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134 Cards in this Set
- Front
- Back
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What makes red blood cells red? |
Red blood cells are very abundant, carries oxygen to tissue and CO2 away from tissues and the membrane is colourless.
The red comes from hemoglobin which is a protein molecule that carries oxygen to tissues and very abundant . Hemoblogin contains a heme, an iron binding component of hemoglobin, which gives it its red colour. |
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What is hemoglobin? |
Oxygen transport protein of erythrocytes, is a tetramer composed of 2 alpha-chains and 2 beta-chains
Heme = iron binding component of hemoglobin (Heme is a prosthetic group)
Structure was Found in 1950s
-Heme:
4 subunits, tetramer
The spirals are alpha helices
Some aa have very little affect when you change while some causes diseases |
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What is sickle cell anemia? |
A single amino acid change (Glu6Val) in beta-chain of hemoglobin causes sickle cell anemia • A single change in aa causes this change, the Glu protein in the sixth position to a Val which resulted in a human diseases
Hb S forms long polymers that change the shape of red blood cells
Anemia means lower red cell count
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What is a protein? |
Proteins are polymers of amino acids linked in series by peptide bonds. • Linear sequence of amino acids
Proteins fold into specific 3D structures • Not linear chain but folds into 3D structures and different structures/fold have different functions or not working properly when misfolded • When proteins are misfolded they turn back into aa's and are chopped up and recycled again
Agents of biological function, often enzymes • Enzymes are catalyzes and speed up reactions, carbonic anhydrase catalyzes into H2O a million times per second per molecule
Encoded by genes
Mutations in genes lead to defective proteins and inherited diseases |
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What is H2O and its properties? |
Water = H2O
• Water is polarized where H is EN+ while O is EN-, it is dipole
• Oxygen loves to interact with itself using hydrogen bonds
• Water is attracted to each other and hydrogen bonds are constantly being broken and created, especially depending temperature
• The oxygen can form weak hydrogen bonds where it can also accept hydrogen bonds because it is EN-
• Without water, proteins won't be able to fold up properly.
• Proteins are designed to fold in aqueous solutions
• Proteins will NOT fold up in oil
• Most important molecule in biology and essential for life as we know it
• Water has a higher boiling point, melting point, heat of vaporization and surface tension compared to other hydrides
• The potential to form 4 H bonds per water molecule is the source of the strong intermolecular attractions that endow this substance with its anomalously high boiling point, melting point, heat of vaporization and surface tension
• Each H2O molecule has 4 nearest neighbours to which it is hydrogen bonded: Each H atom donates a H bond to the O of a neighbor and the O atom serves as a H-bond acceptor from H atoms bound to 2 different molecules
• H-bonded water molecule serving as an acceptor is a better H-bond donor than an unbounded molecule and a H2O molecule serving as a H-bond donor becomes a better H-bond acceptor
• The directional preference of H bonds leads to an open lattice structure
• The presence of strain creates a kinetic situation in which H2O molecules can switch H-bond allegiances; fluidity ensues |
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What are hydrophobic interactions? |
Non-polar groups are driven together not so much because they have a high affinity for each other but because water hydrogen bonds strong to itself
Hydrophobic interactions result from the strong tendency of water to exclude non-polar groups or molecules
They arise because water molecule prefer the stronger interactions that they share with one another compared to their interaction with non-polar molecules
Non-polar solutes do not readily H bond to H2O and as a result, such compounds tend to be only sparingly soluble in water
The process of dissolving such substances is accompanied by significant reorganization of the water surrounding the solute so that the response of the solvent water to such solutes can be equated to "structure making"
Consequently, the H-bonded water network rearranges towards formation of a local cage-like (clathrate) structure surrounding each solute molecule
This fixed orientation of water molecules around a hydrophobic "solute" molecule results in a hydration shell
Clathrate formation is accompanied by significant ordering of structure or negative entropy
Hydrophobic effect happens inside the protein |
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What is ionic interactions? |
Opposite charges attract each other
The result of attractive forces between oppositely charged structures, such as negative carboxyl groups and positive amino groups
Strength of electrostatic interactions is highly dependent on the nature of interacting species and the distance, r, between them
• May involve ions (species possessing discrete charges), Permanent dipoles (having a permanent separation of positive and negative charges) or induced dipoles (having temporary separation of positive and negative charge induced by the environment |
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What are hydrogen bonds? |
Specific
Water can break hydrogen bonds as they are always in constant competition with each other
Hydrogen bonds form between a hydrogen atom covalently bonded to an electronegative atom (such as O or N) and a second EN negative atom that serves as the hydrogen bond acceptor
Stabilizing, attractive interactions between a proton and an electron - donating species |
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What are Van der Waals interactions? |
• Contact
• Protein are tightly packed like a crystal, so packed so tightly and the interaction between the nucleus and electrons of another, very weak and insignificant
• Van der Waals forces are the result of induced electrical interactions between closely approaching atoms or molecules as their negatively charged electron clouds fluctuate instantaneously in time
• They operate only over a very limited interatomic distance (0.3 - 0.6 nm) and are an effective bonding interaction at physiological temperatures only when a number of atoms in a molecule can interact with several atoms in a neighbouring molecule
• Tend to be weak and individually contribute 0.4 - 4.0 kJ/mol of stabilization energy |
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What are olgiosaccharides? |
Polymers of sugars linked in linear and branched series of glycosidic bonds resulting in complex structures |
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What are the four nucleotide bases of DNA? |
2 Purines: -Adenine -Guanine
2 Pyrimidines: -Thymine -Cytosine |
