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structure, functions, folding of proteins
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By_ Saurav K. Rawat M.Sc. Chem.
(Physical special)
Structure Of Proteins & Protein Folding Problems
Saurav K. Rawat Department of Chemistry, St. John’s College, Agra ;
Sat. Dec.14th
,2013
Presentation By_
What the Proteins Are?Importance and Biological
FunctionsClassificationMolecular Masses of Some ProteinsAmino Acids as Monomers of
Proteins20 Types of Amino Acids4 levels of Protein Structure- viz.
Primary, Secondary, Tertiary, and Quaternary Structures
Corey -Pauling RulesStructure of Peptide Bond
Ramachandran Plotα- and β- Pleated Sheet StructuresStability and Folding of ProteinAnfinsen’s Experiment, Levinthal Paradox and
KineticsHsp and Molecular Chaperons in Protein FoldingProbes for Conformational DetectionDo You Know?Disorders Due to Conformational ChangeQuick and Hot ReviewReferencesUniversity Questions
Proteins (Gr. Protiose : first of foremost)Berzelius (1837) and Mulder (1838) coined the term
protein.Proteins are macronutrients that support the growth
and maintenance of body tissues.Chemical composition- C-51%, O- 25%, H- 7%, S- 0.4%, sometimes P- also present in traces.Amino acids are the basic building blocks of proteins
and are classified as essential or non-essential. Essential amino acids are obtained from protein-rich
foods such as meat, legumes and poultry, while non-essential ones are synthesized naturally in your body.
According to the Centers for Disease Control and Prevention, you should obtain 10 percent to 25 percent of your daily calorie needs from proteins
WHAT THE PROTEINS ARE..!
Importance of Proteins and Their Biological Functions
Type Examples Occurrence/function
Contractile Proteins
• Actin• Myosin• Dynein
•Thin filaments in myofibril•Thick filaments in myofibril•Cilia and flagella
Enzymes
• Hexokinase• Lactatae dehydrogenase• Cytoochrome c• DNA Polymerase
•Phosphorylates glucose•Dehydrogenates lactate•Transfer electrons•Replicates and repairs DNA
Hormones
• Insulin•Adrenocorticotrophic hormone• Growth hormone
•Regulates glucose metabolism•Regulates corticosteroid synthesis
•Stimulate growth of bones
Type Examples Occurrence/function
Receptors
•Ion channel receptors•G protein linked receptors•Tyrosine kinase receptors
•Present on cell membrane and cytoplasm and receives the stimulations from the outer environment so as to cell may respond according to them.
Toxins
• Clostridium bolulinum toxin• Diphtheria toxin• Snake venom
• Ricin• Gossypin
•Causes bacterial food poisoning•Bacterial toxin•Enzymes that hydrolyze phosphoglycerides•Toxic protein of castor bean•Toxic protein of cottonseed
Storage proteins
• Ovalbumin• Casein• Ferritin• Gliadin• Zein
•Egg-white protein•A milk protein•Iron storage in spleen•Seed protein of wheat•Seed protein of corn
Type Examples Occurrence/function
Defensive proteins
•Antibodies•Fibrinogen•Thrombin
•Form complexes with foreign proteins•Precursor of fibrin in blood clotting•Component of clotting mechanism
Transport proteins
• Hemoglobin• Hemocyanin
• Myoglobin• Serum albumin• Ceruloplasmin
•Transports O2 in blood of vertebrates•Transports O2 in blood of some invertebrates•Transports O2 in muscle cell •Transports fatty acids in blood•Transports copper in blood
Structural proteins
•Viral coat protein• Glycoprotein•α- keratin• Sclerotin• Fibroin• Collagen• Elastin• Mucoprotein
•Sheath around nucleic acid•Cell coats and walls•Skin, feathers, nails, hoofs etc•Exoskeletons of insects•Silk of cocoons, spider webs•Fibrous connective tissues(tendons,bone,cartilages)•Elastic connective tissue(ligaments)•Mucous secretions,synovial fluid
Classification of ProteinsBased on Conformation Based on Composition
FibrousInsoluble in H2O
GlobularSoluble in H2O
•α-Keratin•β-Keratin •Collagen
•Myoglobin•Hemoglobin•Lysozyme•Ribonuclease•Chymotrypsin•Cytochrome-c•Lactate dehydrogenase•subtilisin
Simple Conjugated Derived
•Albumin•Globulin•Glutalins•Prolamins•Protamines•Histones•Scleroproteins
•Nucleoprotein•Lipoprotein•Phosphoprotein•Metalloprotein•Glycoprotein•Flavoprotein•Hemoprotein•chromoproteins
•Protiose•Peptones•Small peptides•Fibrin•Metaproteins•Coagulated proteins
Based on Nature of MoleculesAcidic Basic
