SCHOOL OF MATHEMATICAL AND NATURAL SCIENCES

BIOCHEMISTRY DEPARTMENT

Protein Biochemistry
BCM 3521

Lecturer: Dr. A. Burger

Protein Biochemistry Outline
TEXT BOOKS
How Proteins Work
Author Mike Williamson
Essential Biochemistry 3rd Edition
Charlotte W. Pratt & Kathleen Cornely

CONSULTATION HOURS
Tuesday 12:00 13:00 Office SF034
Wednesday 12:00 13:00 Office SF034

LECTURE TIMETABLE
TUESDAY 11:00 11:50 Venue A2
WEDNESDAY 11:00 11:50 Venue A1
Protein Biochemistry Outline
ASSESSMENT CRITERIA AND WEIGHTING
Assessment tasks towards calculation of semester mark will include:
Assignment and class tests 25 %
Practical reports and exam 25 %
Semester test 1 25 %
Semester test 2 25 %

Pass requirement is 50 %
Final mark compilation
Semester mark (40 %) + Exam mark (60 %)
A student must get a subminimum mark of 40 % in the Exam
Protein Biochemistry Outline
SEMESTER TEST DATES
Assessment tasks towards calculation of semester mark will include:
Semester Test 1 Wednesday 29th March
Semester Test 2 Wednesday 3rd May
Assignment Wednesday 12th April

Lectures end 18th May

Exams commence 25th May
PROTEIN FOLDING AND ROLE OF
MOLECULAR CHAPERONES
 Cell compartments and folding
eukaryotes
- cytosol ..................................protein synthesis, folding/assembly
- extracellular .........................proteins are exported in folded form
- mitochondria ........................limited protein synthesis; energy production
- chloroplasts ..........................limited protein synthesis; light harvesting
- endoplasmic reticulum.......... import of unfolded proteins; protein processing
- peroxisome ........................... import of folded proteins; anab./catab. pathways
- nucleus ................................. import of folded proteins
- lysosome................................import of unfolded proteins; degradation
bacteria
- cytosol ..................................protein synthesis, etc.
- periplasm .............................import and folding of periplasmic proteins
- extracellular .........................proteins are exported

archaea
- cytosol ..................................protein synthesis, etc.
- extracellular .........................proteins are exported
Protein structure levels

a. Part of primary structure (Blue-hydrophobic, brown-

aromatic; green-polar, olive-sulfur) of glutamate
dehydrogenase; primary is sequence plus any covalent
modifications such as disulfide bonds, phosphorylation, etc

b. Secondary structure (outcome of source prediction), ie

regular elements of backbone geometry e.g alpha and beta
helices, coils

c. Tertiary structure, full three dimensional fold, that is made

up of domains, may contain active sites on the surface

d. Quarternary structure, how proteins pack together

 Secondary structural motifs
Coiled coil of
Four helix bundle Helix turn motif EF hand of Fos-Jun binding
calmodulin to DNA

Four Greek keys

of human b-
crystallin A -- motif of A hairpin of lysozyme
triosephosphate
isomerase barrel
 Native State
Native structure is compact and stabilized by Cytochrome
multiple hydrophobic contacts B562 with Haem

In a folding reaction, the native state has the

lowest free energy
protein folding is spontaneous in principle
in practice, impractically slow

Folding intermediates are flexible, less compact,

with exposed hydrophobicity

Model of folding reaction

Daggett and Fersht
 Folding vs. Aggregation
Folding intermediates can aggregate
with other unfolded polypeptides

Both folding and aggregation depend

on hydrophobic interactions

Normal aggregates are structurally

disordered

Amyloid fibrils are a special type of

aggregate
ordered conformation that is not the
native state
pathogenic
 Folding in vitro vs. in vivo

in vitro in vivo

protein denatured
in a chaotrope
Differences:
1. One has all of the
information immediately
available for folding; the
folding by dilution folding
other process is gradual
in buffer
2. the cellular
environment is very
different (much more
crowded)
folded folded
protein protein
 Folding Thermodynamics

Each point on curve represents a different polypeptide

conformation

Each conformation has a different DG

unfolded

A curve is only one of many possible folding paths

DG intermediate

native

number of internal contacts

compactness
coverage of hydrophobicity
 Folding Landscape
Folding Landscape
many different unfolded and partially
folded states

different folding pathways lead to one

native state

intermediates can persist in local free

energy minima
kinetically trapped, requires
energy to escape minimum
Local Minimum

Native State:
Global
Minimum
 Folding / Aggregation Landscape

multiple intermolecular contacts in

aggregates can make them more stable
than individual native state

amyloid aggregates are the most

stable
 Co-translational protein folding

Fact:
- first ~30 amino acids of the polypeptide chain present
within the ribosome is constrained
(the N-terminus emerges first)
folding

Assumption:
as soon as the nascent chain is extruded, it will start
assembly
to fold co-translationally
i.e., acquire secondary, super-secondary structures
& domains
until complete polypeptide is produced & extruded
 Macromolecular crowding

In vitro E. coli cytosol

<0.1 mg/ml ~340 mg/ml

ribosome
Ellis and Hartl (1996)
FASEB J. 10:20-26 proteins other
chaperonin macromolecules
nucleic acids

When doing experiments in vitro, we should all be thinking about this:

proteins in isolated (pure) systems may not behave as they do in the cell
- binding partner(s) might be missing - cell conditions (pH, salts, etc.
- post-translational modifications might be missing may be dramatically different
 Effects of crowding
Definition:
Molecular crowding is a generic term for the condition where a significant volume of a
solution (cytoplasm) is occupied with things other than water

Fact:
- association constants (ka) increase significantly
- dissociation constants (kd) decrease significantly (kd=1/ka)
- increased on-rates for protein-protein interactions
(see for example Rohwer et al. (2000) J. Biol. Chem. 275, 34909)

Assumption:
- non-native polypeptides will have greater tendency to associate inter-molecularly,
enhancing the propensity of aggregation