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What is another name for alpha-carbons and what is special about them? |
Central carbon
Alpha-carbon are asymmetric or chiral and the 2 possible configurations for the alpha constitute non-superimposable mirror-image isomers, or enantiomers |
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How are peptide bonds formed and what are they? |
Amino acids can join via peptide bonds
The crucial feature of amino acids that allow them to polymerize to form peptides and proteins in the existence of their 2 identifying chemical groups: the amino (-NH3+) and carboxyl (-COO-) groups
They can react in head-to-tail fashion, eliminating a water molecule and forming a covalent amide linkage, which, in the case of peptide and proteins, is typically referred to as a peptide bond
Equilibrium of reaction favours peptide bond hydrolysis
Is planar and trans between carbonyl O and amide H
Dihedral angel w about the peptide C-N bond is fixed at 180 degrees
Although it is 2 planes, when joined together, it can be rotated (turn about alpha carbon) |
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What is zwitterion? |
A dipolar ion
Had negative and positive change creating a neutral molecule (no net charge) |
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How do you know if a beta carbon is NOT beta branched? |
It has 2 or more hydrogens bonded to it |
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What is Glycine structure, short form and properties? |
Glycine, Gly, G
• Only has a hydrogen atom as a side chain
• Aliphatic side chain
• Does not have an L or D forms
• Very and maybe most flexible aa and the smallest
• Allows close packing and van de Waal forces
• Simplest amino acid, has only a single hydrogen for an R group and this hydrogen is not a good hydrogen bond former,
• Glycine's solubility properties are influenced mainly by its polar amino and carboxyl groups and thus glycine is best considered a member of the polar, uncharged group
• Except for glycine, all the amino acids isolated from proteins have 4 different groups attached to the alpha-carbon atom
• Glycine is sterically the most adaptable of the amino acids and it accommodates conveniently to other steric constraints in the beta-turn |
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What is Alanine structure, short form and properties? |
Alanine, Ala, A
Has a methly group for R-chain
Aliphatic side chain
Most generic
Non-polar
Hydrophobic |
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What is Valine structure, short form and properties? |
Valine, Val, V
• Beta branched
• Large aliphatic chain
• Non-polar
• Hydrophobic due to aliphatic chain
• Terrible alpha-helix former due to beta branch |
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What is Leucine structure, short form and properties? |
Leucine, Leu, L
Aliphatic side chain
Most common amino acid in proteins
Great alpha-helix former
Hydrophobic
Non-polar |
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What is Isoleucine structure, short form and properties? |
Isoleucine, Ile, I
• Aliphatic side chain
• Non-polar
• Hydrophobic |
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What is Serine structure, short form and properties? |
Serine, Ser, S
• Has a hydroxyl group: -Form hydrogen bonds with water where the Oxygen can be a hydrogen acceptor which makes it polar
• Aliphatic hydroxyl side chain
• Good hydrogen bond-forming moeities
• Hydrophilic
• Polar
• EN negative |
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What is Threonine structure, short form and properties? |
Threonine, Thr, T
• Beta branched
• Aliphatic hydroxyl side chain
• Polar
• EN negative
• Hydroxyl group AND methyl group
• Good hydrogen bond-forming moieties
• Hydrophilic |
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What is Phenyalanine structure, short form and properties? |
Phenyalanine, Phe, F
• Aromatic side chain
• Benzyl group R-side chain
• Hydrophobic
• Absorb ultraviolet light above 250nm |
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What is Tyrosine structure, short form and properties? |
Tyrosine, Tyr, Y
• Aromatic side chain
• Amphipathic
• Hydrophobic with polar properties
• Good hydrogen-bond forming moieties
• Also has non-polar characteristics due to its aromatic ring and could be arguably be placed in the non-polar group (Has pKa of 10.1, its phenolic hydroxyl is a charged, polar entity at high pH)
• Absorb ultraviolet light above 250nm |
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What is Tryptophan structure, short form and properties? |
Tryptophan, Trp, W
• Aromatic side chain
• Has indole ring R-side chain which gives it absorption of 290nm light
• Nitrogen on indole ring give it hydrogen donor potential
• Considered a borderline member of aromatic side chain group because it can interact favourable with water via the N-H moiety of indole ring |
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What is Beer-Lambert Law? |
Concentration of a give target protein solution is directly related to its UV absorption reading
A=ecl
Where A is absorbance, e is moral extinction coefficient, c is concentration of target protein and l is length of optical path |
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What is Cysteine structure, short form and properties? |
Cysteine, Cys, C
Contains sulfur
EN negative
Can deprotonate at pH values greater than 7
Hydrophilic
Can also be considered hydrophobic because of its sulfide and found buried inside the protein
Can create disulfide bridges with other cysteines |
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What is Methionine structure, short form and properties? |
Methionine, Met M
Contains sulfur
EN negative
Often the first protein to be cut off as it is the initiator protein
Amphipathic (least polar of the amphipathic amino acids but its thioether sulfur can be an effective metal ligand in proteins)
Hydrophobic |
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What is a disulfide bridge and what is its properties? |
• It is a link between two cysteine amino acids put together in a protein
• There is an enzyme that breaks the disulfide bridge over and over again till protein gets it right
• Disulfide bridges exist outside and inside of cells
• Increase stability of proteins
• This is a reduced form, if you oxidize it, you create a disulfide bridge and you can reduce it to break the bridge
• Formed during protein folding process
• Two cysteine from other sides of protein can bind together and help anchor the protein |
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What is Asparate structure, short form and properties? |
Aspartate, Asp, D
• Polar
• Acidic
• Hydrophilic |
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What is Glutamate structure, short form and properties? |
Glutamate, Glu, E
• Polar
• Acidic |
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What is Asparagine structure, short form and properties? |
Asparagine, Asn, N
• Good hydrogen-bonding forming moieties
• Hydrophilic
• To test what a D is doing in a protein, you can change it to N to see if it does anything or if it kills the protein