•Blood proteins •Histones
Molecular Mass of Some Proteins
Protein Relative molecular mass
Insulin 5,700
Hemoglobin 64,500
Myoglobin 16,900
Hexokinase 102,000
Glycogen phosphorylase
370,000
Glutamine synthetase 592,000
Protein synthesis (DNA transcription, translation and folding).MP4
VIDEO SHOWING PROTEIN SYNTHESIS
AND FOLDING
Proteins are Linear Polymers of Amino Acids
R1
NH3+ C CO
H
R2
NH C CO
H
R3
NH C CO
H
R2
NH3+ C COO ー
H
+
R1
NH3+ C COO ー
H
+
H2OH2O
Peptide bond
Peptide bond
The amino acid sequence is called
as primary structure A AF
NGG
S TS
DK
A carboxylic acid condenses with an amino group with the release of a water
Amino Acid: Basic Unit of Protein
Different side chains, R, determin the properties of 20 amino acids.
COO-NH3+ C
R
HAmino group Carboxylic
acid group
Facts About Amino Acids
Though approximately 300 amino acids occur in nature but only 20 make the composition of proteins.
All amino acids, apart from the simplest one (glycine) show optical isomerism.
This can result in two different arrangements viz. D- amino acid and L- amino acid.
With a few minor exceptions, e.g., bacterial cell wall contains D- amino acids only the L- forms are found in living organisms.
Gamma Amino Butyric Acid (GABA), Histamine serotonin, Ornithine, Citruline and β- alanine are the amino acids, which are not found in proteins.
20 Types of Amino Acids
Glycine (G)
Glutamic acid (E)Asparatic acid (D)
Methionine (M)
Threonine (T)Serine (S)
Glutamine (Q)
Asparagine (N)
Tryptophan (W)Phenylalanine (F)
Cysteine (C)
Proline (P)
Leucine (L)Isoleucine (I)Valine (V)
Alanine (A)
Histidine (H)Lysine (K)
Tyrosine (Y)
Arginine (R)
White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic
Hierarchical Nature of Protein Structure
Primary structure (Amino acid sequence)↓
Secondary structure ( α-helix, β-sheet )↓
Tertiary structure ( Three-dimensional structure formed by assembly of
secondary structures )↓
Quaternary structure ( Structure formed by more than one polypeptide chains )
Definitions of the Four Levels of Structure Primary structure- refers to the covalent backbone of the
polypeptide chain and the sequence of its amino acid residues. The enzyme ribonuclease and the protein myoglobin function
only in their primary structure. Secondary structure- refers to a regular recurring
arrangement in space of the polypeptide chain along one dimension.
Secondary structures are stabilized by H-bonds. Keratin (a fibrous protein found in skin) is composed of almost
entirely of α- helices, while Fibrion (silk protein) is almost entirely composed of β- sheets.
Tertiary structure- refers to how the polypeptide chain is bent or folded in three dimensions, to form the compact, tightly folded structure of globular proteins.
The interactions involved in folding include weak ionic bonds, H-bonds, hydrophobic interactions and strong disulphide bonds b/w neighbouring cysteine amino acids.
Enzymes are functional with a tertiary structure only. Quaternary structure- refers to how individual polypeptide
chains of a protein having two or more chains are arranged in relation to each other. Most larger proteins contain two or more polypeptide chains b/w which there are usually no covalent linkage.
Primary Structure of Protein
• It is a globular protein
• It contains two polypeptide chains
• Alpha unit has 21 amino acid residues
• Beta subunit has 30 amino acid residues
• Neighbouring cysteines are linked by disulphide bond
Introduction to Structure of Proteins
• Unlike most organic polymers, protein molecules adopt a specific 3-dimensional conformation in the aqueous solution.
• This structure is able to fulfill a specific biological function
• This structure is called the native fold• The native fold has a large number of favorable
interactions within the protein• There is a cost in conformational entropy of folding
the protein into one specific native fold
Corey- Pauling RulesA set of rules, formulated by Robert Corey and Linus
Pauling in 1951, that govern the secondary nature of proteins. The Corey-Pauling rules are concerned with the stability of structures provided by hydrogen bonds associated with the –CO-NH– peptide link. The Corey-Pauling rules state that:
(1) All the atoms in the peptide link lie in the same plane.
