L14 - Protein Structure & Function Flashcards
Cells and protein
Cells jam packed with all kinds of protein (and other things)
- human cell ~1-3 billion
Protein function
Drive (almost) everything in the cell - DNA replication - Cell division - Synthesis of the cell membrane - Metabolism - Transport of molecules - Generating energy - Structure And much more…
Roles of proteins
- digestive enzyme/catalytic
- transport
- structural
- hormone signalling
- immunological
- contractile
- storage
- toxins: used by pathogens
- regulatory: physiological processes
Peptides
Short (approx. < 50 aa) polypeptides
___peptide
Very short peptides
- dipeptide, tripeptide, tetrapeptide
Residue
Individual amino acid
Protein composition
A polypeptide: chain of amino acids linked by peptide bonds
Amino acid forms
- unionised form
- ionised form (protonated to NH3+ and deprotonated to COO-): more prevalent as present at physiological/normal pH (~7.4)
Glycine
Simplest amino acid
Proline
Commonly seen at sharp turns of protein structure
Amino acids with S
Cysteine and methionine
Peptide (amide) bond formation
Condensation/dehydration synthesis reaction (release of H2O)
Peptide bond rotation
- rotation at single bonds between alpha carbon and its neighbouring atoms
- no rotation at peptide bonds due to resonance (O-C N-H of bonds are essentially co-planar)
Peptide bond configuration
- typically in trans orientation with alternating side chains
- very rarely in cis as less stable due to steric repulsion of side chains
Protein structure
- complex structure to facilitate varied functions as shape critical to its function
- shape driven by chemical properties and sequences of amino acids in protein
Enzymes
- Long ridged interconnecting structural proteins
- Active site
- Binding causes conformational change
- provide function or strengthen interaction
- lock & key vs induce fit
Primary structure
- unique sequence of amino acids of protein
Primary structure driven by
DNA sequence of gene coding protein
= structure can be deduced from known DNA sequence
Secondary structure
Localised folding of polypeptide
Secondary structure driven by
Hydrogen bonding interactions within polypeptide backbone
Alpha helices
- right handed helix
- normally each turn is 3.6aa with pitch of 5.4 (0.54nm)
- H-bond between N-H and C=O 3 or 4 residues earlier
- tightly packed; almost no free space within
- side chains protrude out from helix
Amino acids in alpha helix
Ethiosine, alanine, leucine, glutamine, lysine
NOT proline, glycine
Beta pleated sheets
H-bond between N-H on one strand and C=O on another strand
Type of beta pleated sheet structures
Parallel, anti parallel
- alternating R groups
Amino acids in beta pleated sheets
- large aromatic residues: tryrosine, phenylalanine, tryptophan
- beta-branche amino acids: threonine, valine, isoleucine
Secondary structure prediction
Different amino acids have propensity to favour structures
= can fairly accurately predict regions from sequence
Tertiary structure
3D shape of protein
Tertiary structure driven by
Chemistry of side chains and interactions between them
Tertiary noncovalent interactions
- ionic bonds
- hydrophobic interactions
- hydrophilic interactions
- dipole-dipole interactions
- Van der Waals forces
Tertiary covalent bonds
Disulphide bond
- formed by cystines
- thiol oxidised, H removed, covalent linkage formed between two sulphur atoms
Tertiary interaction strengths
Disulphide > ionic > hydrogen > Van der Waals
Cofactors
Aka prosthetic groups
- Within protein and coordinated in some proteins (particularly enzymes) using R groups
- may be essential for structure and/or function of protein
- metal ions (Mg, Mn, Zn, Fe, Ca), organic molecules (heme), or vitamins
Quarternary structure
Multiple polypeptide chains - multiple folded protein subunits
- may be dynamic: come together to perform function then separate when not needed or to release something
Quarternary structure driven by
Ionic interactions, H-bonding, hydrophobic interactions (Disulphide possible but uncommon)
Quarternary structure types
- homooligomer: 2 of the same subunits
- heterooligomer: 2 different subunits
Types of proteins
Globular, fibrous, membrane
Globular solubility
Typically soluble in water
Globular function
Functional; metabolic functions (enzyme, transport, immune)
Globular structure
- spherical/globular in shape
- irregular sequence and secondary structure
Globular repetition
Not many repeating elements (structure, sequence within primary or secondary sequence)
Globular and quarternary structures
Moderate to none
Globular stability
Lower stability
Globular example
Enzymes, hemoglobin, antibodies
Fibrous solubility
Typically insoluble in water
Fibrous function
Structural
Fibrous repetition
Often repetitive primary and secondary structure (repeated alpha helices)
Fibrous and quarternary structures
High level
Fibrous stability
Highly stable (to heat, pH etc.)
