Proteins

Attila Ambrus

versatile functions in biological systems

linear polymers of amino acids

spontaneous folding to 3D structures that eventually determines function

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

structure dictates function

(DNA replication machinery)

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)

“side chain”

L=S (except for cysteine)

absolute configuration (Cahn–Ingold–Prelog [CIP] system)

CORN rule: if COOH, R, NH2 are clockwise: D-form,

anticlockwise: L-form

Ionization state of amino acids as a function of pH(without side chain contributions)

side chainsSide 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 evolutionarytree (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 itsrigid 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 of a protein

Polar/uncharged amino acids

reactiveadditionalasymmetric center

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

Cysteine is also special in a way…

Polar/charged amino acids

at near neutral pH, depending on local environment (catalytic effects,

enzyme active centers)

Asparticacid

Glutamicacid

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)

(amide bond)

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 is only ~3:1!; similar state of energy)

two resonance forms, Ea=~20 kcal/mol, less reactive than esters, detection

of peptide bond: at 190-230 nm (UV spectrometry)

Peptide bond residue

dihedral angle: =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)

condensation

steric clashes in cis configuration

with proline the magnitudes of theeffects are similar

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)

60% 40%

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 oligo-peptide with indeed differing properties)

distinctive side chains (variable parts)

main chain or backbone (repeating/constant)

Backbone and side chains

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 chainsand 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 oligo-

peptides 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 (intracel-lular 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 periplasmicprotein 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 veryfirst 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 mechanismof an enzyme)

proteins with novel properties can be generated by varying the sequencesof known proteins (the science of protein engineering)

the primary sequence determines the 3D structure of the protein and itis 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 (searchingfor mutations that determines predisposition to various diseases – alter-ations in amino acid sequence may result in abnormal function and disease)

sequence of a protein reveals much about its evolutionary history, proteinsequences that resemble to one another likely to have a common ancestor,hence molecular events in evolution can be traced down (phylogenetics – “relatedness”, molecular paleontology)