2-2

Amino acid structures

methionine (M) isoleucine (I) valine (V) leucine (L) aspartic glutamic
acid (D) acid (E)

phenylalanine (F) tyrosine (Y) tryptophan (W) aspargine (N) glutamine (Q)

serine (S) threonine (T)

lysine (K) arginine (R) histidine (H) glycine (G)

alanine (A) cysteine proline

+
2
+
3
 Peptide bond formation 2-4

©Alberts et al. (1998)

 You should know the structure of a polypeptide chain (protein)!

 2-6

Protein structure: overview

Structural element Description
primary structure amino acid sequence of protein
secondary structure helices, sheets, turns/loops
tertiary structure folded structure of whole protein
• includes disulfide bonds
quaternary structure assembled complex (oligomer)
• homo-oligomeric (1 protein type)
• hetero-oligomeric (>1 type)
 2-9

Protein structure: sheets (detail)

- notice the difference

in H-bonding pattern
between parallel and
anti-parallel beta-sheets

- also notice orientation

of side chains relative
to the sheets

‘twisted’
 2-12

Types of non-covalent interactions

bond “bond”
interaction nature length strength example
ionic electrostatic 1.8-4.0 Å 1-6 positive: K, R, H,
(salt bridge) (3.0-10 Å kcal/mol N-terminus
for like negative: D, E,
charges) C-terminus
hydrophobic entropy - 2-3 hydrophobic side chains
(M,I,L,V,F,W,Y,A,C,P)
H-bond H-bonding 2.6-3.5 2-10 H donor, O acceptor
van der attraction/ 2.8-4.0 <1 closely-spaced atoms;
Waals repulsion if too close, repulsion
aromatic- p-p 4.5-7.0 1-2 F,W,Y (stacked)
aromatic
aromatic- H-bonding 2.9-3.6 2.7-4.9 N-H donor to F,W,Y
amino group

these all contribute to some extent to protein structure & stability;

- important to understand extremophilic (or any other) proteins
 2-13
Ikatan pada Struktur tersier
hydrophilic amino acids (D, E, K, R, H, N, Q)
- these amino acids tend to interact extensively with solvent in
context of the folded protein; the interaction is mostly ionic and H-
bonding
- there are instances of hydrophilic residues being buried in the
interior of the protein; often, pairs of these residues form salt
bridges

hydrophobic amino acids (M, I, L, V, F, W, Y, A*, C, P)

- these tend to form the ‘core’ of the protein, i.e., are buried
within the folded protein; some hydrophobic residues can be
entirely (or partially) exposed

small neutral amino acids (G, A*, S, T)

- less preference for being solvent-exposed or not
 2-14

The disulfide bond

oxidation
protein protein protein protein
+ + 2 H+ + 2 e-
reduction

• disulfide bond formation is a covalent modification; the

oxidation reaction can either be intramolecular (within the same
protein) or inter-molecular (within different proteins, e.g.,
antibody light and heavy chains). The reaction is reversible.

- most disulfide-bonded proteins are extracellular

(e.g. lysozyme contains four disulfide bonds);
the conditions inside the cytosol are reducing,
meaning that the cysteines are usually in reduced form

- cellular enzymes (protein disulfide isomerases) assist

many proteins in forming proper disulfide bond(s)
 2-16

Folding of RNAse A in the test tube

denaturation renaturation

Incubate protein 100-fold

in guanidine dilution of protein
hydrochloride into physiological
(GuHCl) buffer
or urea (aggregation)
- the amino acid sequence of a polypeptide is sufficient to
specify its three-dimensional conformation

Thus: “protein folding is a spontaneous process that does not

require the assistance of extraneous factors”
 2-25

Antibiotics & protein synthesis

antibiotics can be useful tools for manipulating translation, folding
antibiotic effect
inhibits the eukaryotic peptidyltransferase;
prevents release of the polypeptide chain. Can
cyclohexamide
be used to isolate ribosome-nascent chain
complexes
chloramphenicol inhibits the prokaryotic peptidyltransferase
causes premature chain termination and release
from ribosome. Puromycin is similar to a
puromycin tyrosyl-tRNA and acts as a substrate during
elongation. Once added to the carboxyl end of
the nascent chain, protein synthesis is aborted
tetracycline inhibits aminoacyl tRNA binding to the A-site
kanamycin causes misreading of the mRNA
streptomycin causes misreading of the mRNA
 2-32

Protein denaturants
• high temperatures
- cause protein unfolding, aggregation

• low temperatures
- some proteins are sensitive to cold denaturation

• heavy metals (e.g., lead, cadmium, etc.)

- highly toxic; efficiently induce the ‘stress response’

• proteotoxic agents (e.g., alcohols, cross-linking agents, etc.)

• oxygen radicals, ionizing radiation

- cause permanent protein damage

• chaotropes (urea, guanidine hydrochloride, etc.)

- highly potent at denaturing proteins;
often used in protein folding studies