The planarity of the link is due to delocalization of pi electrons over the O ,C and N atoms and the maintenance of maximum overlap of their p- orbitals. (2) The N, H, and O atoms in a hydrogen bond are approximately on a straight line. (3) All the CO and NH groups are involved in bonding.
Two important structures in which the Corey-Pauling rules are obeyed are the alpha helix and the beta sheet.
Scheme Showing Peptide Structure
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 bondsbe less reactive compared to e.g. esters
be quite rigid and nearly planar
exhibit large dipole moment in the favored trans configuration
The Rigid Peptide Plane and the Partially Free Rotations
Rotation around the peptide bond is not permittedRotation around bonds connected to the alpha
carbon is permitted f (phi): angle around the -carbon—amide
nitrogen bond y (psi): angle around the -carbon— carbonyl
carbon bondIn a fully extended polypeptide, both y and f are
180°
Distribution of f and y Dihedral Angles
• Some f and y combinations are very unfavorable because
of steric crowding of backbone atoms with other atoms in
the backbone or side-chains
• Some f and y combinations are more favorable because of
chance to form favorable H-bonding interactions along the
backbone
• Ramachandran plot shows the distribution of f and y
dihedral angles that are found in a protein
• shows the common secondary structure elements
• reveals regions with unusual backbone structure
Ramachandran Plot
PROTEIN SECONDARY STRUCTURES
Secondary structure refers to a local spatial arrangement of the polypeptide chain
Two regular arrangements are common: The helix
stabilized by hydrogen bonds between nearby residues
The sheetstabilized by hydrogen bonds between adjacent
segments that may not be nearby
Irregular arrangement of the polypeptide chain is called the random coil
Basic structural units of proteins: Secondary structure
α-helix β-sheet
Secondary structures, α-helix and β-sheet, have regular hydrogen-bonding patterns.
The helix
The backbone is more compact with the y dihedral (N–C—C–N) in the range
( 0 < < -70)y Helical backbone is held
together by hydrogen bonds between the nearby backbone amides
Right-handed helix with 3.6 residues (5.4 Å) per turn
Peptide bonds are aligned roughly parallel with the helical axis
Side chains point out and are roughly perpendicular with the helical axis
alpha helix.MP4
VIDEO SHOWING ALPHA HELIX
The helix: Top View
• The inner diameter of the helix (no side-chains) is about 4 – 5 Å• Too small for anything to fit
“inside” • The outer diameter of the
helix (with side chains) is 10 – 12 Å• Happens to fit well into the
major groove of dsDNA• Residues 1 and 8 align nicely
on top of each other• What kind of sequence
gives an helix with one hydrophobic face?
Sequence Affects Helix Stability
Not all polypeptide
sequences adopt -helical
structures
Small hydrophobic residues
such as Ala and Leu are
strong helix formers
Pro acts as a helix breaker
because the rotation around
the N-Ca bond is impossible
Gly acts as a helix breaker
because the tiny R-group
supports other conformations
The Helix Macro-Dipole
Peptide bond has a strong dipole momentCarbonyl O negativeAmide H positive
All peptide bonds in the helix have a similar orientation
The helix has a large macroscopic dipole moment
Negatively charged residues often occur near the positive end of the helix dipole
Sheets
The backbone is more extended with the y dihedral
(N–C—C–N) in the range ( 90 < < 180)y
The planarity of the peptide bond and tetrahedral geometry of the -carbon create a pleated sheet-like structure
Sheet-like arrangement of backbone is held together by hydrogen bonds between the more distal backbone amides
Side chains protrude from the sheet alternating in up and down direction
Beta sheet.MP4
VIDEO SHOWING BETA SHEET
Parallel and Antiparallel b Sheets
Parallel or antiparallel orientation of two chains
within a sheet are possible
In parallel b sheets the H-bonded strands run in
the same direction
In antiparallel b sheets the H-bonded strands
run in opposite directions
Structure of -Keratin in Hair
Chemistry of Curly Hair
Structure of Collagen Collagen is an important constituent of connective tissue: tendons, cartilage, bones,
cornea of the eye
Each collagen chain is a long Gly- and Pro-rich left-handed helix
Three collagen chains intertwine into a right-handed superhelical triple helix
The triple helix has higher tensile strength than a steel wire of equal cross section
Many triple-helixes assemble into a collagen fibril
Collagen Fibrils
Silk Fibroin Fibroin is the main protein in silk from moths and spiders
Antiparallel b sheet structure
Small side chains (Ala and Gly) allow the close packing of sheets
