Biol 2012: Exploring Proteins

Biol 2012: Exploring Proteins

The Structures of Life

Instructor: Jörn Werner

How Do We Study Proteins?

Primary Structure

• Definition: The primary structure refers to the linear sequence of amino acids in a protein.

Techniques for Studying Primary Structure

• Edman Degradation: A method used chemically to sequence amino acids.

• Mass Spectrometry: A technique used for identifying protein fragments and determining their mass.

Secondary and Tertiary Structures

• There are various methods available for studying both secondary and tertiary structures of proteins.

Investigating Matter at Different Scales

Scale of Measurement

• Scale Range: 0.1 nm (atomic scale) to 1 m (macroscale).

• Objects of Study: Includes atoms, molecules, proteins, bacteria, eukaryotic cells, C. elegans, organs, and humans.

Methods of Investigation

• Light Microscopy: Used for observing larger structures such as cells and tissues.

• Electron Microscopy (EM): Offers higher resolution for visualizing proteins and cellular structures.

• X-ray Scattering, X-ray Diffraction: Techniques for examining the atomic arrangements in proteins.

• Nuclear Magnetic Resonance (NMR): Characterizes the physical and chemical properties of molecules.

Spectroscopic Methods

Beer-Lambert Law

• Law: A = e l c

• Definitions:

  • A: Absorbance

  • e: Molar Extinction Coefficient (specific to each molecule)

  • l: Path length in the cuvette

  • c: Concentration of the solute (e.g., protein, DNA)

Circular Dichroism (CD) and Second Protein Structure

Measurement Technique

• Measures the difference between the absorbance of left and right circularly polarized light as a function of wavelength.

• Produces a CD spectrum used for characterizing secondary structure.

Properties of Light

1. Intensity

2. Wavelength

3. Polarization

Polarization Process

• Linear Polarization: The electric field (E-field) oscillates in one plane.

• Circular Polarization: Results from the combination of linear polarization directions into a circular form (corkscrew orientation).

Protein Conformation and Circular Dichroism

Importance of Peptide Bonds

• Peptide bonds are optically active groups responsible for circular dichroism, which is sensitive to protein conformation.

Characteristic CD Spectra

• Red: Represents alpha-helical structure.

• Blue: Represents beta-strand structure.

• Yellow: Represents random coil structure.

Structural Transition of Prion Protein Using Circular Dichroism

Native Prion Protein

• Primarily in an alpha-helical conformation.

• Not associated with disease.

Solubilized Fibrillar Form

• Mainly exhibits a beta-strand structure.

• Associated with prion diseases.

Electron Microscopy (EM)

Techniques and Resolutions

• Resolution Limits: Single particle imaging resolution ~20 Å; averaging of crystalline samples can achieve ~4 Å.

• Sample Preparation: Metal staining enhances contrast; cryo-EM allows for fewer artifacts and better preservation.

Imaging Capabilities

• Whole cells can be imaged, allowing analysis of protein distribution inside the cell.

Limitations

• Radiation damage is a significant concern.

• Transmission EM requires very thin samples (~<100 nm).

Cryo Electron Microscopy (Cryo EM)

Sample Preservation Techniques

• Flash Freezing: Samples are rapidly frozen (e.g., using liquid ethane) to preserve structure without forming ice.

Preparation of Samples

• Need for very thin samples (<30-50 nm), achieved via cutting or ion-beam ablation.

Enhancing Signal and Resolution

• EM Crystallography: Allows obtaining 3D and 2D crystals through averaging of many particles and using periodicities of these objects.

• Single Particle Reconstruction: Accumulates images of individual particles to enhance the signal-to-noise ratio.

• EM Tomography: Involves rotating the object and recording a series of 2D images (projections) to reconstruct a 3D object.

Scattering and Molecular Shape

Principles of Scattering

• Incident Radiation: Scattered off the sample, captured by a detector.

• q Parameter: Defined as the angle of scattering.

Experimental Output

• I(q): Intensity of radiation as a function of q. The interpretation of I(q) reveals structural information about scattering molecules, and understanding the individual domains allows for assembling the correct shape.

Example: Small-Angle X-ray Scattering (SAXS) Analysis

Data Presentation

• Graph of I(q): Displays intensity against q values, demonstrating how data is presented.

• Pair Correlation Function (P(r)): The Fourier Transform of I(q), reveals distance relationships between points in the molecule.

