Aminoacids, Peptides and Proteins

1
Amino acids, peptides and

proteins
Johanna Marcela Flórez Castillo,

Ph.D.
2
Performance criteria
Recognizes amino acids, their

classification and physicochemical
properties, the nature of the peptide bond
and the structural levels of proteins.
J.M. Flórez-Castillo 2
Amino acids
3
Carboxyl
group
Amino
group
44
Amino acids are enantiomers in which the chiral center is found in C-

alpha.
"The biosynthesis of a substance that has asymmetric

centers almost always produces a pure stereoisomer."
This Photo by Unknown Author is licensed under

CC BY-SA
This Photo by Unknown Author is licensed under CC BY-SA
J.M. Flórez-Castillo
Voet, D., Voet, J.G. & Pratt, C.W. (2007)
5
Amino acids can act as acids and bases (amphoteric)
Zwitterion
imagen tomada de Lehninger, D.L. Nelson, M., & Cox, M.M. 6 Edición (2014).
6
Amino acids can be classified by

R group)
7
Properties of
amino acids found
in proteins
8
Titration curve of Gly
Imagen tomada de Lehninger, D.L. Nelson, M., & Cox, M.M. 6 edición.(2014).
Peptides and proteins
9
A peptidic bond is a covalent bond formed between the amino group of the
first amino acid and the carboxylic group of the second amino acid.
Imagen tomada de Lodish, Berk, et al. 5 Edición (2005).
10
10
Imagen tomada de Lehninger, D.L. Nelson, M., & Cox, M.M. 6 edición.(2014).
11
11
12
De las siguientes secuencias de péptidos responda:
a)¿Cuántos aminoácidos básicos y ácidos presentan su

estructura
b)¿Presenta puentes disulfuros? ¿Cuántos?
c)¿Cuál es la carga neta del péptido a pH 3, 7 y 9?
1. EWGRRCCGWGPGRRYCRRWC
2. KKLFKKILKYL
3. EWGRRMMGRGPGRRMMRWWR 12
13
13
Imagen tomada de Lodish, Berk, et al. 5 Edición (2005).
14
14
Imagen tomada de https://commons.wikimedia.org/wiki/File:Bsheet-es.jpg (20/02/18)
15
15
16
J. H. F Redondo Ortega1, J. M. Flórez-

Castillo2*, J. L. Ropero-Vega2, P. Rondón-
Villarreal
17
2LYF: High resolution NMR solution structure of the theta-

defensin RTD-1 (imagen tomada de https://www.rcsb.org/3d-
view/2LYF/0)
18
Hemoglobin
Imagen tomada de https://commons.wikimedia.org/wiki/File:1GZX_Haemoglobin.png (20/02/18)
19
Protein stability
pH changes
Temperature
Enzymatic degradation
Imagen tomada de J.M. Flórez-Castillo

https://commons.wikimedia.org/wiki/File:Proteinas-desnaturalizacion.jpg
Protein solubility 20
• Presence or absence of charges and protonated groups

pH
Solvent polarity
Temperature • Kinetic energy of molecules
Salt
concentration
Imagen tomada de Voet, D., Voet, J.G. & Pratt, C.W. (2007)
21
Solvent Dielectric
constant
Water 79
Acetone and Ethanol: proteins precipitation Ethanol 24
DMSO and DMF: solubilty of proteins
Acetone 21
DMSO 47
DMF 38
22
ionic strength:

1 2
𝐼 = ∑ 𝐶𝑖 𝑍𝑖
2
Ci= molar concentration

Zi= ionic charge
Solubility of some proteins in ammonium

sulfate solutions
Imagen tomada de Voet, D., Voet, J.G. & Pratt, C.W. (2007)
23
• Salting in: incremento

de la solubilidad por
sales
• Salting out: reducción

de la solubilidad por
sales
Purification thecniques 24
Característica Procedimiento
1. Salting in
Solubility
2. Salting out
1. Ion Exchange chromatography

Ionic charge 2. Electrophoresis
3. Isoelectrofocusing
1. Adsortion chromatography
Polarity 2. Reversed phase chromatography
3. Hydrophobic interaction chromatography
1. Dialysis and ultrafiltration

2. Electrophoresis
Molecular sizw
3. Gel filtration chromatography
4. Ultrafiltration
Binding specificity 1. Afinity chromatography

25
Dialysis
• Used to purify the sample of

ions and small molecules
• This technique not differentiate

between active and inactive
proteins.
Imagen tomada de https://www.ncbi.nlm.nih.gov/books/NBK22410/figure/A445/?report=objectonly

26
Gel filtration chromatography
Large molecules flow faster through the column than small molecules.
Imagen tomada de
https://www.ncbi.nlm.nih.gov/books/NBK22410/figu
re/A447/?report=objectonly
27
Ion Exchange chromatography
Proteins are separated based on their net charge.

Salts that compete for binding to the column can be used
for elusion.
Imagen tomada de https://www.ncbi.nlm.nih.gov/books/NBK22410/

28
Afinity chromatography
• It takes advantage of the affinity that some proteins have for specific
chemical groups.
• It is widely used to isolate transcription factors or proteins that

regulate gene expression.
Imagen tomada de
28
https://www.ncbi.nlm.nih.gov/books/NBK22410/figure/A451/?rep
ort=objectonly
http://www.labome.es/method/Protein-Purification.html J.M. Flórez-Castillo
Polyacrilamide gel electrophoresis (PAGE) 29
29
30
Isoelectrofocusing 31
31
Patrón proteíco de E. coli en presencia 32
Patrón proteíco de E. coli
de IBM2/SPIONs@Chi
32
33
FhuA: Captación de Fe en E. coli
33

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1

Amino acids, peptides and

Johanna Marcela Flórez Castillo,

Recognizes amino acids, their

Amino acids are enantiomers in which the chiral center is found in C-

"The biosynthesis of a substance that has asymmetric

This Photo by Unknown Author is licensed under

This Photo by Unknown Author is licensed under CC BY-SA

Amino acids can be classified by

Titration curve of Gly

De las siguientes secuencias de péptidos responda:

a)¿Cuántos aminoácidos básicos y ácidos presentan su

J. H. F Redondo Ortega1, J. M. Flórez-

2LYF: High resolution NMR solution structure of the theta-

Imagen tomada de J.M. Flórez-Castillo

• Presence or absence of charges and protonated groups

Temperature • Kinetic energy of molecules

Ci= molar concentration

Solubility of some proteins in ammonium

• Salting in: incremento

• Salting out: reducción

1. Ion Exchange chromatography

1. Dialysis and ultrafiltration

Binding specificity 1. Afinity chromatography

• Used to purify the sample of

• This technique not differentiate

Imagen tomada de https://www.ncbi.nlm.nih.gov/books/NBK22410/figure/A445/?report=objectonly

Proteins are separated based on their net charge.

Imagen tomada de https://www.ncbi.nlm.nih.gov/books/NBK22410/

• It is widely used to isolate transcription factors or proteins that

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