Explaination: Homology modeling is a part of comparative modeling. It is used when there is a clear relationship between the sequence of a protein of unknown structure to that of a sequence of a known structure. It is based on the assumption that two homologous proteins will share very similar structures. It is most accurate when the target and template have similar sequences.
Explaination: Threading is also a part of comparative modeling. This technique deals with finding relationships between sequence and structure that do exist, but are not immediately obvious, that is, a successful model will be proven to have structural similarity to a known fold, but no immediately obvious sequence similarity. A scoring function is used to assess the compatibility of the sequence to the structure, thus yielding possible three-dimensional models.
Methods used: Integer programming based fold recognition, Profile-profile alignment and secondary structure, Evolutionary information recognition
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Ab Initio prediction
Explaination: This method seek to build 3D protein models from scratch, that is based on physical principles rather than directly using previously solved structures or sequence data.
Methods used: Reduced modeling tool, A computational protocol for modeling and predicting protein structures at the atomic level, Molecular Dynamics folding
Part 1: Describe in great detail the two basic types of transmembrane proteins based on secondary structure
Transmembrane proteins: Membranes are permeable to nonpolar compounds and impermeable to polar compounds. A bilayer is formed by phospholipids in which nonpolar areas of lipid molecules are embedded in the bilayer and polar heads face outwards interacting with the aqueous phase on either side. Proteins are held in this bilayer sheet by hydrophobic interactions between membrane lipids and hydrophobic domains in the proteins. Intergral membrane proteins are embedded in the lipid bilayer which can be dissociated from the membrane only by physically disrupting the bilayer. They are amphipathic molecules that are in contact with the lipid bilayer as well as the aqueous environment. Peripheral membrane proteins can be dissociated from the membrane leaving the membrane intact as they are loosely bound by associations with integral membrane proteins or phospholipids.
Some integral membrane proteins are called transmembrane proteins because they are located all over the lipid bilayer with their surfaces exposed to cytosol and extracellular fluid. The two basic types of transmembrane proteins are alpha helices and beta barrels.
(Fluid mosaic model for membrane structure, Source: Lehninger - Principles of Biochemistry)
Î±-helical membrane proteins are responsible for interactions between most cells and their environment. They are typically encoded by stretches of 17-25 residues, which provide sufficient length to cross the membrane. Within alpha helices, the electronegative N and carbonyl O of the fourth amino acid on the amino-terminal side of that peptide bond are hydrogen bonded. This stabilizes the structure. Every peptide bond participates in such hydrogen bonding. They held successive turns of the helix. The helix has 3.6 amino acid residues per turn. The helices make complementary interactions with the hydrophobic lipid bilayer.
Beta sheets are formed by hydrogen bonding between adjacent segments of polypeptide chain. These adjacent polypeptide chains can be parallel or antiparallel. When the first strand of a large beta sheet forms hydrogen bond with the last strand of the sheet due to twisting of beta sheet, a characteristic structure is formed called beta-barrel in which beta strands are arranged in antiparallel fashion. Beta-barrels are found in the outer membrane of Gram-negative bacteria, mitochondria and chloroplasts and in the mitochondrial membrane. They help in active ion transport and passive nutrient intake. They also act as membrane anchors and defense against attack proteins.
Part 2: Find two examples of each transmembrane protein
Single transmembrane alpha-helix
(bitopic membrane protein)
2)Receptor tyrosine kinase
Polytopic alpha helical protein
Transmembrane Beta barrel
1) BtuB and FhuA (22 Î²-strands) Â active transporters for ferrichrom iron and vitamin B12Â uptake, respectively.
2) FadL (14 Î²-strands) Â long chain fatty acid transporter
Part 3: Please discuss thoroughly the difficulties membrane proteins pose to crystallization and the ramifications of this, and any novel crystallization methods being used to overcome this challenge.
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Problem: For any molecule to crystallize, it is necessary that is completely dissolves in the solvent. It is said to be dissolved when it is free to roam around in the solvent. Membrane proteins are not soluble in aqueous environment since they are amphiphilic that is they have hydrophobic and hydrophilic regions. Membrane proteins form hydrophobic cover by aggregating in aqueous environment. Most crystallization techniques try to reduce the solubility of membrane proteins in an aqueous environment, but they aggregate or denature so rapidly that the crystals are never formed.
To overcome this problem, detergents are added to aqueous solution, so that the hydrophobic region of the detergent binds to the protein's hydrophobic region and the detergents' polar heads face the aqueous solution. Hence, the hydrophobic regions are covered with hydrophilic polar heads. Even then, x-ray sources have shown that membrane proteins yield crystals that diffract x-rays to either high resolution or very low resolution. This could be because the monomers in the detergent solubilized proteins are not able to interact much with each other.
Many novel crystallization methods are being used to overcome this challenge like,
new improved detergents, antibodies, Bicelle crystallization and Cubic lipid phases.
Part 4: Address how ion channels can have such amazing specificity and ion conductance. I would like you to explain in your discussion the following observations "The K+ leak channels are highly selective for K+ ions by a factor of 10,000 over Na+ . What is the chemical basis for this selectivity for K+ ions since both ions are featureless spheres? Steric occlusions cannot account for the selectivity since Na+ is a smaller ion than K+(0.95Å and 1.35Å respectively ). Finally, How can K+ channels be so selective and at the same time exhibit a throughput of 108 ions per second?"
