Protein Quaternary Structure and Analytical Ultracentrifugation Notes

Protein Quaternary Structure and Analytical Ultracentrifugation (AUC)

Introduction

• Protein Quaternary structure involves protein complexes, where individual proteins come together as oligomers.
• These oligomers can be homo-complexes (same protein subunits) or heterocomplexes (different protein subunits).
• Analytical Ultracentrifugation (AUC) is used to analyze proteins and determine their Quaternary structure.

Levels of Protein Structure

• Primary Structure: The amino acid sequence.
• Secondary Structure: Local folding, such as alpha helices and beta strands.
• Tertiary Structure: The overall three-dimensional structure of a single protein molecule (covered in crystallography).
• Quaternary Structure: The arrangement of multiple protein subunits in a complex.

Analytical Ultracentrifuge (AUC)

• An AUC resembles a normal analytical centrifuge but with key differences.
• It spins a sample in a cell, causing the sample to move from the center to the outside due to centrifugal force.
• The distance traveled by the molecule is dictated by its size.
• AUC is used to determine the sizes of proteins and protein complexes.
• Two primary types of experiments can be performed:
  • Sedimentation Velocity
  • Sedimentation Equilibrium

AUC Rotor and Cells

• The rotor contains cells, each with two sectors:
  • Sample Sector: Contains the protein sample in a specific buffer.
  • Reference Sector: Contains the buffer alone.
• A monochromator directs light of a certain wavelength through the sample.
• The absorbance of the protein is measured as it sediments down over time during spinning.
• The reference is subtracted from the sample to account for the buffer's absorbance.
• Sectors are not completely filled to avoid interference from the air-liquid interface.
  • Reference sector is filled higher than the sample sector.
• Air-liquid interface causes scattering, resulting in peaks.
  • Reference meniscus shows a negative peak due to subtraction.
  • Sample meniscus shows a positive peak.
• The protein absorbance forms a diffusion gradient as the protein moves down the sector.
• The peaks indicate where the sample started and ended, allowing for distance measurement.
• Knowing the distance and time, the size of the protein can be determined (larger proteins sediment faster).
• The boundary indicates where the protein is at a specific time, and the plateau demonstrates constant sedimentation.

History and Units of AUC

• The technique was first developed in 1926.
• The first commercial AUC was built in 1947.
• The founder of AUC was Theodore Svedberg.
• AUC is measured in Svedberg units (S), where $1S = 10^{-13}$ seconds.

Sedimentation Velocity

• High speeds are used to observe protein sedimentation over time.
• The time and distance taken for sedimentation are measured.
• The sedimentation coefficient is calculated and converted into mass.
• Larger oligomers sediment faster than monomers.
• The technique provides information on protein shape (round vs. elongated) and rate constants.

Sedimentation Equilibrium

• Lower speeds are used, causing slower protein sedimentation.
• As the protein reaches the bottom of the cell, back diffusion occurs due to the concentration gradient.
• A balance between sedimentation and diffusion is achieved.
• This provides information on protein mass, oligomeric state, and affinity of the oligomer.
• It also reveals the stoichiometry (e.g., 1:1 or 1:2 complex) and stability of the sample.
• Sedimentation velocity experiments are preferred because they can be completed in a few hours, whereas equilibrium experiments take a few days.

AUC Setup

• The sample in the cell is placed in the rotor.
• A Xenon lamp and monochromator direct light through the sample.
• A photomultiplier tube detects the light.
• Wavelengths of 280 nm are commonly used because proteins absorb well at this wavelength.

Svedberg Equation

• The Svedberg equation is used to derive mass values from Svedberg units.
• $M = \frac{S \cdot R \cdot T}{D \cdot (1 - \bar{v} \cdot \rho)}$
  • $M$ = Mass
  • $S$ = Svedberg coefficient (directly measured in AUC)
  • $D$ = Diffusion coefficient (measured in AUC)
  • $\bar{v}$ = Partial specific volume of the protein
  • $\rho$ = Density of the buffer
  • $R$ = Gas constant
  • $T$ = Temperature (controlled in AUC)
• The density of, of the sample $(\rho)$, and the partial specific volume $(\bar{v})$ can be calculated. The gas constant $\R$ can be derived due to the pressurised environment. The temperature $T$ can be controlled, and the diffusion coefficient $D$ and Svedberg coefficient $S$ are both measured values allowing for mass to be calculated.