Fibrous examples
Keratin, actin, collagen, silk, cytoskeleton proteins
Membrane location
Transverse through lipid bilayer
Membrane function
Transport, receptors, signalling, adhesion
Membrane structure by region
- transmembrane region: single alpha helix or alpha helical bundle
- mitochondria and gram negative bacterial: beta barrel transmembrane proteins
Beta barrel
Lots of beta sheets that stack around to form barrel (like sheet of paper)
Membrane polarity
- high degree of non-polar (hydrophobic) amino acids where side chains face out towards membrane
- polar (hydrophilic) side chains face inwards except at top or bottom where no farting membrane and towards soluble environment
Resolution
Distance corresponding to smallest observable feature
- closer than this = one combine blob not two separate objects
ABBE’s diffraction limit
Can only resolve objects that are about half the wavelength of imaging light
ABBE’s diffraction limit visual light example
Visual light: 400-700 nm
= theoretically can only see/resolve items 200-300 nm apart under perfect optical microscope (but this is not common too)
Studying protein structure
Experimental lab determination
- very time consuming
- often ‘impossible’
Ways of studying protein structure
1) X-ray crystallography/diffraction
2) electron microscopy
- single particle cryogenic electron microscopy
X-ray crystallography/diffraction
Very pure protein crystallised into lattice (left in condition where evaporation will take place very slowly)
X-ray crystallography/diffraction calculations
Short wavelength X-rays and basic principles of diffraction to deduce atomic structure of protein
- Bragg’s law (2dsinø = n.wavelength) and some complex math
- use predictable diffractions and back-calculate
X-ray crystallography/diffraction usage
Most common/widely used
- used for ~50 years; revolutionised understanding of biology
X-ray crystallography/diffraction resolution
Atomic resolution: generally highest resolution
X-ray crystallography/diffraction limitations
- slow crystallisation; may take years
- prone to failure; most proteins don’t crystallise, crystallisation may damage protein structure
- doesn’t tell much about how protein moves (somewhat artificial state - not same as when in human body)
X-ray crystallography/diffraction protein
- any size macromolecule
- good for soluble proteins (and SOME membrane proteins)
Electron microscopy radiation
Illuminating radiation type with smaller wavelength
- wavelength of accelerated electron beam ~2.5 pm (>150,000 shorter than light)
Electron microscopy resolution
Theoretical ~1.5 pm (C atom ~ 70 pm)
Practical ~ 0.5 nm
- electron optics complex and hard to control
Electron microscopy images
Inherently noisy thus thousands of images must be combined to increase S/N (signal:noise ratio)
Single particle cryogenic electron microscopy
Freeze suspension of particles in thin layer of virtuous ice in native state
- rapidly emerging technique
Single particle cryogenic electron microscopy advantages over X-ray crystallography/diffraction
- Fast determination (early as a week)
- insight to protein dynamics/movements
- tease out multiple conformations in one sample
- extract active/inactive state or ligand-bound/unbound separately, freeze individually and treat separately
- big protein complexes with lots of subunits (and symmetry) that would never crystallise
Single particle cryogenic electron microscopy proteins
Soluble proteins, MEMBRANE PROTEINS, large protein complexes, dynamic proteins
Levinthal’s paradox
- very large number of degrees of freedom in unfolded polypeptide chain
- 100 aa protein will have 3^198 different conformations
- Require time longer than age of universe to sample all conformations; simple computational prediction of structures under same basis is infeasible
Protein folding time taken
Most correctly fold in ms-µs scale (instantaneously) - how is not known
Influences on protein folding
- Environment: solute, salt conc., pH, temp., macromolecular crowding etc.
- Temporal: co-translational folding as polypeptide is coming off ribosome (N folds before C term)
- Chaperones: other proteins which bind to/prevent misfolding of parts of protein
Protein folding current technology
G enerally quite bad at predicting (historically)
- constantly improving
- if structure of related protein known, better
Computational de novo prediction of tertiary protein structure is improving rapidly
- Google DeepMind - AlphaFold
improving but not accurate enough so typically still need to experimentally investigate structure