The K+ channel is formed by 4 subunits arranged around a central ion pore. There are lots of negative charged residues at both ends of the pore. Obviously, these will attract positively charged ions. A narrow selectivity filter connects this cavity to the external solution. This filter is lined by carbonyl oxygen atoms of their polypeptide backbones. Hence, these provide close binding sites for dehydrated K+ ions which will have to cover very short distance to diffuse from one site to the next within the selectivity filter since the dimensions of these binding sites fit K+ ions exactly to balance out the energy loss caused during dehydration. This doesn't happen with Na+ ions. The side chain refrains the carbonyl oxygen atoms to bind with Na+ ions by compensating its energy loss caused during dehydration.
Also, the concentration of K+Â is high inside the filter and low outside. So K+Â will tend to leave the cell through the K+Â leak channels, driven by its concentration gradient. As K+Â moves out, it will leave behind unbalanced negative charge, thereby creating an electrical field, or membrane potential, which will tend to oppose the further outflow of K+. The net outflow of K+Â will stop when the electrochemical gradient for K+Â is zero.
Part 5: I would like you guys to do a little study of glutamate receptors. There are three types of human glutamate receptors. I will give you two out of the three (NMDA, AMPA) I need you to tell me
The different types of glutamate receptors in human beings
There are two main types of glutamate receptors in human beings:
Ionotropic receptors and Metabotropic receptors.
Ionotropic receptors are ligand gated ion channels. They are divided into 3 groups NMDA (N-methyl D- aspartate receptor), AMPA (Î±-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) and Kainate receptors, orÂ KARs
Metabotropic receptors are are G-protein coupled receptors.
What is the overall quaternary, tertiary, and secondary structures for each type of receptor?
The basic structure of ionotropic receptor is like this:
The long N-terminus is extracellular while short C-terminus in intracellular. There are 4 hydrophobic domanis. TMIV lies within the central portion of the sequence; TMII forms a re-entrant loop, and the intracellular loop between TMIII and TMIV is exposed to the cell surface and forms part of the binding domain with the C-terminal half of the N-terminus.
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NMDA: NMDA is composed of two subunit families - GluN1 and GluN2. GluN2 has four subunits GluN2A, GluN2B, GluN2C and GluN2D. These subunits are a product of different genes. Thus, NMDA receptor forms aÂ heterotetramerÂ between GluN1 and two GluN2 subunits. These different subunits of GluN2 get combined with GluN1 to form many different NMDA receptors that have different properties and functions.
AMPA: AMPA are composed of four types of subunits GluA1Â ,Â GluA2Â ,Â GluR3Â , andÂ GluA4 which combine to formÂ tetramers. The intracellular C-terminus contains binding sites for AP2, NSF and PDZ proteins. AMPAs also have 2 splice variants.
Kainate receptors: Kainate receptor is a tetramer composed of 5 subunits:Â GluK1, GluK2, GluK3, GluK4 and GluK5. GluK1, GluK2 and GluK3 can combine to form functional heteromeric and homomeric assemblies. Kainate receptors also have splice variants and undergo RNA editing to form many different receptors with different properties and functions.
For each type of receptor tell me what are the functions of each different domain
NMDA: Is responsible for variety of post synaptic functions. They are permeable to Calcium ions as well as other ions. They are responsible for activation of CaMKII and phosphorylation of the GluA2 AMPA receptor subunit resulting in LTP (HFS), activation of PICK1 to drive the PKC-dependent synaptic insertion of AMPA receptors during LTP, activation of hippocalcin, a Ca2+-binding protein that recruits AP2 to the GluA2 AMPA receptor subunit prior to internalisation during LTD (LFS) and activation of PI3K/Akt/GSK3 to modulate LTP.
AMPA: AMPA receptors are responsible for fast excitatory synaptic transmission. Increasing the post-synaptic response to a stimulus is achieved through increasing the number of AMPA receptors at the post-synaptic surface or by increasing the single channel conductance of the receptors expressed.
Kainate : Kainate has been used as a model for temporal lobe seizures. They are not only synaptically activated but are crucial for synaptic plasticity as well. They are critical for the induction ofÂ NMDA receptor-independent LTPÂ at theÂ mossy fibre synapseÂ in the CA3 region of the hippocampus. Outside of the hippocampus, a major role for kainate receptors in synaptic plasticity in theÂ somatosensory cortexÂ has recently been revealed; during LTP, while AMPA receptor-mediated synaptic transmission is increased, kainate receptor-mediated transmission is reduced.
What are the differences between each receptor on the basis of amino acid sequence?
How do these differences account for observed physiological differences between these receptors?
When glutamate binds to AMPA or kainite receptoprs, a fast EPSP is produced by sodium movement into the cell.
When glutamate binds to NMDA receptors, calcium channels open, resulting in an influx of calcium. Calcium acts as a second messenger, producing biochemical changes in the postsynaptic neuron.