PROTEIN Oleh Dr. Ir. Ani Suryani, DEA

Presentasi berjudul: "PROTEIN Oleh Dr. Ir. Ani Suryani, DEA"— Transcript presentasi:

1 PROTEIN Oleh Dr. Ir. Ani Suryani, DEA
DEPARTEMEN TEKNOLOGI INDUSTRI PERTANIAN FAKULTAS TEKNOLOGI PERTANIAN INSTITUT PERTANIAN BOGOR

2 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)

3 Biology/Chemistry of Protein Structure
Primary Secondary Tertiary Quaternary Assembly Folding Packing Interaction S T R U C T U R E P R O C E S S

4 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 thermodynamically unfavorable, with E = +10kJ/mol, thus coupled to reactions that act as sources of free energy yields primary structure 2 n 3 + -

5 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 thermo-dynamically favorable) yields secondary structure

6 Ramachandran Plot Pauling built models based on the following principles, codified by Ramachandran: 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:  (phi) angle = rotation about N – C  (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!

7 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

8 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

9 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

10 Amino Acids

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15 Peptide Linkage

16 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

17 Primary Structure linear ordered 1 dimensional
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!

18 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

19 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!

20 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

21 Three Levels of Protein Structure

22 Quaternary Protein Structure

23 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

24 Protein Denaturation

25 Agen Penyebab Denaturasi :
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

26 Protein Denaturation. organized molecular configuration is disturbed

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28 Analogy between benzene solubility in water and protein denaturation

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30 Denaturation of proteins. Since many of the bonds holding a protein ...

31 if an intramolecular hydrogen bond in a protein is broken or deleted ...

32 Protein denaturation and refolding
Protein denaturation and refolding. An external file that holds a picture, ...

33 The zone of protein denaturation

34 Counteraction of urea-induced protein denaturation by trimethylamine

35 function of protein being by shape, denaturation

36 Four levels of Organization of Protein

37 by water molecules, no further desiccation or denaturation occurs.

38 protein denaturation by coagulation (e.g., acetone and methanol);

39 Counteraction of urea-induced protein denaturation by trimethylamine ...

40 . out of the cells and osmosis pressure as well as protein denaturation.

41 DENATURATION OF PROTEIN:

42  Emulsion formation: During the formation of a meat emulsion, meat proteins ...

43 Protein structure can be simple chains (primary) or helical or pleated ...

44 Protein denaturation kinetics and glass transition conditions are predicted ...

45 This excessive thermal denaturation could explain the lower values of

46 Some Protein Applications

47 CD spectra of the protein at these pH. FIG. 8
CD spectra of the protein at these pH. FIG. 8. Effect of denaturation on ...

48 At high concentrations of urea, protein denaturation occurred and

49 Fraction of unfolded protein is plotted versus temperature.

50 protein denaturation occurs at lower pressures using lower

51 Effect of Temperature on Rate of Enzyme Action
denaturant

52 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

53 Chain Entropy One native state Increased chain entropy
Many denatured states

54 Why is Denaturation Sudden?
COOPERATIVE PROCESS Partly denatured structure is weaker so begins to change faster 100% Native Structure 0% Critical value Concentration of denaturant or temperature

55 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

56 Types of Denaturation Temperature Organic solvents Surface pH Shear

57 Reversibility? One native form
Refolding is a complex process – particularly for large proteins or complex proteins Many denatured forms

58 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.

59 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

60 Protein Change On Heating
Native Protein Denaturated Protein Degradated Protein Predenaturated Protein Self interaction & product interaction with other compounds Product interaction with other compounds

61 Modification of Functional Properties of Protein
Process Extraction Fractionation Separation Drying Dispersion Solubility Heating Extrusion Spinning Isolation Processing Structural Modification Dissociation Agregation Denaturation Hydrolysis Degradation Complexing Affecting Parameter pH Temperature Pressure Organic solvents Neutral salts Lipids Carbohydrates Functional Properties

62 Hydrolytic Modification
Removing impurities of protein substrate by hydrolysis, purification and resynthesis by means of the plastein reaction

63 Hydrolytic Modification of Protein
Transformation of reactive protein side chain to lysinoalanine side chains via elimination and cross-linking formation

64 Derivative Modification
B Acylation reaction of (A) acetic and (B) succinic anhydride to form acylated derivatives

65 Synthesis of Amino Acids
Strecker Synthesis: recall reductive amination

66 Reactions of Amino Acids
Amino acids will undergo reactions characteristic of the amino (amide formation) and carboxylic acid (ester formation) groups Ester Formation of Carboxylic Group Amide Formation of Amino Group

67 Derivatization with Ninhydrin
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.

68 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 : R-CH-COOH NH2 R-CH=O Oxidizing agents ½ O2 -CO2 -NH3 Agen yang dapat menyebabkan degradasi asam amino : bahan organik & anorganik

69 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

70 Enzymatic Breakdown of Amino Acid

71 TERIMA KASIH