Structure is stabilized byhydrogen bonding within sheetsLondon dispersion interactions between sheets
b Turns (Hairpins) b-turns occur frequently whenever strands in b sheets change the
direction The 180° turn is accomplished over four amino acids The turn is stabilized by a hydrogen bond from a carbonyl oxygen to
amide proton three residues down the sequence Proline in position 2 or glycine in position 3 are common in b-turns
• Tertiary structure refers to the overall spatial arrangement of atoms in a polypeptide chain or in a protein
• One can distinguish two major classes– fibrous proteins
¤ typically insoluble; made from a single secondary structure– globular proteins
¤ water-soluble globular proteins¤ lipid-soluble membraneous proteins
PROTEIN TERTIARY STRUCTURE
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 -helixes and -sheets
• London dispersion – 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
Motifs (folds)
Arrangements of several secondary structure elements
Three-dimensional structure of proteins
zzzzz
Tertiary structure
Quaternary structure
• Quaternary structure is formed by spontaneous assembly of individual polypeptides into a larger functional cluster together. Proteins with two or more polypeptide chains are known as oligomeric proteins.
PROTEIN QUATERNARY STRUCTURE
Close relationship between protein structure and its function
enzyme
A
B
A
Binding to A
Digestion of A!
enzyme
Matching the shape to A
Hormone receptor AntibodyExample of enzyme reaction
enzyme
substrates
The Four Levels of Protein Structure.MP4
VIDEO SHOWING
FOUR LEVELS OF
STRUCTURE
Protein Stability and Folding•A protein’s function depends on its three-dimensional 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
• Ribonuclease is a small protein that
contains 8 cysteins 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
Ribonuclease Refolding/Anfinsen’s Experiment
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?
Protein folding is a very finely tuned process. Hydrogen bonding between different atoms provides the force required. Hydrophobic interactions between hydrophobic amino acids pack the hydrophobic residues.
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
The Levinthal Paradox and KineticsLevinthal's paradox is a thought experiment, also
constituting a self-reference in the theory of protein folding. In 1969, Cyrus Levinthal noted that, because of the very large number of degrees of freedom in an unfolded polypeptide chain, the molecule has an astronomical number of possible conformations. An estimate of 3300 or 10143 was made in one of his papers.
The Levinthal paradox observes that if a protein were folded by sequentially sampling of all possible conformations, it would take an astronomical amount of time to do so, even if the conformations were sampled at a rapid rate
(on the nanosecond or picosecond scale). Based upon the observation that proteins fold much faster than this, Levinthal then proposed that a random conformational search does not occur, and the protein must, therefore, fold through a series of meta-stable intermediate states.
If we assume that a protein molecule has n amino acid residues, that each residue has 2 bonds capable of rotation, and that there are 3 possible conformations (ϕ or ψ angles) for each rotatable bond in he backbone, the maximum number of possible conformations is 32n , which is approximately equal to 10n . Since each single bond can rotate completely in about 10-13 s, the total time required for every formal single bond in the backbone to rotate once is about 2×10-13s. Therefore the time required for a peptide chain to try out every possible conformation it can assume that t=10n (2n×10-13) . For a polypeptide chain of 6 residues t is in the range of microseconds, for a chain of 11 residues, about 0.2s, but for a chain of 100 residues it would be about 2×10 89s. or longer than the age of the earth. Yet staphylococcal nuclease, which has 149 residues, requires at most 0.1 to 0.2 s. How…? Why the chain fold so quickly into native conformation?
Why it is not trying out all its possible conformations?This question is a major problem in biochemistry and researches are going
on..This is only a hypothesis that it works on The Principle of cooperativety- once a
weak bonds (hydrogen bonds or hydrophobic interactions) have correctly formed in a part of polypeptide chain, they greatly increase the probability of the formation of further correct bonds without requiring the chain to try out all possible conformations.
Heat shocked proteins (Hsp) – These proteins are being synthesize vigorously when the cell is on the heat, or the environment where they have high heat.
High heat can trigger the translation of more and more Hsp.