X-ray Crystallography for Structure Determination

Steps Involved

1. Crystal Preparation: Need to generate protein crystals from saturated solutions.

2. Data Collection: Gather diffraction data.

3. Electron Density Map Creation: Convert diffraction data into a usable electron density map.

Phasing Problem

• A significant challenge in X-ray crystallography where additional information needs to be sourced for comparison.

Building the Model

• Fit the molecular model into the created electron density map.

Diamond Light Source

• Located near Didcot in the UK, is a significant research investment.

• Opened recently, producing X-rays for research.

Nuclear Magnetic Resonance (NMR)

NMR Principles

• Nuclei (especially protons) produce a spectrum when subjected to a magnetic field.

Chemical Shift

• Recorded in parts per million (ppm), indicating the environment surrounding a nucleus.

Detecting Peaks

• Each peak corresponds to a distinct proton in a protein, showing the diversity within a structure.

3D Structure Insights from NMR

Correlations between Protons

• Correlation peaks indicate protons close in space, aiding in determining distance relations.

Structure Determination from Peaks

• Convert pairs' proximity data into a comprehensive 3D structure using triangulation principles.

Family of Structures

• NMR results typically yield families of compatible structures based on distance constraints, with well-defined regions and ill-defined regions depending on constraint density.

Strengths and Limitations of Techniques

Electron Microscopy (EM)

Variants

1. Negative Stain EM

   • Strengths: Requires small amounts of sample, capable of imaging a variety of samples.

   • Limitations: Resolution about 20 Å, can generate artifacts.

2. Cryo EM

   • Strengths: No staining required increases accuracy; enhanced resolution obtainable.

   • Limitations: Requires averaging techniques, object size restrictions.

3. Electron Diffraction

   • Strengths: Useful for specific 2D crystals like membrane proteins; potentially high resolution (~3-4 Å).

Strengths and Limitations of Scattering Techniques

• X-ray and neutron scattering provide shape and size information with large applicability but suffer from resolution limits (~20 Å) and material requirements.

Strengths and Limitations of X-ray Crystallography

• Highest resolution under 1 Å but requires protein crystallization, which can be challenging due to the protein environment influencing the structure.

Strengths and Limitations of NMR

• Versatile for many macromolecules, provides dynamic and interaction insights but limited to smaller proteins and requires isotope labeling for accurate measurements.

How Do We Study Proteins?

Primary Structure

• Definition: The primary structure refers to the linear sequence of amino acids in a polypeptide chain, linked by covalent peptide bonds. This sequence dictates the eventual three-dimensional fold of the protein.

Techniques for Studying Primary Structure

• Edman Degradation: A chemical sequencing method where the N-terminal amino acid is selectively labeled (using phenylisothiocyanate) and cleaved from the peptide without disrupting the remaining bonds. This process is repeated in cycles to determine the full sequence.

• Mass Spectrometry (MS): A high-throughput technique used for protein identification. Proteins are typically digested into peptides using enzymes like trypsin. The MS measures the mass-to-charge ratio ($m/z$) of these fragments, allowing for the determination of the amino acid sequence by comparing observed masses to genomic databases.

Secondary and Tertiary Structures

• Secondary structure involves local folding patterns like $\alpha$-helices and $\beta$-sheets stabilized by hydrogen bonds. Tertiary structure refers to the overall 3D arrangement of a single polypeptide chain.

Investigating Matter at Different Scales

Scale of Measurement

• Scale Range: $0.1$ nm (atomic scale) to $1$ m (macroscale).

• Structural Hierarchy: Atomic details involve understanding bond lengths ($1.5$ Å), while cellular levels involve organelles ($1-10 \mu m$).

Methods of Investigation

• Light Microscopy: Limited by the diffraction of light ($\sim 200$ nm resolution). Used for observing live cells and tissue architecture.

• Electron Microscopy (EM): Uses electron beams with much shorter wavelengths than light, allowing for resolution at the molecular level ($1-20$ Å).

• X-ray Scattering/Diffraction: Provides information on atomic arrangements based on how X-rays interfere with the electron clouds of the atoms.

• Nuclear Magnetic Resonance (NMR): Probes the magnetic properties of atomic nuclei to determine proximity and environment within a molecule in solution.

Spectroscopic Methods

Beer-Lambert Law

• The Equation: $A = \epsilon \cdot l \cdot c$

• Definitions:

  • A: Absorbance (unitless).

  • $\epsilon$: Molar Extinction Coefficient ($M^{-1} cm^{-1}$). For proteins, this is usually measured at $280$ nm, where Tryptophan and Tyrosine residues absorb light.

  • l: Path length of the sample (typically $1$ cm).

  • c: Concentration ($mol/L$).

• Application: Crucial for quantifying protein concentration in a purified sample.

