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Amino acids structures & properties. Peptides. Proteins primary, secondary, tertiary & quaternary structures. protein folding
Proteins group based on solubility & shape: Fibrous proteins Globular proteins Membrane proteins
Cathode (-)
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Anode (+)
Buffer
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Modified Karp
INTEGRAL PROTEINS
Receptors, transporters
Tightly folded into compact globular shape with hydrophobic residues inside and hydrophilic residues on surface. Structurally complex - contains several types of 20 structures. Soluble in aqueous medium. Diffuse readily. Mobile or dynamic functions. Enzymes, transport proteins, antibodies.
Insoluble in aqueous medium Extended structures with hydrophobic residues on the outside Consist largely of one type of 20 structure Structural or protective role a-keratin, collagen, fibroin (silk)
Simple protein contains only polypeptide chain (sometimes with modified amino acids). Some proteins have other modifications - Conjugated proteins (polypeptide chain + prosthetic group organic or inorganic moiety). Classification based on nature of prosthetic group
PRIMARY SEQUENCES
Refers to number and sequence of amino acids number - amino acid composition sequence - amino acid sequence Also location of S-S bond(s), if any.
Glia Cell-line Derived Neurotrophic Factor (GDNF) different way to visualize a protein by bioinformatics
Cartoon representation
Wire-frame
Ball-stick
animation
Secondary structures
-Turns -Sheets
Loops
-Helix
H-bonds Hydrophobic interactions Electrostatic interactions (ionic) Van der Waals interactions
Amide plane
2 degree of rotation - and angles
N- terminus C- terminus
Calculate the & angles for every residue Ramachandran plot (conformation plot)
+180
(deg)
-180 -180
+180
(deg)
(deg)
Cytochrome C
(deg)
-180 -180
+180
(deg)
(deg)
Plastocyanin
(deg)
(deg)
Because of the constraints of how the amide planes can twist, there MUST only be a limited number of favorable secondary structures.
Few types of secondary structures are stable and occur widely in proteins - helices and sheets
Side view
Front view
Animation. ..\..\..\rasmol\rw32b2a.exe
Rod-like structure
H-bonding
R-groups extend outward in a helical array Intra-H bond spacing is 4 residues apart 3.6 amino acid residues/turn Proline residues interrupt a-helix Commonly found in globular proteins
Lodish et al
-SHEET
Extended sheet-like structure Intermolecular H-bonds Parallel or antiparallel -sheets Favour amino acids with compact R groups -(Gly-Ser-Gly-Ala-Gly-Ala)n- sequence motif in the silk protein, fibroin
-sheet antiparallel
C O
PARALLEL
H N
ANTIPARALLEL
OTHER STRUCTURES
Helices/-sheets: ~50% of regular 20 structures of globular proteins Remaining : coil or loop conformation Examples : reverse turns, -bends (connect successive strands of antiparallel -sheets
-Turns
Coil or Loop
TRIPLE HELIX
Limited to tropocollagen molecule ( 3 strands of molecules subunit)
Sequence motif of (Gly-X-Pro/Hypro)n3 left-handed helices wound together to give a right-handed superhelix. Stable superhelix : glycines located on the central axis (small R group) of triple helix. One interchain H-bond for each triplet of amino acid between NH of Gly and CO of X in adjacent chain.
C
N O
C C
In large proteins, some recognizable structures observed groups of unique combinations of folds (secondary structures). Combinations of a few secondary structures (helices and -strands) with specific geometric arrangements in some proteins Four classes: All All / ( and segments are interspersed or alternate) & ( and regions are somewhat segregated) Number of folds are finite (< 1,000 different types of motifs in all proteins).
-helical
-Sheets
/ motif interspersed structures of & + motif. and are segregated and distinct.
Many large proteins contain several discreet, independently folded globular units domains Each domain typically consists of about 100-200 aa residues and a combination of motifs Have a specific function has two distinct domains, and NAD+ binds to the first domain while G-3-P, the second.
Glyceraldehyde-3-phosphate dehydrogenase
tetramer with a total molecular weight of 145 kD
Many domains are independent structural units and often have distinct functions
Kringle domain
Fibronectin domain
Kringle domain
QUATERNARY STRUCTURE
Stabilize by non-covalent interactions between subunits Multimeric proteins can have 2 100s of subunits For example, mammalian haemoglobin has 2 (141 aas) and 2 (146 aas) polypeptide chains but earthworm Hbs have > 100 subunits If subunits are composed of a number of nonidentical subunits, overall structure is asymmetric and quite complicated If identical subunits or repeating groups of nonidentical subunits, usually symmetrical
Structure-rasmol
Oligomers more stable than dissociated subunits prolong life of protein in vivo. Active sites formed by residues from adjacent chains Error of synthesis is greater for longer/bigger chains.
Subunit interactions : cooperativity/ allosteric effects function of single subunit is influenced (positively or negatively) by other associating subunits.
PROTEIN FAMILIES
Proteins sharing similar primary sequences and/or have similar structures/functions same family.
