PROTEIN

Oleh
Dr. Ir. Ani Suryani, DEA

DEPARTEMEN TEKNOLOGI INDUSTRI PERTANIAN

FAKULTAS TEKNOLOGI PERTANIAN
INSTITUT PERTANIAN BOGOR
Protein  Homoprotein (hanya mengandung asam amino)
Heteroprotein (asam amino dan senyawa non-protein)
contoh : nukleoprotein, lipoprotein, fosfoprotein,
glikoprotein, dll

Protein berdasarkan konformasi atau organisasi tiga dimensi

terdiri dari :
- fibrous protein (contoh : kolagen, keratin, dll)
- globular protein (contoh : actin, fibrinogen)

• Struktur primer  susunan asam amino

dalam protein
• Struktur sekunder dan tertier  berhubungan
dengan bentuk tiga dimensi
• Struktur kuartener  penyusunan geometrik
diantara rantai polipeptida, dan rantai-
rantai tersebut saling berikatan
(ikatan non-kovalen)
 Biology/Chemistry of Protein Structure

Primary Assembly
STRUCTURE

PROCESS
Secondary Folding

Tertiary Packing

Quaternary Interaction
 Protein Assembly

• occurs at the ribosome

• involves dehydration
synthesis and
polymerization of amino
acids attached to tRNA:

NH +- {A + B  A-B + H O} -COO -
3 2 n

• thermodynamically
unfavorable, with E =
+10kJ/mol, thus coupled
to reactions that act as
sources of free energy
• yields primary structure
 Protein Folding
• occurs in the cytosol • tumbles towards
• involves localized spatial conformations that reduce
interaction among primary E (this process is thermo-
structure elements, i.e. the dynamically favorable)
amino acids • yields secondary structure
• may or may not involve
chaperone proteins
 Ramachandran Plot
• Pauling built models based on the following
principles, codified by Ramachandran:

(1) bond lengths and angles – should be similar

to those found in individual amino acids and
small peptides
(2) peptide bond – should be planer
(3) overlaps – not permitted, pairs of atoms no
closer than sum of their covalent radii
(4) stabilization – have sterics that permit
hydrogen bonding

• Two degrees of freedom:

(1)  (phi) angle = rotation about N – C
(2)  (psi) angle = rotation about C – C

• A linear amino acid polymer with some folds is

better but still not functional nor completely
energetically favorable  packing!
 Protein Packing
• occurs in the cytosol (~60% bulk
water, ~40% water of hydration)
• involves interaction between
secondary structure elements
and solvent
• may be promoted by
chaperones, membrane proteins
• tumbles into molten globule
states
• overall entropy loss is small
enough so enthalpy determines
sign of E, which decreases
(loss in entropy from packing
counteracted by gain from
desolvation and reorganization
of water, i.e. hydrophobic effect)
• yields tertiary structure
 Protein Interaction
• occurs in the cytosol, in close proximity to other
folded and packed proteins
• involves interaction among tertiary structure
elements of separate polymer chains
• may be promoted by chaperones, membrane
proteins, cytosolic and extracellular elements as
well as the proteins’ own propensities
• E decreases further due to further
desolvation and reduction of surface area
• globular proteins, e.g. hemoglobin,
largely involved in catalytic roles
• fibrous proteins, e.g. collagen,
largely involved in structural roles
• yields quaternary structure
 Proteins
• Composed of building blocks called amino acids

• Amino acids have at least one amino (-NH2) group

and one acidic carboxyl (-COOH) group

• Each amino acid is distinguishable by a different

chemical group (R group)

• Peptide bonds: covalent bond that links an amino

group of one amino acid to carboxyl group of
another
Amino Acids
Peptide Linkage
 The Structure of Proteins
• Primary structure: consists of the specific
amino acids in a polypeptide chain