Hsp help to fold protein properly.There are two major classes of Hsp viz.Hsp 70- also called Chaparones (DnaJ-
DnaK)Hsp 60- also called Chaparonins (GroEL-
GroES)
MOLECULAR CHAPARONES
Chaperones Assisted Protein folding
Chaperones Prevent Misfolding
Chaperonins Facilitate Folding
Probes of Protein Conformation
X-Ray AnalysisORD- optical rotatory dispersionCD- circular dichroismFluorescenceFluorescence polarizationNMR- nuclear magnetic resonance spectroscopy
Protein Structure Methods: X-Ray Crystallography
Steps needed: Purify the protein Crystallize the protein Collect diffraction data Calculate electron density Fit residues into densityPros: No size limits Well-establishedCons: Difficult for membrane
proteins Cannot see hydrogens
Circular Dichroism (CD) Analysis
CD measures the molar
absorption difference of left-
and right- circularly polarized
light: = L – R
Chromophores in the chiral
environment produce
characteristic signals
CD signals from peptide
bonds depend on the chain
conformation
Proton NMR spectrum of a protein
Amides Aromatics Alphas Aliphatics Methyls
Structure Methods: Biomolecular NMR
Steps needed: Purify the protein Dissolve the protein Collect NMR data Assign NMR signals Calculate the structure
Pros: No need to crystallize the protein Can see many hydrogens
Cons: Difficult for insoluble proteins Works best with small proteins
Do You Know…..?Collagen is the most abundant protein
in animal world and RibUlose BISphosphate Carboxylase Oxygenase (RUBISCO) is the most abundant protein in the whole biosphere.
Monellin, a Protein is the sweetest chemical obtained from an African Berry.
3 billion base pair => 6 G letters &
1 letter => 1 byte
The whole genome can be recorded in just 10 CD-ROMs!
In 2003, Human genome sequence was deciphered!
Genome is the complete set of genes of a living thing.
In 2003, the human genome sequencing was completed.
The human genome contains about 3 billion base pairs.
The number of genes is estimated to be between 20,000 to 25,000.
The difference between the genome of human and that of chimpanzee is only 1.23%!
Some Common Diseases Caused by Conformational Change in Protein Structure
Proteopathy (Proteo- [pref. protein]; -pathy [suff. disease]; refers to a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body. Often the proteins fail to fold into their normal configuration; in this misfolded state, the proteins can become toxic in some way (a gain of toxic function) or they can lose their normal function.The proteopathies (also known as proteinopathies, protein conformational disorders, or protein misfolding diseases), include such diseases as Alzheimer’s disease, Parkinson's disease, Prion disease, Type 2 Diabetes, Amyloidosis,and a wide range of other disorders
Mutations are because of this abnormality.
Sickle cell Disease- in sickle cell hemoglobin (Hb-S) the glutamic acid residue in the 6th position of the β- chains are replaced by valine.
Sodium cyanate injections are given to recovery from sickle cell anemia
Proteopathy Major aggregating protein
Alzheimer's disease Amyloid β peptide (Aβ);
Tau Protein
Prion diseases (multiple) Prion protein
Parkinson's disease and other synucleinopathies (multiple) α-Synuclein
Familial British dementia ABri
Familial Danish dementia ADan
Type II diabetes Islet amyloid polypeptide (IAPP; amylin)
Cataracts Crystallins
Retinitis pigmentosa with rhodopsin mutations Rhodopsin
REFERENCESHarper’s Illustrated BiochemistryBiochemistry by Albert L. LehningerBiophysical Chemistry by Gurtu & GurtuPrinciples of Physical Chemistry by
Puri,Sharma & PathaniaAtkins’ Physical ChemistryMolecular Biology by Dr. Virbala RastogiCompetitive Biology by K.N. Bhatia & K.
BhatiaText book of biology by S. ChakrabartyNCERT text books of Chemistry and Biology
Frequently Asked University Questions-Explain the structure of Protein.Describe the folding problems in
protein.How protein fold?
The truth shall make you free….!!!
Tribute to Deptt. Of Chemistry
Thanks A Lot-Our HOD SirDr. Susan Ma’m,Who Gave Me
This OpportunityAnd All Respected Teachers Special Thank Goes To-Dr. Girish Maheshwary SirDr. Jyoti Zack Ma’m (Deptt. of Zoology, St. John’s
College)
Rawat’s [email protected]@yahoo.co.uk
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