week 2 - 5 Flashcards
Protein Stability and Folding
- A protein’s function depends on its 3D-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
How can proteins fold so fast?
Proteins fold to the lowest-energy fold in the microsecond to second time scales.
Search for the minimum is not random because the direction toward the native structure is thermodynamically most favorable
Ken Dill’s folding funnel: hypothesis
Unfolded structures lie around the top. As the protein folds, it falls down the wall of the energy funnel to more stable conformations.
The native, folded structure is at the bottom
Thermodynamics of protein folding
Proteins fold into 20 and 30 structures that possess the lowest possible free energy
A protein’s internal residues direct its folding into native conformation- Hydrophobic Effect
*Non polar groups aggregate and water molecules are released *Increase in entropy owing to the release of water molecules into bulk water.
*Decrease in entropy of protein; increase in entropy of water
Protein Folding and Disease
Cells expend a substantial amount of ATP to enable correct protein folding preventing protein aggregation or, failing that, to rid cells of misfolded forms. In some cases, ordered aggregates found in prion and other amyloid diseases, result in severe consequences for the organism as a whole.
Protein misfolding is the basis of numerous human diseases
Alzheimer’s disease: amyloid b- protein
Motifs (folds)
Specific arrangement of several secondary structure elements
– All alpha-helix – All beta-sheet – Both
* Motifs can be found as reoccurring structures in numerous proteins
* Proteins are made of different motifs folded together
Fibrous Proteins: From Structure to Function
Collagen: ~ 12 major types; types I-III assemble in fibrils, type IV assembles in laminar network Elastin: crosslinked random coiled protein; gives elasticity to tissues
Alpha Keratin: A helix of helices
Hard alpha keratin (occur in birds and reptiles)– shells, fingernails, claws
* Soft alpha keratin (occur in mammals)–skin, hair, wool
Elastin
- Majorcomponentofconnective tissue of lung and arteries allows these to resume their shape after stretching or contracting
- Hydrophobic,insoluble,forms3D elastic network; Formed from loose and unstructured polypeptide chains
- Conformation that of random coil àpermits the protein to stretch and recoil. A variety of random coil conformations possible
- Canstretchinanydirection,
structure more elastic than rubber
EdmanDegradation(Classicalmethod)
Successive rounds of N-terminal modification, cleavage, and
identification
– Can be used to identify protein with known sequence
MassSpectrometry(Modernmethod)
MALDI MS and ESI MS can precisely identify the mass of a
peptide, and thus the amino acid sequence
– Can be used to determine post-translational modifications
Determining the amino acid sequence of a protein
- Separate chains.
* Cleavage of Disulfide Bridges (Performic acid oxidation; Sulfhydryl reducing agents (mercaptoethanol, dithiothreitol).
* Separation of chains: Subunit interactions depend on weak forces. Separation is achieved with (extreme pH; 8M urea; 6M guanidine HCl; high salt concentration -usually ammonium sulfate). Purify the chains - Acid hydrolysis + ion exchange chromatography.
Determines the AA composition of the protein. - Identify N- and C-terminal residues
N-terminal analysis:
* Dinitrofluorobenzene (DNFB): Sanger reagent
* Dansyl chloride forms a fluorescent derivative
* Edman’s reagent (phenylisothiocyanate),
– Cleaves one amino acid at N-terminus at a time. Helps to identify the number of distinct polypeptides
* eg Insulin has equal amounts of N-terminal residues (Phe and Gly). Two non identical polypeptide chains.
Dinitrofluorobenzene (DNFB): Sanger reagent
ReactwithDNFB
* HydrolysewithHCl
* RunonTLCwith standards
* IdentifytheN- terminus AA
5 Stages of Protein Synthesis
- Activation of amino acids
– tRNA aminoacylated - Initiation of translation
– mRNA and aminoacylated tRNA bind to the ribosome - Elongation
– cycles of aminoacyl-tRNA binding and peptide bond formation…until a STOP codon is reached - Termination and ribosome recycling
– mRNA and protein dissociate, ribosome recycled - Folding and post translational processing
– catalyzed by a variety of enzymes
All proteins are initially synthesized with methionine at their N/C-terminus?