• Secondary structure: consists of the folding

or coiling or amino acids chains into a
particular pattern

• Tertiary structure: folding of the protein into

globular shapes or fibrous threadlike strands

• Quaternary structure: the association of

several tertiary-structured polypeptide chains
 Primary Structure
primary structure of human insulin
CHAIN 1: GIVEQ CCTSI CSLYQ LENYC N
• linear
CHAIN 2: FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT • ordered
• 1 dimensional
• sequence of amino
acid polymer
• by convention, written
from amino end to
carboxyl end
• a perfectly linear
amino acid polymer is
neither functional nor
energetically
favorable  folding!
Secondary Structure
• non-linear
• 3 dimensional
• localized to regions of an
amino acid chain
• formed and stabilized by
hydrogen bonding,
electrostatic and van der
Waals interactions
 Tertiary Structure

• non-linear
• 3 dimensional
• global but restricted to the
amino acid polymer
• formed and stabilized by
hydrogen bonding, covalent
(e.g. disulfide) bonding,
hydrophobic packing toward
core and hydrophilic
exposure to solvent
• A globular amino acid
polymer folded and
compacted is somewhat
functional (catalytic) and
energetically favorable 
interaction!
Quaternary Structure
• non-linear
• 3 dimensional
• global, and across
distinct amino acid
polymers
• formed by hydrogen
bonding, covalent
bonding, hydrophobic
packing and hydrophilic
exposure
• favorable, functional
structures occur
frequently and have been
categorized
Three Levels of Protein Structure
Quaternary Protein Structure
 Classification of Proteins
• Structural proteins: contribute to the three-
dimensional structure of cells, cell parts,
and membranes

• Enzymes: protein catalysts – substances

that control the rate of chemical reactions in
cells
Protein Denaturation
Denaturasi Protein :
Perubahan konfigurasi protein dari bentuk struktur sekunder dan
tertier yang rapuh. Bentuk struktur primer tidak berubah

Agen Penyebab Denaturasi :

Agen Fisik : panas, dingin, perlakuan mekanis,
tekanan hidrostatis
Agen kimiawi : asam, basa, logam, pelarut organik,
persenyawaan organik
 Protein Denaturation.
organized molecular
configuration is disturbed
 Analogy between benzene
solubility in water and protein
denaturation
Denaturation of proteins. Since
many of the bonds holding a
protein ...
if an intramolecular hydrogen
bond in a protein is broken or
deleted ...
 Protein denaturation and
refolding. An external file that
holds a picture, ...
The zone of protein
denaturation
Counteraction of urea-induced
protein denaturation by
trimethylamine
function of protein being by
shape, denaturation
Four levels of Organization of
Protein
 by water molecules, no further
desiccation or denaturation occurs.
 protein denaturation by
coagulation (e.g., acetone and
methanol);
Counteraction of urea-induced protein
denaturation by trimethylamine ...
. out of the cells and osmosis pressure as
well as protein denaturation.
DENATURATION OF
PROTEIN:
 Emulsion formation: During the
formation of a meat emulsion, meat
proteins ...
 Protein structure can be simple
chains (primary) or helical or pleated
...
Protein denaturation kinetics and glass
transition conditions are predicted ...
This excessive thermal denaturation
could explain the lower values of
Some Protein Applications
CD spectra of the protein at these pH.
FIG. 8. Effect of denaturation on ...
At high concentrations of urea, protein
denaturation occurred and
Fraction of unfolded protein is plotted
versus temperature.
protein denaturation occurs at
lower pressures using lower
 Effect of Temperature on Rate of
Enzyme Action
rate

denaturant
 Thermal Denaturation
• Trypsinogen 55°C
• Pepsinogen 60°C
• Lysozyme 72°C Affected by pH, water, solutes

• Myoglobin 79°C
• Soy Glycinin 92°C
• Oat globulin 108°C

Table 11
 Chain Entropy
One native state

Increased chain entropy

Many denatured states

 Why is Denaturation Sudden?
COOPERATIVE
100% PROCESS
Native Structure

Partly denatured structure

is weaker so begins to
change faster

0%
Critical value

Concentration of denaturant or temperature

 Behavior of Denatured Protein
Hydrophobic core

Hydrophilic surface
DENATURED
Fast under non-physiological conditions

Slow under physiological conditions

NATIVE

Unfolding forces some

hydrophobic AA to
surface
AGGREGATED
or other ingredient interactions
 Types of Denaturation
• Temperature
• Organic solvents
• Surface
• pH
• Shear
 Reversibility?
One native form