• Polar
• Amide R-side chain
• Acidic |
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What is Glutamine structure, short form and properties? |
Glutamine, Gln, Q
Hydrophilic
Acidic |
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What is Lysine structure, short form and properties? |
Lysine, Lys, K
Has a 4 methyl group in a row making it hydrophobic • Head poking out interacting with water while the chain is hiding inside the protein, called snorkeling • Head is different from rest of protein
Basic
Lysine contains a protonated alkyl amino group
Side chains are protonated under physiological conditions and participate in electrostatic interactions in proteins
Amphipathic
Can be considered amphipathic because its R group consists of an aliphatic side chain which can interact with hydrophobic amino acids in protein and normally charged at neutral pH
Polar |
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What is Arginine structure, short form and properties? |
Arginine, Arg, R
Very positively charge and never lose it under natural circumstances
Very important for binding substances
Basic
Arginine contains aguanidiniumgroup
Side chains are protonated under physiological conditions and participate in electrostatic interactions in proteins
Hydrophilic
Has resonance structure due to double bond
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What is Histidine structure, short form and properties? |
Histidine, His, H
Has an imidazole ring where resonance structure where the positive charge can change. It can lose it proton easily where it can take or lose easily
Basic
Side chains fully protonated at pH7 but histidine with a side chainpKaof 6 which means it is only 10% protonated at pH 7
With a pKa near neutrality, histidine side chains plays important roles as proton donors and acceptors in many enzyme reaction
Hydrophilic |
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What is Proline structure, short form and properties? |
Proline, Pro, P
Is an imino acid, not really an amino acid but is because it is cyclic
Side chain is cyclic and forms a ring via a covalent bond with the backbone nitrogen atom.
Cyclic ring makes it a very rigid structure and makes the kink in chains
If you put in alpha helix, it would break it and bend it
For the protein to fold, it is often found that proline is found in the bends and folds
Non-polar
Hydrophobic
Proline has a cyclic structure and a fixed phi angel, so, to some extent, it forces the formation of a beta-turn |
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What are the hydrophobic amino acids? |
Ala, Cys, Ile, Leu, Met, Phe, Val |
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What are the hydrophilic amino acids? |
Basic: Arg, Lys
Acidic: Asp, Glu
Polar: Asn, Gln, His |
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What is the most common amino acid? |
Leu |
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What is pH? |
Soren Sorensen devised the pH scale by defining pH as the negative logarithm of the hydrogen ion concentration:
pH = -log[H+] pKw = pH + pOH = 14
Low pH values represent the highest H+ concentration and lowest OH- concentrations
Point of neutrality is at pH 7 and this is called neutral pH
Substances that are almost completely dissociated to form ions in solutions are called strong electrolytes
Electrolyte describes substances capable of generating ions in solution and thereby causing an increase in the electrical conductivity of the solution
Acids are proton donors and bases are proton acceptors
Substances with only slight tendency to dissociate to form ions in solution are called weak electrolytes
Ka is also termed an ionization constant because it states the extent to which a substance forms ions in water
Relatively low values of Ka for acetic acid reveals that un-ionized form, CH3COOH, predominates over H+ and CH3COO- in aqueous solutions of acetic acid |
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What is the Henderson-Hasselbalch equation? |
Describe the Dissociation of a Weak Acid in the Presence of its conjugate base
pH = pKa + log([A-]/[HA])
The pH of a solution can be calculated provided Ka and the concentration of the weak acid HA and its conjugate based A- are known |
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What is the pKa's of R, K, C, H, E, D? |
R = 12.5
K = 10.5
C = 8.3
H = 6.0
E = 4.3
D = 3.9 |
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What is Trypsin? |
Digestive enzyme trypsin is the most commonly used reagent for specific proteolysis
Will only hydrolyze peptide bonds in which the carbonyl function is contributed by an arginine or a lysine residue
Trypsin cleaves on the C-side of Arg or Lys, generating a set of peptide fragments having Arg or Lys at their C-termini
Number of small peptides resulting from trypsin action is equal to the total number of Arg and Lys residues n the protein plus one - the protein's C-terminal peptide fragment |
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What is protein primary structure? |
Amino acid sequence |
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What is protein secondary stucture? |
Periodic, repetitive units (alpha-helix, beta-sheet, reverse turn)
In the alpha-helix, the N-H group of residue (i) donates a H-bond back to C=O group of residue (i - 4)
Need to pack side chains and minimize side-chain steric clashes
Through hydrogen-bonding interactions between adjacent amino acid residues
Is the 3D arrangement |
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What is protein tertiary structure? |
Assembly of secondary + unstructured segments into functional protein
Defines the final folded functional structure of the protein
Represents the assembly of alpha helices, beta sheets, beta turns, intervening and disulfide bonds
Segments without periodicity are described as 'random' or 'unstructured'
Structure defined by sequence
Proteins are dynamic and exist in different conformational states |
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What is protein quaternary structure? |
Assembly of protein sub-units
Described proteins where function depends upon the assembly of two or more subunits
Subunits interact through the same forces as occur within individual subunits
Subunits can be identical, or may all be different
Key role played by van der Waals packing and hydrophobic effect |
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What is an alpha-helix? |
Intramolecular H-bonds stabilize the structure:
N-H group of residues i donates hydrogen back to C = O of residue i - 4, instead of H-bonding with water
Secondary structure
Each peptide bond posses a dipole moment that arises from the polarities of the N-H and C=O groups and because these groups are all aligned along the helix axis, the helix itself has a substantial dipole moment with a partial positive charge at the N-terminus and a partial negative charge at the C-terminus
In a typical alpha helix, there are ( n - 4 ) hydrogen bonds
3.6 residues per 360 degrees turn (3 aa during single turn)
100 degrees between adjacent residues.