All proteins are initially synthesized with methionine at their N-terminus?
Collagen is a protein in which the polypeptides are mainly folded into a ….
triple helix
Plasma membrane is a
single bilayer
with 2 faces (sides):
- Interior cytosol with its face;
- Exterior environment with its
exoplasmic face
Single membrane organelles
(lysosome, Golgi and ER): internal
aqueous space (matrix) is topologically
equivalent to exoplasmic face
Double membrane organelles
nucleus,
mitochondria, and chloroplast)
contains an inner and outer membrane with the Intermembrane space topologically equivalent to the exterior of the cell. Their matrix is equivalent to the cytosol.
Biological Functions of Lipids
- Storage of energy
– Hydrophobic nature: good packing - Insulation from environment
– Low thermal conductivity
– High heat capacity (can “absorb” heat)
– Mechanical protection (can absorb shocks) - Water repellant
– Hydrophobic nature: keeps surface of the organism dry
– Prevents loss of water via evaporation - Membrane structure
– Main structure of cell membranes - Cofactors for enzymes
– Vitamin K: blood clot formation
– Coenzyme Q: ATP synthesis in mitochondria - Signaling molecules
– Paracrine hormones (act locally)
– Steroid hormones (act body-wide)
– Growth factors
– Vitamins A and D (hormone precursors) - Pigments
– Color of tomatoes, carrots, pumpkins, some birds - Antioxidants
– Vitamin E
Sterols and Cholesterol
Sterol:
– Steroid nucleus: four fused rings (almost planar)
– Hydroxyl group (polar head) in the A-ring
* Cholesterol and related sterols are present in the
membranes of most eukaryotic cells (but not in bacteria):
-Modulate fluidity and permeability
-Thicken the plasma membrane
* Mammals obtain cholesterol from:
-food or,
-de novo synthesis by the liver.
Structural Lipids in Membrane
Contain polar head groups and nonpolar tails (fatty acids)
* Diversification comes from:
* changing the fatty acids
* modifying the head groups
● The properties of head groups determine the surface
properties of membranes
● Different organisms have different membrane lipid head
group compositions
● Different tissues have different membrane lipid head
group compositions
Classification of Lipids
Lipids that do not contain fatty acids: cholesterol, terpenes, …
* Lipids that contain fatty acids: storage lipids and membrane
lipids
Fatty Acids and Melting Temperature
Saturated fatty acids pack in an orderly way
- extensive favorable interactions (high melting point)
Unsaturated cis fatty acids pack less orderly due to the kink
- less-extensive favorable interactions (lower melting point)
Unsaturated trans fatty acids adopt an extended conformation.
- extensive favorable interactions (highest melting point
micelle
A micelle is a spherical aggregate of amphiphilic molecules, such as surfactants or lipids, in a liquid.
Hydrophobic Core:
The hydrophobic tails of the amphiphilic molecules (e.g., hydrocarbon chains) orient themselves inward, away from the surrounding water.
This creates a nonpolar interior that can trap hydrophobic substances like oils or fats.
Hydrophilic Shell:
The hydrophilic heads of the molecules face outward, interacting with the surrounding water and stabilizing the structure.
Cleansing Agents:
Soaps and detergents form micelles to trap grease and dirt, which can then be rinsed away with water.
Vesicle (Liposome)
A vesicle (liposome) is a spherical structure with one or more lipid bilayers enclosing an aqueous core. These structures form when amphipathic lipids self-assemble in water.
Key Features:
Bilayer Structure: Hydrophilic heads face outward, while hydrophobic tails face inward, forming the bilayer.
Aqueous Core: Can encapsulate water-soluble substances.