Refolding is a complex process –

particularly for large proteins or complex
proteins

Many denatured forms

 Denaturation
• The conversion of a biologically functional
molecule into a non-functional form
• There are many denatured states but one
native state
• Proteins can regenerate to their native state
but slowly
• Denatured proteins have a greater tendency
to aggregate.
Pengaruh denaturasi :
• Penurunan kelarutan
• Mengubah kapasitas pengikatan air
• Kehilangan aktivitas biologis (enzim, bahan imunologi)
• Meningkatkan kemampuan bahan untuk dihidrolisis oleh
protease
• Meningkatkan viskositas intrinsik
• Tidak dapat dikristalisasi
 Protein Change On Heating

Native Protein Denaturated Protein Degradated Protein

Predenaturated Protein Self interaction &

product
interaction with
other compounds

Product interaction with

other compounds
 Modification of Functional Properties of
Protein

Process
Extraction Affecting
Isolation

Parameter Structural
Fractionation
pH Modification
Separation
Temperature Dissociation
Drying
Pressure Agregation Functional
Dispersion Properties
Organic Denaturation
Solubility solvents
Processing

Hydrolysis
Heating Neutral salts Degradation
Extrusion Lipids Complexing
Spinning Carbohydrates
Drying
 Hydrolytic Modification
Removing impurities of protein substrate by hydrolysis,
purification and resynthesis by means of the plastein reaction
 Hydrolytic Modification of Protein
Transformation of reactive protein side chain to lysinoalanine
side chains via elimination and cross-linking formation
 Derivative Modification

Acylation reaction of (A) acetic and (B) succinic anhydride

to form acylated derivatives
Synthesis of Amino Acids
Br NH2
Br2, PBr3 NH3
R-CH2-CO2H R C CO2H R C CO2H
H H

Strecker Synthesis: recall reductive amination

NaB(CN)H3 NH2
O NH3 NH2
R C CO2H
R C CO2H R C CO2H
H
H

NH2 NH2
O NH3 NH2 NaCN H3O+
R C CN R C CO2H
R C H R C H -or-
H NaOH, H2O H
NC:
Reactions of Amino Acids

Amino acids will undergo reactions characteristic of the amino

(amide formation) and carboxylic acid (ester formation) groups

H3C
O O O
H2N H H3N H HN H
HOCH2CH3 H3C O CH3

R CO2CH2CH3 H+ R CO2 base R CO2H

Ester Formation of Carboxylic Group Amide Formation of Amino Group

Derivatization with Ninhydrin

O O
OH H O
2 + H3N C CO N
OH O
R(any)
O O O

Ninhydrin (2 mol) reacts with one mol of ANY amino acid to give
the SAME blue colored product. This reaction is performed post-column,
after Ion Exchange Chromatography separation of a mixture of amino
acids. The area of each peak in the chromatogram is proportional
to the relative molar amount of the amino acid of that retention time.
Reaksi Deaminasi dan Dekarboksilasi
1. Secara kimiawi : dekarboksilasi (degradasi strecker)
Pemecahan asam amino α- dengan gugus karbonil dan bahan
pengoksidasi lainnya, menghasilkan evolusi CO2, aldehid, amino,
dan senyawa lain.

Mekanisme reaksi :

Oxidizing agents ½ O2
R-CH-COOH R-CH=O
-CO2 -NH3
NH2

Agen yang dapat menyebabkan degradasi asam amino :

bahan organik & anorganik
2. Secara Enzimatis

Sumber utama dekarboksilasi adalah kontaminasi

(spoilage) mikroorganisme (genera Achromobacter,
Micrococcus, Staphylococcus, Sarcina,
Pseudomonas, dll) yang menghasilkan enzim
tertentu untuk asam amino tertentu

Contoh : kontaminasi (spoilage) produk perikanan

oleh mikroorganisme, flavor khas dari
produk susu
Enzymatic Breakdown of Amino Acid