Side chains exposed around the periphery of the structure
Most helices are amphipathic |
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What is a pitch in an alpha-helix? |
Translation distance of the helix
If ignoring side chains, the helix is about 6 A in diameter
Side chains, extending outward from the core structure of the helix, are removed from steric interference with polypeptide backbone
Each peptide carbonyl is hydrogen bonded to the peptide N-H group 4 residues farther up the chain
All H bonds lie parallel to the helix axis and all the carbonyl groups are pointing in one direction along the helix axis while the N-H groups are pointing in the opposite direction |
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What is helix capping? |
Providing H-bonds partners for the otherwise bare N-H and C=O groups and folding other parts of the protein to foster hydrophobic contacts with exposed non-polar residues at the helix termini |
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What are beta-pleated sheets? |
Not planar
Important feature of protein secondary structure
Formed from extended chains of amino acids, bonded to each other through alternating intermolecule H-bonds
Every second aa is hydrophobic making one side hydrophobic and other hydrophilic
Forms because of local, cooperative formation of hydrogen bonds
Pleated sheet can exist in both parallel and antiparallel forms
3.0 residues per translation |
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What is a parallel beta-pleated sheet? |
Adjacent chains run in same direction
Tend to be more regular than antiparallel
Typically large structures; those composed of less than 5 strands are rare
Distribute hydrophobic side chains on both sides of sheet |
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What is an antiparallel beta-pleated sheet? |
Adjacent strands run in opposite direction
Can contain as few as 2 strands
Usually arranged with all their hydrophobic residues on one side of the sheet |
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What are beta turns? |
Designates the 4-residue segment through which the protein chain turns 180 degrees (fold back on itself, usually surface of protein)
AKA Reverse turns
Can link two beta strands, helices or strand to helix
Peptide chain forms a tight loop with carbonyl oxygen of 1 residue hydrogen bonded with the amide proton of the residue 3 position down the chain
Because it lacks a side chain, glycine is more steric the most adaptable of the amino acids and it accommodates conveniently to other steric constraints in the beta-turn
Proline, however, has a cyclic structure and a fixed phi angel, so, to some extent, it forces the formation of a beta-turn |
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What are phi/psi/omega/chi angels? |
Dictate the secondary protein structures (alpha helix/beta sheets), thus those angles define secondary structures
The angle about the C alpha - N bond is denoted by the Greek letter phi and that about the C alpha - C is denoted by psi
Omega is around the C-N peptide bond
Chi describes the side chain rotation |
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What are protein backbone angles? |
Dihedral angle at each backbone bond determines the actual three-dimensional progress of the protein chain
Helices and sheets have characteristic backbone angles |
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What is the Ramachandran Plot? |
Plotting group of proteins by phi and psi angles |
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What is Poly-proline helix? |
Put a bunch of Pro together to form a helix
Pro doesn't have protons, thus Pro cannot form H-bond
It has carboxyl group which acts as a proton acceptor BUT not a proton donor
Thus Pro can't be a H-bond donor, but it can work as a H-bond acceptor
When you put bunch of Pro's together, there's no NH groups, so it's not H-bonded at all
Polyprolinehelix has NO H-bonds due to its conformational restriction of Pro residue |
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Describe the fibrous protein, silk: |
Most common residue is Gly
Has proton, conformational flexibility, can be in turns, alpha helix, beta sheets
Repeating unit of: - G - S - G - A - G - A - which is conducive to beta-pleated sheets
Inter-chain H-bonds are formed while side chains are above and below the plane of H-bond network
Spider silk (a form of keratin) is synthesized in special glands in the spider's abdomen
Strong and elastic
Dragline silk (from which the spider hangs) has a tensile strength of 200000 psi (pounds per square inch)
Small residue Gly allows tight packing
Hydrogen bond network link beta-strands
Fibers strong: resistance to tension is borne by covalent bonds |
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Describe the fibrous protein wool: |
Mostly Glu + Gln
Is a keratins
Much lower in Gly vs silk, fairly even distribution of helix-forming residues (Ala, Glu)
7 amino acid heptad repeat
Cystine crosslinks
Can be stretched to twice its length
In stretched state, intra-helix H-bonds may be broken, and inter-strand H-bonds may form: via an alpha-helix to beta-sheet transition (structure NOT static, changes depends on humidity)
Due to sulfur content, disulfide crosslinks constitute the main restoring force |
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Describe the fibrous protein keratins: |
Highly-complex macromolecular structure: each single alpha-helix is twisted with others into a "protofibril"
The alpha helices are bundled/packed together into protofibril which packs together to form microfilbril with 9-11 protofibrils, to form a fiber resembling rope |
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Describe the fibrous protein collagen |
High in Gly
Lots of Pro and modified version of Pro, hydroxy-proline
Can form turns and bents
Put bunch of proline together to form polyproline helix (no H-bonds to stabilize it)
About 1/3 of our protein is collagen
Universal occurrence
Rigid, inert material, resistant to stretching
Used for connective tissue, skin, tendons
Collagen contains large proportions of glycine and proline and much of its structure is composed of (Gly-x-Pro) repeating units where x is any amino acid
Three unusual modified amino acids are also found in collagen: 4-hydroxyproline (Hyp) 3-hydroxyproline 5-hydroxylysine (Hyl) |
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What is the structure, short form and properties of Hydroxyproline |
Hyp
OH group used to form H-bond cross links between collagen strands
Enzyme proline hydroxylase adds -OH group to proline side chain in pro-collagen protein
