Lecture 8: Protein 3D-Structure and Levels of Protein Structure

Function of a Protein

Depends on its amino acid sequence

Amino acid sequence confers 3D structure

3D structure confers function

Polymorphic

Having amino acid sequence variants

Most human proteins

Edman Degradation

Classic method of sequencing amino acids

Method to Study Protein Primary Structure

Traditional methods are labeling proteins and or breaking proteins into their parts

Studying Protein Structure through Breaking Bonds

Oxidation with performic acid or reduction by dithiothreitol (breaks disulfide bonds, denatures proteins)

Mass Spectrometry

Provides information on molecular mass, amino acid sequence, and entire proteomes

Measures molecular mass with high accuracy

Can sequence short amino acid sequences (20 to 30 amino acids residues)

Can document the entire cellular proteome

Steps involved in Mass Spectrometry

1. Ionize analytes in a vacuum

2. Introduce charge molecules to electric and or magnetic field

3. Charge molecules move through field as a function of the mass to charge ratio m/z

4. Deduce mass (m) of analyte

Tandem MS

A way to extract amino acid sequence information

Two filters in tandem

The first sorts peptides produced by cleavage

The second measures m/z ratios of charged fragments

Liquid Chromatography

Liquid chromatography tandem MS

Analyzes Complex protein mixtures

Chromatography on complex mixture of peptides

Resolved peptides introduced to MS successively

Identifies proteins and protein abundance

Types of Bonds in Protein Structure

Covalent bonds are the strongest, then electrostatic forces, H-bonds, then Van der Waals interaction forces are the weakest

Relationship between Structure and Function

Proteins can assume an uncountable number of special arrangements (conformations)

Chemical or Structural functions relate to unique three dimensional structure (native structure)

Protein Conformations

Limited number of conformations predominate under biological conditions

Conformations are thermodynamically the most stable, that is lowest free energy

Native

Proteins in any functional, folded conformations

Stability

Tendency of a protein to maintain a native conformation

Unfolded proteins have high conformational entropy

Intrahelical Hydrogen Bonds

Between hydrogen atom attached to the electronegative nitrogen atom of residue n and the electronegative carbonyl oxygen atom of residue n+4

Confers significant stability

Beta Conformation

Backbone extends into a zigzag

Organizes polypeptide into sheets

The beta strand is a single protein segment

The beta sheet is several strands in beta conformation side by side

Adjacent Polypeptide

Chains in a beta sheet can be antiparallel or parallel

Antiparallel (opposite conformation) occurs more frequently

Parallel is the same orientation

H bonds form between the backbone atoms of adjacent segments

Tertiary Structure

Overall, three-dimensional arrangement of all the atoms in a protein

Weak interaction and covalent bonds hold interacting segments in position

Quaternary Structure

Arrangement of 2+ separate polypeptide chains in three dimensional complexes

Structural Biology

Study of three-dimensional structures of biomolecules including proteins, nucleic acids, lipid membranes, and oligosaccharides

Employs biochemical approaches, physical tools and computational methods

X Ray Crystallography

Pattern of diffracted x-rays is collected directly, and an image is reconstructed by mathematical techniques

Limited to molecules that can be crystallized

Infromation obtained depends on the degree of structural order in the sample

Protein Crystallography Steps

1. X ray diffraction patterns from protein crystals

2. Three-dimensional electron density map using a Fourier transform

3. Localized atomic nuclei

4. Complete protein structure

Physical Environment in a Crystal

Not identical to the physical environment in solution or in a living cell

Imposes a space and time average on the structure

Provides little information about molecular movement of proteins

Crystal derived structure usually represents a functional conformation

Nuclear Magnetic Resonance NMR

Carried out on molecules in solution

Can measure the distance between protein atoms

Captures dynamics of protein structure like conformational change, protein folding, interactions with other molecules

Nuclear Spin

NMR measures nuclear spin angular momentum a quantum mechanical property of atomic nuclei

Nuclear spin of H1, C13, N15, F19, and P31 gives rise to an NMR signal

Nuclear spin generated a magnetic dipole

Cryo-electron Microscopy (cryo-EM)

Sample of the structure of interest is quick frozen in vitreous (or noncrystalline) ice and kept frozen while being observed in two dimensions with the EM

Greatly reduces damage to the specimen

Useful for determining the molecular structure of large dynamic, macromolecular complexes and integral membrane proteins

AlphaFold

A revolutionary approach to protein structure determination

An AI program from google and deepmind

Will take the amino acid sequence and give you the 3D structure

It solves two problems, Sequence Structure gap and Protein folding