TEMA 4 (I) : Aminoácidos. Enlace peptídico.

Análisis de la estructura
primaria de proteínas.

Learning goals:
• Structure and naming of amino acids
• Structure and properties of peptides
• Ionization behavior of amino acids and peptides
• Methods to characterize peptides and proteins

Proteins:
Main Agents of Biological Function

• Catalysis
– enolase (in the glycolytic pathway)
– DNA polymerase (in DNA
replication)
• Transport
– hemoglobin (transports O2 in the
blood)
– lactose permease (transports
lactose across the cell membrane)
• Structure
– collagen (connective tissue)
– keratin (hair, nails, feathers, horns)
• Motion
– myosin (muscle tissue)
– actin (muscle tissue, cell motility)

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 Amino Acids:
Building Blocks of Protein
• Proteins are linear heteropolymers of -amino acids

• Amino acids have properties that are well-suited to

carry out a variety of biological functions
– Capacity to polymerize
– Useful acid-base properties
– Varied physical properties
– Varied chemical functionality

Amino acids share many features, differing only at the R substituent

All amino acids are chiral (except glycine): Proteins only contain L amino
acids

• The -carbon always has four

substituents and is tetrahedral
• All (except proline) have:
– an acidic carboxyl group
– a basic amino group
– an -hydrogen connected to
the -carbon
• The fourth substituent (R) is
unique
– In glycine, the fourth
substituent is also hydrogen

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 L-Alanine

AMINOÁCIDOS

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 Amino Acids: Atom Naming
• Organic nomenclature: start from one end
• Biochemical designation:
– start from ‐carbon and go down the R‐group

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 ESCALAS DE HIDROFOBICIDAD

Octanol Coctanol
K=
Cagua
Agua

Coctanol n AA int
ΔGa-o = - RT ln K = - RT ln ΔGe-i = - RT ln K = - RT ln
Cagua n AA ext.

Amino Acids: Classification

Common amino acids can be placed in five

basic groups depending on their R substituents:
• Nonpolar, aliphatic (7)
• Aromatic (3)
• Polar, uncharged (5)
• Positively charged (3)
• Negatively charged (2)

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 Amino Acids 21 & 22:
(descubierto en) (1986) (2002)

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 G A
P
V
Flexibilidad, presente en Iminoácido, rígidez
acodamientos

L I M

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F
Y

Fosforilación por W
Tyr kinasas

These amino acid side chains absorb UV light at 270–280 nm

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 Puentes disulfuro
S C
T estabiliza la conformación

Fosforilación por kinasas

O-glicosilación

N-glicosilación N Q

These amino acids side chains can form hydrogen bonds.

Cysteine can form disulfide bonds.

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 D
E

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AA 21: Selenocisteina en codón UGA

(pro- y eucariotas)
AA 22: Pirrolisina en codón UAG
(procariotas)

Modified Amino Acids

Found in Proteins
(irreversible modifications)

Reversible Modifications

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 Aminoácidos no proteinogénicos

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Protein Sequences as Clues to

Evolutionary Relationships
• Sequences of homologous proteins from a wide
range of species can be aligned and analyzed for
differences
• Differences indicate evolutionary divergences
• Analysis of multiple protein families can indicate
evolutionary relationships between organisms,
ultimately the history of life on Earth
• Highlights the critical amino acids for the
biological function of the protein

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 Sustituciones conservativas de aminoácidos en la naturaleza
(no repercuten sobre la actividad biológica de la proteína)

G=A=S (aas pequeños)

A=V (aa pequeño, ramificado)

V=I=L=M (cadena alifática hidrofóbica)

I=L=M=Y=F=W (hidrofobicidad alifática o aromática)

K=R=H (carga positiva)

D=E=Q=N (carga o puentes de H)

S=T=Q=N (puentes de H)

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Chemical Environment Affects pKa Values

‐carboxy group is much more acidic than in carboxylic acids
‐amino group is slightly less basic than in amines

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 Disociación de un ácido. Ec. de Henderson-Hasselbach

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Ionization of Amino Acids Cation  Zwitterion  Anion

At acidic pH, the carboxyl group is
protonated and the amino acid is in the
cationic form.
At neutral pH, the carboxyl group is
deprotonated but the amino group is
protonated. The net charge is zero; such
ions are called Zwitterions.
At alkaline pH, the amino group is neutral –
NH2 and the amino acid is in the anionic
form.

Amino acids with uncharged side chains, such

as glycine, have two pKa values:
The pKa of the ‐carboxyl group is 2.34
The pKa of the ‐amino group is 9.6
It can act as a buffer in two pH regimes. 1 equivalent of OH– = 0.1 M NaOH added.