Thus no CODON for Hyp since it can only be made by the enzyme, proline hydroxylase
more hydrophilic because it has H-bond donor (the OH group is present in Hyp not Pro)
Hyp likes to interact with water |
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What is basic coiled-coil structure of collagen? |
Three left-handed single-chain helices wrap around one another with a right-handed twist |
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What are 3 fibers inside cells that make up cytoskeleton and what is cytoskeleton? |
Actin
Microfilaments
Intermediate filaments
they are long rod-like structures
vesicles are carried onto cytoskeletons
Help move vesicles around (from ER togolgi)
Motor proteins usually use ATP to move vesicles along the cytoskeleton |
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What is G-actin? |
Single actin subunit
Single polypeptide (monomer)
G for globular protein
Actin is important in forming long fibers |
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What is F-actin? |
F for filament
We can shrink/expand actin filaments by adding/removing single actin subunits
Involved in cell motion
Actin filament is added to one end to make longer or fall off another end |
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What are globular protiens? |
Have folded, roughly spherical structures
With right phi/psi angles, it can form alpha helices and beta strands
Many are enzymes, with active sites embedded in the protein core
Consists largely of secondary structure elements, including helices, sheets and turns
Polypeptide chain is compactly folded so that hydrophobic amino acid side chains are in the interior of the molecule and the hydrophilic side chains are on outside exposed to solvent, water
Usually very soluble in aqueous solutions
Globular proteins exist in an enormous variety of 3-dimensional structures but nearly all contain substantial amounts of alpha-helices and beta-sheets folded into a compact structure that is stabilized by both polar and non-polar group
Space between the helices and sheets in protein interior is filled efficiently and tightly with mostly hydrophobic amind acid side chains |
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What is the globular protein: myoglobin? |
Richest source of it: muscles of aquatic vertebrates like whales.
First protein experimented for X-ray crystallography in 1958
'Box' for a heme group: heme binds oxygen, protein stores it until required for metabolic oxidation
153 amino acids: 121/153 in alpha helices
8 connect helix segments connected by turns and loops
Strongly hydrophobic core
Helices fold via beta-turns: several turns contain Pro or Gly residues
The glu for the oxygen binding site is the His side chains
2 His residue shelp stabilize heme molecule in a very specific position |
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What is the globular protein: ribonuclease A? |
Chain-cutting enzyme, cleaves the polyribonucleotide chains of RNA but not DNA
Taco-shaped molecule with active site in crevice running across the molecule
Has significant segments of beta-sheet and alpha-helix
Four disulfide bonds to help stabilize the protein once outside the cell
Active site located by binding inhibitor which includes His 12, His 119 and Lys 41
Antiparallel structure
His119 and His12 right beside each other => critical for their activity |
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What is the mechanism of ribonuclease? |
1) N on His12 has on proton, thus acts as nucleophile and pulls H+ from ribose hydroxyl group
2) the O has 2 e- now since deprotonated and forms H-bond with phosphate
3) now this O (from phosphate group) takes proton from His119 and His119 becomes deprotonated
Lysine-41 is not directly involved in this mechanism, but it is there to stabilize the intermediate state
NOTE: DNA does not have OH at carbon 2 (unlike RNA's ribose sugar), thus rxn with deoxyribose sugar wouldn't work |
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What are membrane proteins? |
Are responsible for key cellular processes
Functions: transporters, receptors, enzymes, cellular morphology
Bilayer maintains native conformation of proteins
Have to be compatible with hydrophobic phase of lipid bilayer |
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What are multi-spanning proteins: transporters? |
Carry out exchange of substrates in and out of the cell across the plasma membrane
Channels and pumps
Energy (ATP) required for pumps
Electro-chemical gradient |
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Which transports do not require energy? |
Proteins which use gradients across the membrane of the substance they transport
Example) Bind 3 anion transport of red cells, the glucose carrier protein |
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What is the uracil transporter? |
Found in bacteria
Almost 100% alpha helix
Dynamic as it open and closed, bringing uracil in and out
14 transmembrane protein sections
Uracil is bound in middle, H exists in number of states:
Closes up and releases uracil on other side, then opens up again and repeats this process
Recall: membrane proteins are highly helical |
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What are receptor proteins? |
Receptors transmit signals across membranes (nothing is transported)
Ligand-activation fairly general mechanism (binding outside the cell induces signal)
Receptors may be single-spanning (insulin receptor) or multi-spanning (G-protein-coupled receptors) |
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What are insulin receptor signals? |
Bind insulin -> change conformation of protein -> transmit info -> activate the kinase and it puts phosphate groups on tyrosine
Made of 4 subunits
Linked together by disulphide bonds |
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What is Anfinsen's experiment? |
Anfinsen found that reduced RNase (using beta-mercaptoethanol) proceeds to completion only when the protein is partially unfolded by denaturing agents such as urea or guanidine hydrochloride
Anfinsen discovered that removing themercaptoethanl but not the urea restored only 1% of enzyme activity
If anfinsen removed mercaptoethanol and urea at the same time, the polypeptide was able to fold into its native structure, the correct set of 4 disulfides re-formed and full enzyme activity was recovered
This experiment demonstrated that the information needed for protien folding resided entirely within the amino acid sequence of the protein itself
The urea in this situation was able to form H-bonds with protein backbone.