Sedimentation Velocity Experiment

• The radial position indicates the center and outside of the rotor.
• Protein samples move toward the outside during the spinning process.
• The meniscus represents scattering due to the buffer, and the sample peak shows protein scattering.
• Boundaries form as the sample moves down, which is measured over time.
• Boundaries are measured due to absorbance using a monochromator.

Raw Data of Sedimentation Velocity Profile

• Reference meniscus and sample meniscus are identified.
• The solution plateau and zero value are indicated.
• The boundary shows individual time points as the protein moves down the sector.
• The sample plateau represents the protein sedimenting down.
• The bottom of the cell shows scattering when the protein reaches it.
• The number of boundaries indicates the number of protein species in solution (one or two or more).

Computational Programs for Data Fitting

• SEFIT is a program used to fit complex data with multiple species.
• It breaks down data into blocks and models the data within each block, rather than trying to fit all of the data together at once.
• This approach allows to derive much more accurate data for more complex mixtures of proteins.
• A good fit is indicated by a small root mean square deviation (RMSD) between the data and the predicted model.
• Residuals represent bias in the data; tight residuals without scattering are desired.

Models and Data Accuracy

• Model 1 and Model 2 data sets are compared.
• The data with the lower RMSD and tighter residuals is considered more accurate.
• Data points fitting the model well indicate a reliable model representative of the raw data.

Complex Mixtures and Oligomeric States

• Data from an AUC experiment with a complex mixture of proteins, such as APOE3 and APOE4, are analyzed.
• The proteins form various oligomeric states, including monomers, tetramers, octamers, 12-mers, and 16-mers.
• APOE4 is shown to be more prone to forming oligomers than APOE3.

Shape Information

• Shape information can be derived from AUC experiments.
• A spherical protein sediments faster than an elongated protein due to less friction.
• This data can be used to model protein shape and provide more accurate information about protein mass.
• The ribosome example is given, with 30S and 50S subunits combining to form a 70S complex due to shape changes.

Frictional Ratio

• The frictional ratio changes as APOE3 and APOE4 form oligomers.
• The monomer is elongated, but the overall complex becomes more spherical as more proteins bind together, resulting in a change in the frictional ratio.
• A value of one represents a perfect sphere.

Case Studies

• AUC is valuable for understanding protein behavior and interactions.
• Example 1: Antigen 43
  • Antigen 43, found on the bacterial cell surface, allows bacteria to stick together and form biofilms.
  • AUC experiments showed that antigen 43 forms dimers in solution.
  • Oligomerization tends to be concentration-specific, with higher concentrations favoring oligomer formation.
  • Mutations causing the protein to straighten out resulted in a change in frictional ratio.
• Example 2: Antibody-Based Inhibitor (FAB) binding to antigen 43.
  • Differentiation of an antigen 43 dimer from an antigen 43-FAB complex can be determined using AUC due to their different shapes, even if they have similar molecular weights.

Sedimentation Velocity Experiments Summary

• Quaternary state of a protein (monomer, dimer, tetramer).
• Protein complexes and heterozymer formation.
• Protein binding to inhibitors or ligands.
• Shape information.

Other Methods for Determining Quaternary Structure

• SAXS (Small-Angle X-ray Scattering)
  • Requires a synchrotron.
  • Provides information about the shape and size of the protein in solution.
  • Generates an envelope of the protein.
  • Information obtained is approximate.
• Blue Native PAGE
  • Kumasi brilliant blue is added to the protein, which gives the protein charge, and then the native protein is run on a gel to determine the protein quaternary structure.
  • Kumasi brilliant blue gives the protein a charge.
  • Sizes obtained are approximate because the markers may have different shapes than the protein.
• Size Exclusion Chromatography
  • Proteins are passed through a resin column, with larger proteins moving faster than smaller proteins.
  • The column is calibrated with proteins of known molecular weight.
  • The shape of the proteins can affect the results, making it necessary to match the shape of the reference proteins to the sample proteins for decent information.
  • AUC does not have this limitation because it can derive shape information directly from the sample.