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 Amino acids carry a net charge of zero
at a specific pH (the pI)

• Zwitterions predominate at pH values between the pKa

values of the amino and carboxyl groups
• For amino acids without ionizable side chains, the Isoelectric
Point (equivalence point, pI) is
pK1  pK 2
pI 
2

• Ionizable side chains can be also titrated

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Aminoácidos diamino
monocarboxílicos

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Aminoácidos monoamino
dicarboxílicos
+1
0

pI=3,22
-1
-2

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 Reacciones químicas de los aminoácidos:
- COOH:
- Amidas: R-COOH + R’-NH2 R-CO-NH2

- Ésteres: R-COOH + R’-OH R-CO-O-R’ Protección durante la síntesis química (R’, fenol)

- NH2:
- Protección grupo amino:

- Bases de Schiff: R’-COH + R-NH2 R-CO-NH2

Oxidación de tioles/reducción de puentes disulfuro:

β-mercaptoetanol
(rompe los S-S)

Protein disulfuro
isomerasa (PDI)
(forma los S-S en el RE)

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 Enlace peptídico:

Recordad, en la célula necesita energía y un molde !!!

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Peptides: A Variety of Functions

• Hormones and pheromones
– insulin (think sugar)
– oxytocin (think childbirth)
– sex-peptide (think fruit fly mating)

• Neuropeptides
– substance P (pain mediator)

• Antibiotics
– polymyxin B (for Gram – bacteria)
– bacitracin (for Gram + bacteria)

• Protection, e.g., toxins

– amanitin (mushrooms)
– conotoxin (cone snails)
– chlorotoxin (scorpions)

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 Péptido

Using full amino acid names: Serylglycyltyrosylalanylleucine

Using the three-letter code abbreviation: Ser-Gly-Tyr-Ala-Leu
For longer peptides (like proteins) the one- letter code can be used: SGYAL

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Dimensiones enlace peptídico:

Pauling y Corey

Enlace Sencillo Doble E. Péptídico

C–N 1.46 Å 1.27 Å 1.33 Å
C–O 1.43 Å 1.20 Å 1.24 Å
C–C 1.51 Å 1.51 Å

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 Enlace peptídico : Híbrido resonancia entre 2 estructuras electrónicas extremas

Consecuencias: Es polar, está en un plano y está estabilizado

por resonancia (isomería cis-trans)

Peptidil-prolil cis-trans isomerasas (PPI)

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Se sintetiza trans:

Dador de H

Aceptor de H

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 Structure of the Peptide Bond
• Structure of the protein is partially dictated by the
properties of the peptide bond
• The peptide bond is a resonance hybrid of two
canonical structures
• The resonance causes the peptide bonds
– to be less reactive compared to esters, for
example
– to be quite rigid and nearly planar
– to exhibit a large dipole moment in the favored
trans configuration

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Restricciones conformacionales de la cadena polipeptídica

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 The Rigid Peptide Plane and
the Partially Free Rotations

• Rotation around the peptide bond is not permitted

• Rotation around bonds connected to the alpha
carbon is permitted
•  (phi): angle around the -carbon—amide
nitrogen bond
•  (psi): angle around the -carbon—carbonyl
carbon bond
• In a fully extended polypeptide, both  and  are
180°

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Distribution of  and  Dihedral Angles

• Some  and  combinations are very unfavorable because of

steric crowding of backbone atoms with other atoms in the
backbone or side chains

• Some  and  combinations are more favorable because of

chance to form favorable H‐bonding interactions along the
backbone

• A Ramachandran plot shows the distribution of  and 

dihedral angles that are found in a protein

• shows the common secondary structure elements

• reveals regions with unusual backbone structure

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 Ramachandran Plot

Oi-1 Oi

Oi-1 Ni-1

Ni Hi+1
Hi+1 Oi-1

Oi-1 Oi

values of phi and psi for amino acid residues (except

Gly) in the enzyme pyruvate kinase overlaid on the
plot of theoretically allowed conformations

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Formas de representar la estructura de proteínas

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 The coalescence of hydrophobic residues in the core of the protein:

Figure 4-5 Essential Cell Biology (© Garland Science 2010)

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Non-covalent interactions:

Figure 4-4 Essential Cell Biology (© Garland Science 2010)

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Figure 4-6 Essential Cell Biology (© Garland Science 2010)

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Favorable Interactions in Proteins