C-type cytochrome of different species same protein family (homologues) same functions (electron carrier) low primary sequence similarity structurally similar (specially in the interior of the protein)
Most abundant protein (25-35% of total protein in mammals) Found in bones, tendons, skin, blood vessels, and cornea of the eye
~30 genes encode collagen. >19 distinct types of collagen in human. Tropocollagen (basic structural unit of collagen) is a triple helix of 3 polypeptides (1050 aa residues each) Sequence motif of chain: Gly-X-Pro/Hypro Undergoes extensive post-translational modifications Hydroxylation of Pro and Lys residues (requires vitamin C/hydroxylase enzyme) Hydoxyproline additional H-bond for stability of helix structure Hydroxylysine attachment sites for carbohydrate moieties (glycosylation)
Organization of collagen
Collagen fibrils
Each helix
(Vitamin C)
Oxidation of side chains of (NH2)Lys to form (OH)allysine (an aldehyde) (OH)Allysine-lysine x-links : intermolecular covalent x-links between tropocollagen molecules Allysine-allysine x-links : intramolecular covalent x-links within a tropocollagen unit (unlike keratin in hair where disulphide forms (cys) x-links, resulting in strong fibrils). Essential for formation of strong collagen fibrils Collagen diseases : mutation of glycine is lethal (gly-X-pro) Covalently X- linked
Osteogenesis imperfecta results in abnormal bone formation in babies Ehlers-Danlos syndrome is characterized by hyperextensibility of skin, abnormal tissue fragility, and increased joint mobility. Scuvy (not a genetic disease) affects collagen xlinking. Deficiency of ascorbic acid. Bleeding in gums, poor wound healing. Both can be lethal: due to substitution of a Cys and Ser residue respectively for a Gly (different Gly for each) in collagen. This substitution (especially in C-terminus) produces catastropic effect since it disrupts the Gly-X-Pro (repeat) helical structure of collagen ED syndrome
PROTEIN DENATURATION
Loss of biological activity Conformational/structural change Heat, pH, organic solvents, urea, guanidine hydrochloride Destruction of 2o, 3o and 4o structures, but 1o structure remain intact Ease of proteolytic digestion, aggregation Reversible or irreversible
DENATURATION OF RIBONUCLEASE A
Enzyme (124 aa) digest RNA. Denatured by: 6 8M urea + 2 mercaptoethanol (reducing agent) No activity Two routes for dialysis: either to remove urea or 2mercaptoethanol first Removal of urea first led to recovery of activity
To unravel protein (re)folding is a major area of research (Ribonuclease A study won Anfinsen a Nobel prize 1972).
DENATURATIONRENATURATION OF RIBONUCLEASE A
8 Cys. Probability of any 4 Cys forming the native form of disulphide randomly- 1:105. Renaturation results in fully active protein refolding results in mostly correct native form of the protein. Conclusion: primary sequence dictate 3-D structure for this protein.
preproinsulin
proinsulin
Denaturation/refolding/oxidize
Enzymatic cleavages In vitro.
proinsulin
Notarandomprocess A100residueprotein:randomsearchofall possibleconformationswilltake1087 seconds (manytimestheageoftheuniverse) Acooperativeandsequentialprocess: formationofonepartofthestructureleads totheformationoftheremainingstructure Foldingpatternandfinal conformation dependonprimarystructure
ENERGETICS IN FOLDING
Least stable
Most stable
Nucleus
Unfolded proteins usually possess numerous solvent-exposed hydrophobic regions Tendency to self-aggregate especially in concentrated solution (e.g, inside a cell) Function to prevent or reverse such improper associations by binding to these hydrophobic areas Subsequent release of these proteins will facilitate proper folding based on their amino acid sequence. These include peptidyl prolyl cistrans-isomerases (PPIs) and protein disulphide isomerases (PDIs) PPIs: catalyze the isomerization of incorrect Xaa-Pro bonds which is a slow rate determining step PDIs: facilitates the formation of correct set of S-S bonds Molecular chaperone
Misfolding can result in death of cells E.Coli deal by forming inclusion bodies. Mammalian cells cannot form insoluble products inside. They then commit suicide.
Native proteins
Aggregated proteins
Released protein
ATP
Proteosome
Ubiquitin released (Nobel prize 2004)
Degraded protein
MISFOLDING CAN RESULT IN DEATH OF CELLS e.g., Prion, Alzheimers disease & other neurodegenerative diseases. Prion (proteinaceous infectious agent) Diseases [Transmissible spongiform encephalopathies (TSEs)]
Kuru was once found among the Fore tribe in Papua New Guinea. (laughing deaths eating the dead ritual).
Misfolded protein cause several rare degenerative brain diseases. Mad cow disease outbreaks cow feeds. Fatal.
Madcow.mov
Prion (protein), PrP, has a molecular mass of 28,000 dalton. Found in the brain tissue of all mammals, but function unknown.
Protease sensitive
Protease insensitive
Misfoldingofprotein
Myostatinproteinmutation