protein secondary structure Secondary Structure Signal (+/-) WL (nm)  -helix  -sheet random-200 coil+220

Secondary structure estimation program Optical constants are automatically calculated in the multivariate SSE program after imputing parameters as the path length and mean residue molar concentration.

CD spectra measurement Path length: 1 mm Concentration: 0.2mg/ml Temp.: 20ºC Wavelength: nm Scan speed: 100 nm/min Data interval: 0.1 nm Bandwidth: 1 nm Measurement: 90 sec. per sample

Secondary structure estimation program

Sample name Helix (%) Sheet (%)Turn (%) Other (%)Reference Lysozyme (Lyz) PLS X-ray Cytochrome C (CytC) PLS X-ray Concanavalin A (ConA) PLS X-ray β-Lactoglobulin PLS X-ray Trypsin Inhibitor PLS X-ray Ribonuclease A (RNaseA) PLS X-ray Human Serum Albumin (HSA) PLS X-ray Hemoglobin (Hb) PLS X-ray

Near-UV ( nm) CD is dominated by aromatic amino acids and disulfide bonds  The near-UV CD spectrum (>250 nm) of proteins provides information on the tertiary structure.tertiary structure  The signals obtained in the 250–300 nm region are due to the absorption, dipole orientation and the nature of the surrounding environment of the phenylalanine, tyrosine, cysteine (or S-S disulfide bridges) and tryptophan.disulfide bridges  Unlike in far-UV CD, the near-UV CD spectrum cannot be assigned to any particular 3D structure.  Rather, near-UV CD spectra provide structural information on the nature of the prosthetic groups in proteins, e.g., the heme groups in hemoglobin and cytochrome c.hemoglobincytochrome c

Near-UV ( nm) CD is dominated by aromatic amino acids and disulfide bonds a.a. residue Abs max. (nm) Phe 254, , 267 Tyr 276 Trp282 Disulfides broad band

Circular Dichrosim (CD) Spectroscopy - Applications 1. Secondary structure content of macromolecules 2. Conformation of proteins and nucleic acids - Effects of salt, pH, and organic solvents 3. Kinetics - Protein folding, unfolding, denaturation or aggregation 4. Thermodynamics - Protein stability to temperature or chemical denaturants

Circular Dichrosim (CD) Spectroscopy

Loss of protein structure results in loss of function Protein Denaturation and Folding

Methods for determining the three-dimensional structure of a protein: X-ray crystallography

Determining the three-dimensional structure of a protein: X-ray crystallography

Protein crystallization techniques

Protein crystallization techniques

Microcapillary Protein Crystallization System (MPCS)

Typical protein crystals

X-ray diffractometer

Methods for determining the three-dimensional structure of a protein: X-ray crystallography Protocols: 1. Protein over-expression and purification 2. Protein crystallization 3. X-ray diffraction 4. Phase determination and electron density maps 5. Model building and refinement Advantages: 1. Best resolution 2. No size limitation (in contrast to NMR) Limitations: Technically very challenging to make crystals of proteins. (heterogeneous samples, membrane proteins, protein complexes)

Methods for determining the three-dimensional structure of a protein: Nuclear magetic resonance, NMR

Protocols: 1. A concentrated aqueous protein sample (0.2-1 mM, 6-30 mg/mL) labeled with 13 C and/or 15 N is placed in a large magnet. 2. An external magnetic field is applied; 13 C and 15 N nuclei will undergo precession (spinning like a cone) with a frequency that depends on the external environment 3. From these frequencies, computer determines the through-bond (J coupling) and through-space (NOE) constants between every pair of NMR-active nuclei. 4. These values provide a set of estimates of distances between specific pairs of atoms, called "constraints“ 5. Build a model for the structure that is consistent with the set of constraints

Methods for determining the three-dimensional structure of a protein: Nuclear magetic resonance, NMR Advantages: 1. Native like conditions – sample is hydrated, not in a crystal lattice 2. Can get dynamic information – observe conformational changes 3. Can look at relative disorder of specific regions of a protein – can see if a loop is static or flexible over time Limitations: 1. Not as high resolution as x-ray 2. Require a lot of protein to get a good signal 3. Require very concentrated samples (can get insoluble aggregates) 4. Limit on protein size measurable, since molecule must tumble rapidly to give sharp peaks. Typically, proteins must be <30kD.

Methods for determining the three-dimensional structure of a protein: Cryo-electronic microscopy, Cryo-EM

X-ray Crystallography vs. Cryo-EM

Protein Data Bank (PDB) Research Collaboration for Structural Bioinformatics