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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 rantairantai tersebut saling berikatan
(ikatan non-kovalen)
Primary
Assembly
Secondary
Folding
Tertiary
Packing
Quaternary
Interaction
PROCESS
STRUCTURE
Biology/Chemistry of Protein Structure
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
• involves localized spatial
interaction among primary
structure elements, i.e. the
amino acids
• may or may not involve
chaperone proteins
• tumbles towards
conformations that reduce
E (this process is thermodynamically favorable)
• yields secondary structure
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
CHAIN 2: FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT
•
•
•
•
linear
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 threedimensional 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
rate
Effect of Temperature on Rate of
Enzyme Action
denaturant
Thermal Denaturation
•
•
•
•
•
•
Trypsinogen 55°C
Pepsinogen 60°C
Lysozyme 72°C
Myoglobin 79°C
Soy Glycinin 92°C
Oat globulin 108°C
Affected by pH, water, solutes
Table 11
Chain Entropy
One native state
Increased chain entropy
Many denatured states
Why is Denaturation Sudden?
COOPERATIVE
PROCESS
Native Structure
100%
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
Predenaturated Protein
Product interaction with
other compounds
Degradated Protein
Self interaction &
product
interaction with
other compounds
Modification of Functional Properties of
Protein
Process
Isolation
Extraction
Fractionation
Separation
Affecting
Parameter
pH
Structural
Modification
Temperature
Dissociation
Pressure
Agregation
Denaturation
Solubility
Organic
solvents
Heating
Neutral salts
Degradation
Extrusion
Lipids
Complexing
Spinning
Carbohydrates
Drying
Dispersion
Processing
Drying
Hydrolysis
Functional
Properties
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
A
B
Acylation reaction of (A) acetic and (B) succinic anhydride
to form acylated derivatives
Synthesis of Amino Acids
R-CH2-CO2H
Br
Br2, PBr3
R C CO2H
NH2
NH3
R C CO2H
H
H
Strecker Synthesis: recall reductive amination
NH3
O
R
C
NH2
CO2H
R
C
NH2
NaB(CN)H3
R
CO2H
C
CO2H
H
H
O
NH3
NH2
R C H
R C H
NC:
NaCN
NH2
R C CN
H
H3O+
-orNaOH, H2O
NH2
R C CO2H
H
Reactions of Amino Acids
Amino acids will undergo reactions characteristic of the amino
(amide formation) and carboxylic acid (ester formation) groups
H3C
O
H2N
R
H
CO2CH2CH3
HOCH2CH3
H3N
H+
R
Ester Formation of Carboxylic Group
H
CO2
H3C
O
O
O
base
CH3
HN
R
H
CO2H
Amide Formation of Amino Group
Derivatization with Ninhydrin
O
O
H O
OH
2
+
OH
O
N
H3N C CO
R(any)
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.
O
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
R-CH-COOH
NH2
½ O2
R-CH=O
-CO2
-NH3
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