Circular Dichroism (CD) and Secondary Structure

Measurement Technique

• Mechanism: CD measures the differential absorption of left-handed vs. right-handed circularly polarized light. This difference ($\Delta A = A<em>L - A</em>R$) occurs because chiral molecules like proteins absorb polarized light differently.

• CD Spectrum: Plotted as molar ellipticity versus wavelength (typically in the far-UV range, $190-250$ nm).

Properties of Light and Polarization

• Linear Polarization: The electric field vector oscillates in a single plane.

• Circular Polarization: Two perpendicular linear waves with a phase shift of $\frac{\pi}{2}$ result in a rotating electric field vector (corkscrew motion).

Protein Conformation Signals

• Peptide Bonds: The arrangement of peptide bonds in chiral environments generates characteristic signals:

  • Alpha-helix: Shows two distinct minima at $208$ nm and $222$ nm and a maximum at $192$ nm.

  • Beta-strand: Typically shows a single minimum at $218$ nm and a maximum at $195$ nm.

  • Random Coil: Displays a deep minimum at $195$ nm.

Structural Transition of Prion Protein

• Misfolding Mechanism: Prion diseases (like Creutzfeldt-Jakob disease) result from the conversion of the cellular prion protein ($PrP^C$), which is mostly $\alpha$-helical, into a scrapie isoform ($PrP^{Sc}$), which is rich in $\beta$-sheets.

• CD Utility: CD is used to monitor this transition in vitro as the spectrum shifts from the "Red" alpha-helical pattern to the "Blue" beta-sheet pattern.

Electron Microscopy (EM) and Cryo-EM

Techniques and Resolution

• Transmission EM (TEM): Electrons pass through thin specimens. Traditional TEM often uses heavy metal stains (negative staining) to coat the background, providing high contrast but limited resolution ($\sim 20$ Å).

Cryo Electron Microscopy (Cryo-EM)

• Vitrification: Samples are rapidly frozen in liquid ethane to form "vitreous ice," avoiding ice crystals that would destroy the protein's native structure.

• Single Particle Reconstruction: Thousands of 2D images of proteins in different orientations are computationally aligned and averaged to create a high-resolution 3D density map (the "Resolution Revolution" allowing for atomic-level detail).

• EM Tomography: Capturing images as the sample is tilted allows for 3D reconstruction of entire cellular volumes.

Scattering and Small-Angle X-ray Scattering (SAXS)

Principles of Scattering

• The q Parameter: Defined as the scattering vector, related to the angle $\theta$ by $q = \frac{4\pi \sin(\theta)}{\lambda}$.

• Information Content: SAXS provides information on the overall shape, size (Radius of Gyration, $R_g$), and conformational flexibility of proteins in solution.

Data Interpretation

• Pair Correlation Function (P(r)): A histogram of all inter-atomic distances within the molecule. The maximum distance ($D_{max}$) provides the largest dimension of the protein.

X-ray Crystallography

The Process

1. Crystallization: Proteins must be forced out of solution into a highly ordered crystalline lattice, often via vapor diffusion.

2. Diffraction: An X-ray beam hits the crystal, producing a diffraction pattern governed by Bragg's Law: $n\lambda = 2d\sin\theta$.

3. Electron Density Map: The diffraction spots (reflections) are mathematically processed using Fourier Transforms to calculate the electron density.

4. Phasing: Solving the "Phasing Problem" is required because detectors only measure intensity, not the phase of the X-rays.

Nuclear Magnetic Resonance (NMR)

Principles and Chemical Shift

• Nuclear Spin: Only certain isotopes ($H^1$, $C^{13}$, $N^{15}$) have a magnetic spin that interacts with an external magnetic field.

• Chemical Shift: The exact resonance frequency of a nucleus depends on its electronic environment, allowing scientists to identify specific amino acids.

3D Structure from NMR

• NOE (Nuclear Overhauser Effect): Detects protons that are close in space (< 5-6 Å), even if they are far apart in the primary sequence. These distance constraints are used to calculate the 3D structure.

• Ensemble of Structures: Since NMR is performed in solution, it often produces a "family" of structural models that represent the protein's inherent flexibility.

Summary of Analytical Strengths

• X-ray Crystallography: Best for high resolution (< 2 Å) but requires crystals.

• NMR: Best for small proteins (< 30 kDa) and studying dynamics/kinetics in liquid state.

• Cryo-EM: Best for large complexes and membrane proteins that are hard to crystallize.

• CD/SAXS: Best for quick assessment of folding state and global shape in various buffers.