Takes H-bond away from alpha helix that has H's, lets it bind with itself
Loss of structural order in these complex macromolecules (denaturation) is accompanied by loss of function
After dialysis to remove small molecules (urea and beta-mercaptoethanol), enzyme slowly regained activity
Sulfyhydral groups become re-oxidized by air to form the refolded, catalytically-active form
Results led to main conclusion that primary sequence determines secondary and tertiary structure
When RNase was refolded in 8M urea by oxidation and then dialyzed to remove urea, only 1% enzymatic activity was obtained. -wrong disulfide pairing occured |
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How does denaturants work? |
By disrupting (competing for) non-covalent interctions
Danaturation over a very small temperature range such as this evidence of a two-state transition between the native and the unfolded states of the protein and this implies that unfolding is an all-or-none process
When weak forces are disrupted in one part of the protein, the entire structure breaks down
Danaturation leads to loss of protein structure and function |
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What is dialysis? |
If solution of proteins is separated from bathing solution by a semipermeable membrane, small molecules and ions can pass through the semipermeable membrane to equilibrate between the protein solution and bathing solution
Method is useful for removing small molecules from macromolecular solutions or for altering the composition of protein-containing solution |
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How would you renature a ribonuclease? |
Once you have denatured reduced ribonuclease
Dialysis to remove urea and beta-mercaptoethanol
Air oxidation of the sulhydryl groups in reduced ribonuclease to reform the disfulphide bridges
Then you will have native ribonuclease |
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What is native state of proteins? |
Native state (with all interactions) is only marginally stable relative to the unfolded (denatured) state
Native state posses considerable "kinetic stability" because it can unfold only by passing through a high-energy transition state in which most native interactions are disrupted simultaneously
If proteins are in native conformation, last thing you want to not randomly just fly open (spontaneous denature)
Only way to unfold you must go through energy transition like losing part of helix, losing hydrogen bonds or van der walls |
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What is the cardboard box model? |
In unfolded state, the "flaps" of the protein are free to move independently
entropy is lost during folding because gone from free moving state to a very restricted state (lose free of motion but you're gaining all non-covalent and covalent interactions to stabilize protein => thus balances the entropy and overall stabilization
Large energy barrier must be surmounted to convert from "almost folded" to "fully folded" This guarantees that proteins do not denature too easily |
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What are isozymes: |
Structural varients
These structure don't have negative effects, sometimes they might have positive effect
These variations results in the population (what makes everyone different) |
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What are restriction sites and enzymes? |
Start with gene of interest within double stranded DNA
"A" and "B" are appropriate flanking endonuclease sites
Restriction endonucleases are enzymes found in procaryotic cells
Many restriction endonucleases recognize 'palindromic' sites in double-stranded DNA
Such sites read the same sequence left-to-right and right-to-left |
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What is insertion of synthetic oligos? |
Many genes contain wild type restriction sites: you choose the endonucleases based on palindromic sequence
Cleave the genes of interest at the restriction sites
Synthesize and purify the desired oligonucleotide (replacement therapy)
Insert the oligo into the chain enzymatically using "DNA ligases" |
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What is Thioredoxin? |
You would add another protein sequence that E.coli loves, allowing your protein of interest to be expressed
Essentially, you're using this to help express your protein of interest
It acts as carrier and helps make your protein soluble
Once you have the fusion protein expressed, you can cleave off the tag (since you're not trying to study it)
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What are His-tags? |
6-10 His residues for affinity chromatopgraphy
His-tags are small sequences that codes for many His residues (6-10) in a row
His binds to metals (Ni and Co) in proteins
This tag on the protein will bind to a resin that has Ni on it
Any protein that has His-tag on it, will bind to the resin
Proteins that don't have His-tag will not bind to resin
Ni will bind to proteins with His-tag, then you wash the proteins with imidazole to separate your protein of interest away from other proteins
Put at ends, not middle, because it can interfere with folding
Nickle affinity columns recognize His-tags and bind this protein exclusively. After other proteins are eluted, imidazole is used to elute protein of interest |
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What are WB-tags? |
Western blot antibody tag
It is used for detection (to detect your protein)
It uses antibody to recognize this particular tag |
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What is protein purification? |
Fusion (or carrier) proteins are often contained in expression vectors, which aid in solubilization of expressed proteins
One-step enzymatic cleavage is used at final steps to cleave off fusion protein
Concurrent with characterization, necessary to demonstrate that you have correct protein
Should appear as an extra band at the expected molecular weight
Use 'Western blot' with antibody to fusion protein to confirm identity |
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Why are plasmids useful in cloning genes? |
