Download of a protein

Proteins
Attila Ambrus
versatile functions in biological systems
linear polymers of amino acids
spontaneous folding to 3D structures that eventually determines function
structure dictates
function
(DNA replication machinery)
wide range of functional groups, most of them are chemically reactive
functional groups account for function (e.g. enzymes)
complexes with other biomacromolecules (proteins, RNA/DNA,
lipids, carbohydrates, inorganics (e.g. ions), etc.) adopt even more
functionalities that proteins alone lack
some proteins are rigid, some are flexible: rigid proteins may work
for connective tissues or cytoskeleton while flexible ones can assemble
with other molecules for more complex functions (e.g. transmit some
kind of information in or between cells)
flexibility and function
(the protein lactoferrin undergoes a substantial conformational change
upon binding Fe3+ ; apo- and holo-enzymes)
Alpha-amino acids
building blocks of proteins
four different substituents around (alpha-)carbon: chirality (except Gly)
CORN rule:
if COOH, R, NH2 are clockwise: D-form,
anticlockwise: L-form
L=S
(except for cysteine)
“side chain”
absolute configuration
(Cahn–Ingold–Prelog [CIP] system)
Ionization state of amino acids as a function of pH
(without side chain contributions)
side
chains
Side
chains
they differ in size, shape, charge, H-bonding capacity, hydrophobic
character and chemical reactivity
twenty amino acids build up all proteins in all species in the evolutionary
tree (with few exceptions; this “alphabet” is several billion years old)
hydrophobic effect in proteins: hydrophobic core resisting
contact with water (apolar character), multimerization
surfaces (protein-protein interactions)
polar side chains prefer being on the surface contacting water
Proline is a special amino acid
the ring structure markedly influences local protein structure due to its
rigid nature (see also cis/trans peptide bonds later)
Aromatic side chains
reactive
Determination of protein concentration
# of Tyr, Trp and S-S bonds count for e of a protein
Polar/uncharged amino acids
reactive
additional
asymmetric
center
Cysteine is also special in a way…
much more reactive than -OH
two Cys –SHs can form disulfide bonds (-S-S-, by oxidation,
forming cystine) that is particularly important in stabilization of
the 3D structure of proteins
Polar/charged amino acids
at near neutral pH, depending on
local environment (catalytic effects,
enzyme active centers)
Aspartic
acid
Glutamic
acid
in special environments/settings in a protein Asp/Glu can be (partially
or transiently) protonated that generally has an important functional
role in enzymatic mechanisms
Why these amino acids (why not others)?
they are versatile enough for structure and function of necessary proteins
/enzymes for life
they were probably available from prebiotic reactions (before the origin
of life)
other possible amino acids may be too reactive for the purpose (e.g.
homoserine or homocysteine)
spontaneous cyclization
(limitations for protein structure)
Peptide bond
residue
condensation
dihedral angle: w
(amide bond)
w=0o for cis, 180o for trans isomer (isomerization is slow [10-100 s], but can be
facilitated by peptidyl prolyl isomerases; normal protein folding is 10-100 ms)
endergonic reaction under most conditions, needs input of free energy
the peptide bond is kinetically stabilized (metastable) since the lifetime of
a peptide bond in water is ~1000 years (in the absence of a catalyst)
in folded proteins overwhelmingly (~1000:1) the trans isomer dominate
(for X-Pro peptide bonds this ratio
only ~3:1!;
with is
proline
the similar state of energy)
magnitudes
ofinthe
steric
clashes
two resonance forms, Ea=~20 kcal/mol,
lesssimilar
reactive than esters, detection
effects
are
cis
configuration
of peptide bond: at 190-230 nm (UV spectrometry)
60%
40%
relatively high dipole moment in the double-bonded form (~3.5 D),
lining up these dipoles e.g. in an alpha-helix produces great net dipole
moments (important in physico-chemical properties of proteins)
peptide bonds (proteins) can be broken down to amino acids (or smaller
peptides) chemically by acids or bases (generally with 6 M HCl, 110 oC,
18-96 h or 2-4 M NaOH, 100 oC, 4-8 h) or enzymatically by peptidases
(proteinases, proteases, see later)
Protein termini
the protein chain has a polarity (the two ends of the chain
are different)
by convention, the –NH2 terminus is put at the start of
writing the sequence (Leu-Phe-Gly-Gly-Tyr is another oligopeptide with indeed differing properties)
Backbone and side chains
distinctive side chains
(variable parts)
main chain or backbone
(repeating/constant)
there are great H-bonding potentials in the backbone: N-H is a good donor,
C=O is a good acceptor
they interact with one another and with functional groups from side chains
and stabilize structural elements in proteins
proteins generally contain 50-2000 amino acids (a muscle
protein contain 27,000 amino acids)
sequences of small numbers of amino acids are called oligopeptides or just peptides (although if they serve already
protein-like functions, they may be called miniproteins)
the average molecular weight of an amino acid is ~110 g/mol,
hence the molecular weight (MW) of a protein generally
ranges from 5,500 to 220,000 g/mol
they also use as a unit of molecular weight of biomacromolecules the Dalton (after John
Dalton [1766-1844] who suggested for the unit of atomic mass the weight of an H atom in
1803; since 1961 we use 12C as a basis of atomic weight especially due to the discovery of
elemental isotopes in 1912). Designation of Dalton as a unit can be Da, D, d and kDa, kD, kd;
practically the same number numerically as the regular MW, so for example:
50 kDa=~50,000 g/mol
Cross-linking disulfide bonds
oxidation occurs especially for extracellular proteins (intracellular environment is generally too reductive for the S-S bond)
periplasm of bacterial strains are also rather oxidative and
may support correct folding if proteins are stabile with
specific –SH groups being oxidized (advantage of a periplasmic
protein over-expression system, see protein purification, later)
rarely there are other side chains participating in cross-links
in proteins, like in collagen fibers in connective tissue or in
fibrin blood clots
Frederick Sanger, 1953
amino acid sequence of insuline (protein hormone, the very
first protein sequence determined)
~2,000,000 protein sequences are known today!
amino acid sequence = primary structure (of a protein)
What is a protein sequence good for?
essential to get to know the mechanism of action (e.g. catalytic mechanism
of an enzyme)
proteins with novel properties can be generated by varying the sequences
of known proteins (the science of protein engineering)
the primary sequence determines the 3D structure of the protein and it
is the link between the genetically encoded information in DNA and the
actual biological function of the protein
analysis of the relation between primary and 3D structures uncovers
mechanisms of folding/unfolding/refolding of proteins
sequence determination is a component of molecular pathology (searching
for mutations that determines predisposition to various diseases – alterations in amino acid sequence may result in abnormal function and disease)
sequence of a protein reveals much about its evolutionary history, protein
sequences that resemble to one another likely to have a common ancestor,
hence molecular events in evolution can be traced down (phylogenetics –
“relatedness”, molecular paleontology)