CH 4 (LD) Flashcards
Peptides and proteins
chains of amino acids strung together in sequence via peptide bonds
- can be very short (dipeptide (2 a.a.), tripeptide, tetrapeptide)
- can be very long (over 2000 aa)
Peptide: 2-50 aa in length
Protein: more than 50 aa in length
What is the relative mass of a 100 aa long polypeptide and a 2000 aa long polypeptide?
100 aa: 100 x 110 = 11,000 Da
2000 aa: 2000 x 110 = 220, 000 Da
Conformation
spatial arrangement of atoms in a 3D space dependent on the rotation of a bond or bonds is a molecule’s conformation
Configuration
change in protein by breaking and reforming bonds
Protein diversity
we can determine the expected sequence and size of almost every polypeptide through analysis of its genome
- e.coli: 4000 different proteins
- fruit fly: 14,000 proteins
- humans: 20,000 different proteins
- diversity in their shapes
Proteomics
study of a large set of proteins
Globular Proteins
water-soluble, compact, roughly spherical macromolecules
ex: hemoglobin
Fibrous Proteins
mechanical support
ex: collagen
2D Electrophoresis
samples are separated by two via two dimensions:
1) by molecular weight
2) by pH - proteins migrate to their isoelectric point
Protein Structure
- proteins come in many shapes and sizes
- 4 levels of organization: primary, secondary, tertiary, and quaternary
Primary Structure
- its amino acid sequence
N-terminus to C-terminus
Secondary structure
- regularities in local conformations maintained by H bonds btwn amide hydrogens and carbonyl oxygen in the peptide backbone
- alpha helices (coils) and beta sheets (arrows)
Tertiary Structure
- completely folded and compacted polypeptide chain
- many proteins consist of multiple distinct globular units called domains
- domains: typically 50-300 aa in length
Quaternary Structure
association of two or more polypeptide chains into a multisubunit or oligomeric protein
Ex: Hemoglobin
- reaches all 4 levels of organization/ structure
- transport of oxygen through the bloodstream within red blood cells
X-ray crystallography
- technique to determine the 3D conformation of proteins
- a beam of collimated x-rays (parallel) is aimed at a crystal of protein molecules
- electrons in the crystal diffract the x-rays and the pattern is recorded
- mathematical analysis is performed on the diffraction pattern produced by the electron clouds surrounding atoms in the crystal
- the density map allows the mapping of each atom in 3D space
Dorothy Crowfoot Hodgkin
pioneer in X-ray crystallography in use for biomolecules
- solved the structure of penicillin in 1947
- determined the structure of Vitamin B12
- published the structure of insulin
Limitations of X-ray crystallography
- number of calculations to determine position of atoms (solution: computers)
- preparing crystals of suitable quality for X-ray diffraction
Solution: robotics minimize human error and increase speed
Protein Crystallization
- similar to NaCl crystallization (solution with protein is brought to a supersaturated state to crystallize)
- Heat is not used. Other factors are used to precipitate: pH of buffer, type of salt, cofactors
- solution gradually is saturated with precipitant to out-compete the protein for water interaction (ammonium sulfate)
- precipitate forms and if the conditions are correct, crystals form
Crystallizing
since protein crystal contain water molecules, ligands (substrates or inhibitors) can be diffused
- proteins will many times retain their ability to bind these
- Why would this be important when resolving structure? confirmation of correct conformation
Protein Data Bank
- where protein structures are widely
shared
• Databases were created in the 1970’s which are public domain and easily accessible
Representations
- There are various ways in which peptides can be represented:
• Space-fill models
• Simplified cartoon emphasizing the backbone
• Emphasizing amino acid side chains and bonds
Space-fill model
- depicts atoms as spheres
- shows how tightly packed these molecules are
- used to show overall shape of a protein and the surface exposed to water
Backbone structure
- typically depicted emphasizing the alpha helices and beta strands
- shows the interior of the protein
- easier to compare and recognize patterns
Molecular Interactions
- structure emphasizes the structure of the amino acid side chains
- covalent bonds and weak bonds are typically shown
Nuclear Magnetic Resonance
- technique to decipher protein structures
- protein is placed in a magnetic field in solution
- certain atomic nuclei absorb electromagnetic radiation
NMR structure
- because the absorbance is influenced by neighboring atoms, these interactions can be recorded
- combination with amino acid structure allows calculations of structures that fit the observations
Domains
- discrete, independently folded regions of proteins
- some can be as small as 25-30 aa and can be greater than 300 aa
- GODLEN RANGE: the proposed ideal for stability is 50-300aa in length
Remember:
- Peptide: 2-49 aa
- Protein: 50+ aa
Zn fingers
- structural motif in DNA binding domain of some proteins
- multiple types
example: C2H2 Zn Finger with secondary structures beta-beta-alpha motif that allows DNA binding
Pyruvate Kinase
PDB 1PKM Residues: 116-219 (top part) - Size of domain: 104 aa stable Residues: 1-115 and 220-388 (middle part) - size of domain: 284 aa stable Residues: 389-530 (bottom part) - size of domain: 142 aa stable
- the proposed ideal for stability is 50-300aa in length
Homology in Structure
- protein structure can provide evidence for evolutionary conservation
- cytochrome c has been studied and is highly conserved among species
Protein families
- proteins can be grouped into families according to similarities in domain structures and amino acid sequences
- all members have descended from a common ancestral protein
- lactate dehydrogenase and malate dehydrogenase belong to the same family
- structure provides support for homology
- they are present in the same species
- protein families contain related proteins that are present in the same species that are derived from a common ancestor
Domain Classification
- domains are classified according to structure:
- > all-α contains alpha helices
- > all-β contains beta strands
- > α/β class contains regions with both that arise from alternations in the peptide chain
- > α + β class have clusters of each that arise from different regions in the peptide
all-α
example:
PDB: 1BJ5
Human serum albumin (in the bloodstream: transport, osmotic pressure)
-> structures: alpha helices and bundles
all-β
example:
PDB: 1VBS
Homo sapiens peptidylpropyl cis/trans isomerase
- catalyzes cis/trans of oligopeptides at prolyl residues (activation/deactivation)
- dominant features is a beta sandwich
example:
PDB: 1A45
Bos taurus gamma-crystallin
- Crystallins are the dominant structural components of the verterbrate eye lens and are believed to be very long-lived
- comprised of two beta barrel structures
example:
PDB: 1GFL
Aequorea victoria green fluorescent protein
-main feature is the beta barrel structure but contains a central alpha helix
- orientations strands anti-parallel
example:
PDB: 1AQB
Sus scrofa retinol-binding protein (RBP)
- RBPs have diverse functions including transport of Vitamin A from the liver in the form of the lipid alcohol retinol
- Retinol is shown binding in the center of the beta barrel
α/β
example:
PDB 1OYA
Saccharomyces carlsbergensis (first pure culture lager yeast strain) old yellow enzyme
- roles in yeast are though to include oxidative stress programmed cell death
- central fold is an alpha/beta barrel with parallel beta strands connected by alpha helices
example:
PDB 1AHN - Escherichia coli flavodoxin
Electron transfer protein that contains the prosthetic group flavin mononucleotide
5 strand parallel twisted beta sheet surrounded by alpha helices
example:
PDB 1ERU Homo sapiens thioredoxin
The major cell protein disulfide oxidoreductase; similar to E.coli flavodoxin except this contains a single antiparallel beta strand
example:
PDB 1ABE E.coli l-arabinose-binding protein
essential component of the active transport of l-arabinose
two domain protein similar to flavodoxin
- l-arabinose is shown binding in the cavity between the two domains