Naturally occuring extra-chromosomal DNA
circular dsDNA (ds = double stranded)
Can be cleaved by restriction enzymes, leaving sticky ends
Can be constructed by linking new DNA fragments to sticky ends of plasmid
These recombinant molecules can be autonomously replicated and propagated
Cloning vectors are plasmids that can be modified to carry new genes
Plasmids are useful as cloning vectors as they have:
Replication (origin of replication, ori) • ori allows DNAP to bind in order to allow it to make more copies of plasmid DNA
Selectable marker (antibiotic resistance gene) • have TT of E.coli and you add DNA into it, the DNA will only go into SOME of the E.coli, not all • you only want the E.coli's with your gene • this gene will allow the bacteria to resist the antibiotic • if the E.coli has the plasmid and it makes lots of it, it will make proteins that will resist to antibiotic • you would take the E.coli and add antibiotic • the only E.colis that will survive are the ones with your plasmid in it • those bacterial cells will be antibiotic resistant because you put the antibiotic resistance gene into the plasmid (hence these are the cells that will express your protein)
Cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers) |
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What are chimeric plasmids? |
After cleave of a plasmid with restriction enzyme, a foreign DNA fragment can be inserted
Ends of the plasmid/fragment are closed to form a "recombinant plasmid"
Plasmid can replication when placed in suitable bacterial host if you have an ori |
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What is In Vitro mutagensis? |
One method of PCR-based site-directed mutagenesis
1) Template DNA strands are separate and amplified by PCR
2) Following many cycles of PCR, the DNA product can be used to transform E. coli cells
3) The plasmid DNA can be isolated and screened for the presence of the unique restriction site by restriction endonuclease cleavage |
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What are expression vectors? |
You want to make the protein
You engineering the DNA where a site that can be recognized and makes a lot of mRNA |
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What is SDS gel electrophoresis? |
SDS-PAGE: sodium dodecyl sulfate - polyacrylamide gel electrophoresis (denaturing detergent - meaning the protein loses its activity)
SDS: denaturing detergent and dissolves membranes and solubilizes proteins
Polyacrylamide: gel matrix to separate proteins (If protein small, they go through mesh easily and migrate faster)
Number of SDS molecules bound by a polypeptide is proportional to the length (number of amino acid residues) of the polypeptide
Each dedecyl sulfate contributes 2 negative charges
SDS-PAGE is usually run in the presence of sulfhydryl -reducing agents such as Beta -mercaptoethanol so that any disulfide links between polypeptide chains are broken
Often used to determine molecular weight of a protein |
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What are the lanes of an expressed SDS-PAGE gels of expressed proteins? |
Lane 1: The lysate from E. coli - contains all proteins
Lane 2: The "flow-through" from the Ni-affinity column
Lane 3: The purified expressed (His-tagged) fusion protein eluted from column with 400 mM imidazole
Lane 1', 2', and 3': Same as Lane 1, 2, and 3, but blotted with an anti-fusion protein antibody. Only the fusion protein appears |
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What is an Western (Immuno) Blot? |
Are used to purify or detect protein
Uses an antibody which is recognizing something particle like a his-tag
Has detection signal that produces fluorescent compounds
Has a primary antibody that recognizies the His-tag and binds to it |
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What is subtilisin? |
It is an endopeptidase, MW 27500 (275 residues)
Secret in large amounts from many Bacillus species
X-ray crystal structure determines there was 2 domains in this protein
Its active site features a charge-relay network involving Asp-32, His-64 and active site Ser-221
For the charge-relay system of subtilisin; Asp-32 carboxylate side chain H-bonds to N-bonded proton on His-64 imidazole ring
The other N-atom on His-64 H-bonds to the other O-H proton of Ser-221
Ser-221 becomes 'activated' by virtue of charge-separation of O-H, and its O-atom becomes more nucleophilic
Ser-221 O-atom attacks incoming substrate, assisted by a neighboring carboxyamide side chain of Asn-155
Substrate is susceptible peptide bond bound into the active site clef
This action causes the substrate peptide bond be cleave (needs water for this mechanism to work)
You can do mutagenesis of subtilisin to:
-Determine residues involved in catalysis (mutate Ser, His, Asp and Asn to see what happens)
- Also to enhance enzyme stability (can only cleave certain peptides)
-Obtain details of substrate specificity
-Determining effect of disulfide bonds on secretion, structure and stability |
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What is the catalytic mechanism of serine proteases? |
Substrate coming in and binds to active site
1) His will take proton offSer
2) now you have intermediate, very unstable, electrons goes back to C and bond between N-C is broken
3) now take H+ off imidazole, the other part of peptide chain is covalently attached to the enzyme (acyl-intermediate)
4) since it was onlybindedby weak H-bond, it floats away
5) water must be added for hydrolysis reaction, the N on the ring takes H from water, and the OH will bind to the group that covalently bonded to enzyme
6) the Asp will come in todistabilizethis structure
7) bond betweenSerand C will be broken
8)Serhas proton back from water |
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What is cassette mutagensis of subtilisin? |
Method: choose a codon in the gene and perform 'randomized mutagenesis' (not site directed, you don't know which codon might work)
Use 25% each of nucleotides A, C, T, G at each of three codon bases during oligonucleotide syntehsis. By this method, any three-base codon can arise
Goal: engineering of oxidative resistance.
