CH 4 (LD)

Question 1

Peptides and proteins

Answer 1

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

Question 2

What is the relative mass of a 100 aa long polypeptide and a 2000 aa long polypeptide?

Answer 2

100 aa: 100 x 110 = 11,000 Da

2000 aa: 2000 x 110 = 220, 000 Da

Question 3

Conformation

Answer 3

spatial arrangement of atoms in a 3D space dependent on the rotation of a bond or bonds is a molecule’s conformation

Question 4

Configuration

Answer 4

change in protein by breaking and reforming bonds

Question 5

Protein diversity

Answer 5

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

Question 6

Proteomics

Answer 6

study of a large set of proteins

Question 7

Globular Proteins

Answer 7

water-soluble, compact, roughly spherical macromolecules

ex: hemoglobin

Question 8

Fibrous Proteins

Answer 8

mechanical support

ex: collagen

Question 9

2D Electrophoresis

Answer 9

samples are separated by two via two dimensions:

1) by molecular weight
2) by pH - proteins migrate to their isoelectric point

Question 10

Protein Structure

Answer 10

• proteins come in many shapes and sizes

- 4 levels of organization: primary, secondary, tertiary, and quaternary

Question 11

Primary Structure

Answer 11

• its amino acid sequence

N-terminus to C-terminus

Question 12

Secondary structure

Answer 12

• 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)

Question 13

Tertiary Structure

Answer 13

• completely folded and compacted polypeptide chain
• many proteins consist of multiple distinct globular units called domains
• domains: typically 50-300 aa in length

Question 14

Quaternary Structure

Answer 14

association of two or more polypeptide chains into a multisubunit or oligomeric protein

Question 15

Ex: Hemoglobin

Answer 15

• reaches all 4 levels of organization/ structure

- transport of oxygen through the bloodstream within red blood cells

Question 16

X-ray crystallography

Answer 16

• 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

Question 17

Dorothy Crowfoot Hodgkin

Answer 17

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

Question 18

Limitations of X-ray crystallography

Answer 18

• 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

Question 19

Protein Crystallization

Answer 19

• 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

Question 20

Crystallizing

Answer 20

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

Question 21

Protein Data Bank

Answer 21

• where protein structures are widely
  shared
  • Databases were created in the 1970’s which are public domain and easily accessible

Question 22

Representations

Answer 22

• 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

Question 23

Space-fill model

Answer 23

• 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

Question 24

Backbone structure

Answer 24

• typically depicted emphasizing the alpha helices and beta strands
• shows the interior of the protein
• easier to compare and recognize patterns

Question 25

Molecular Interactions

Answer 25

- structure emphasizes the structure of the amino acid side chains - covalent bonds and weak bonds are typically shown

Question 26

Nuclear Magnetic Resonance

Answer 26

- technique to decipher protein structures - protein is placed in a magnetic field in solution - certain atomic nuclei absorb electromagnetic radiation

Question 27

NMR structure

Answer 27

- 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

Question 28

Domains

Answer 28

- 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

Question 29

Zn fingers

Answer 29

- 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

Question 30

Pyruvate Kinase

Answer 30

``` 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

Question 31

Homology in Structure

Answer 31

- protein structure can provide evidence for evolutionary conservation - cytochrome c has been studied and is highly conserved among species

Question 32

Protein families

Answer 32

- 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

Question 33

Domain Classification

Answer 33

- 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

Question 34

all-α

Answer 34

example: PDB: 1BJ5 Human serum albumin (in the bloodstream: transport, osmotic pressure) -> structures: alpha helices and bundles

Question 35

all-β

Answer 35

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

Question 36

α/β

Answer 36

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