• Hydrophobic effect
– Release of water molecules from the structured solvation layer
around the molecule as protein folds increases the net entropy
• Hydrogen bonds
– Interaction of N-H and C=O of the peptide bond leads to local
regular structures such as -helices and -sheets
• London dispersion (Van der Waals interactions)
– Medium-range weak attraction between all atoms contributes
significantly to the stability in the interior of the protein
• Electrostatic interactions
– Long-range strong interactions between permanently charged
groups
– Salt-bridges, esp. buried in the hydrophobic environment strongly
stabilize the protein

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 Protein Stability and Folding
• A protein’s function depends on its 3D‐structure
• Loss of structural integrity with accompanying loss of
activity is called denaturation
• Proteins can be denatured by:
• heat or cold
• pH extremes
• organic solvents
• chaotropic agents: urea and guanidinium
hydrochloride

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NH2
O=C
NH2

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 Ribonuclease Refolding Experiment
• Ribonuclease is a small protein that
contains 8 cysteines linked via four
disulfide bonds
• Urea in the presence of 2‐
mercaptoethanol fully denatures
ribonuclease
• When urea and 2‐mercaptoethanol are
removed, the protein spontaneously
refolds, and the correct disulfide bonds
are reformed
• The sequence alone determines the
native conformation
• Quite “simple” experiment, but so
important it earned Chris Anfinsen the
1972 Chemistry Nobel Prize

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How can proteins fold so fast?

• Proteins fold to the lowest-energy fold in the microsecond to second
time scales. How can they find the right fold so fast?
• It is mathematically impossible for protein folding to occur by
randomly trying every conformation until the lowest-energy one is
found (Levinthal’s paradox)
• Search for the minimum is not random because the direction toward
the native structure is thermodynamically most favorable

100 aa 5s tiempo real

1aa ------- 10 conformaciones distintas, 10100 conformaciones para proteína

Explorar cada conformación 10-13s -----------------1077 años

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 Proteins folding follow a distinct path

Nucleación de estructura
secundaria

Colapso hidrofóbico

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Energética del plegamiento espontáneo de las proteínas: embudos de energía

N = estado nativo proteína (mínimo de energía)

Sin intermediarios Intermediarios de

Con intermediarios plegamientomuy
de plegamiento y de plegamiento Muy estable y
muchas vías estables
vía única

Intermediarios de plegamiento

- Plegamiento asistido por chaperonas/chaperoninas (in vivo): Hsp70, Hsp60, TriC,…..

- Catalizadores del plegamiento: PDI, PPI

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Chaperones prevent misfolding

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 Chaperonins facilitate folding

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Protein misfolding is the basis of numerous human diseases

Amiloidosis: formación de fibra amiloide

Extracelular: Alzheimer: β-amiloide, type 2

diabetes: amilina

Intracelular: Huntington: huntingtina, Parkinson:

α-sinucleina, Demencia Fronto-temporal Tau…….

- Enfisema: mutaciones en el gen de la alfa1-

antitripsina que conducen a la agregación de la
proteína, impidiendo su secreción.

- Enfermedad de las vacas locas: causada por

una proteína de plegamiento anómalo (PrP)

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 Google Deepmind’s
AlphaFold proved in 2020 that
it could predict the 3D shapes
of proteins with high accuracy

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 Protein Sequencing
• It is essential to further biochemical analysis that we
know the sequence of the protein we are studying
• Actual sequence generally determined from DNA
sequence
• Edman Degradation (Classical method)
– Successive rounds of N-terminal modification, cleavage, and
identification
– Can be used to identify protein with known sequence
• Mass Spectrometry (Modern method)
– MALDI MS and ESI MS can precisely identify the mass of a
peptide, and thus the amino acid sequence
– Can be used to determine post-translational modifications

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Análisis de la estructura primaria de las proteínas:

1º- secuenciación DNA

2º- secuenciación de péptidos y proteínas:

1ªProteasa
2ªProteasa
3ªProteasa

Secuencia completa

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 Edman’s Degradation

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Mass Spectrometry
Protein
sequence
ALVKEVALGYKIGRFGGKAGVRNTQV
Protein digestion: Trypsin (Cleaves after K or R)

ALVK
EVALGYK
Peptides IGR
obtained FGGK
AGVR
NTQV

Trypsin peptide signature or fingerprint of the investigated protein

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MALDI‐TOF MS
-MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight)

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 ESI‐MS Procedures

TANDEM MS

ESI MS: Electrospray

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Síntesis de péptidos:

9-fluorenilmetoxicarbonil (Fmoc)

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