Substilisin known to be stable to denaturants, but highly susceptible to chemcial oxidation
Peptide mapping studies showed that oxidizig Met-222 (next to catalytic Ser-221) results in 90% loss of enzyme activity.
Methionine Oxidation can be done by oxidization of methionine to methionine sulfoxide to methionine sulfone
Met-222 is conserved residue in all subtilisins, suggesting funcitonal role
This is proved by fact that the enzyme is easily inactivated by oxidizing Met-222
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What happens if you try to engineering disulfide bonds into subtilisin? |
The enzyme has no Cys and no S-S bonds
Engineering problem: potential S-S bond must be chosen to conform geometrically to naturally-occuring S-S bonds, and not cause significant main chain rearrangement
Cys must be placed in proper place otherwise protein would not fold properly and not function
Ser-24 to Cys-24 or Ser-87 to Cys-87 (-CH2OH replaced by -CH2SH)
Both Ser residues in wild type subtilisin are located on back side of molecule distant from active site (24 angstroms away)
Results:
-Double Cys-Cys mutant was successfully expressed and isolated
-Secreted and processed normally by B. subtilis (novel engineering feat)
-Had the same specific activity as wild type enzyme
-X-ray confirmed presence of S-S bond, negligible alteration in protein structure
Studies of thermostability of s-S subtilisin were complicated by autolysis: the enzyme begins to digest itself (as substrate) as the temperature is raised
Rate of autoproteolytic inactivation increased as heating increases
Results showed no basic different between S-S and wild type subtilisin, either in specific activity, pH maximum for catalysis or autoproteolytic inactivation (didn't stabilize any more than the wild type)
Some secreted serine proteases have several S-S bonds, some have none |
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How would you predict a protein structure? |
Minimum energy calculations can locate low energy regions for protein backbone and side chain rotational angles.
phi/psi angles will dictate whether that sequence will form alpha helix or beta strands
These calculations 'predict' the existence of favoured secondary structures
big residues like tryptophan, have big side chains => rotational angles are restricted => inside proteins it would be even more restricted
the linear arrangement allows to fold into alpha helix, allowing them to pack the side chains on surface
in 3D structure, the AA in distance will interact with those side chains
beta strands from other regions can interact
you're only predicting the structures, it may actually turn out differently
The alpha-helix and beta-sheet therefore involve local regions in which each residue repeats stereochemically-desirable angels
These procedures have led to the Ramachandran plots
But these calculations give information only about "short-range interactions"
Thus, protein tertiary structure cannot be predicted on this basis alone
At least two other factors must be considered:
1) Environment:
-Must be taken into account; most calculations are done in a vacuum: small energy increments gained or lost at hundreds of sites contribute to folding
-The E to unfold a protein is very low (5-10kcal which is the energy of one H-bond) because proteins are dynamic and have to move and work which means you cannot have a very stable elements
2) Long range protein/protein interactions must be considered to obtain the overall protein structure
-Especially pertinent to formation of inter-molecularly H-bonded structures such as beta-sheets (from sequentially) distant segments of a protein)
- sometimes the last strands of beta sheets will interact with each other to form beta barrels
- want to predict if these last strands interact or not |
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What are Chou-Fasman rules? |
Conformations of given residue was determined by its psi/phi angels
Consecutive sequence of 4 or more helical residues are 'helix' (to make an alpha helix, each turn is 4 AA)
Consecutive sequences of 3 or more beta-sheet residues are "beta-sheet"
Residues not in either region are assigned "random" independent of phi/psi angels
Leu is good alpha helix former
If Leu in sequence, and it's onlyLeu, then it won't be alpha helix
If you have 4 Leu in a row, you're likely to have alpha helix
2-3 residues minimum for beta strand
3 great beta strand formers in a row, likely to be a beta strand |
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What are parameter designations? |
Propensity (P_) alpha, beta and t refers to values for individual amino acids
To obtain average values, add up individual values and divide by number of residues |
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What was Chou-Fasman results? |
Conformational parameters (P-valueS) were obtained from % occurrences of each residue in each structural form, compared to the overall occurence of that form
Thus is if all Ala residues in the 30 proteins are considered, and 45% of them are in helical regions, then: Ala P(alpha) = 0.45/0.38 = 1.18
1 means no real presence
1.18 means rich in presence, higher than 1 is alpha helix
P-values were developed similarly for the three types of secondary structure:
P-alpha = propensity of a given residue to be helical
P-beta = propensity of given residue to be beta-sheet
P-t = propensity of given residue to be in a beta-turn
Val and Ile are great beta former and are beta branched (beta branched are great Beta formers)
Reason why beta branch not good in Alpha is because they are large and makes it hard to make it compact
Glu worst beta former despite being best alpha former
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What are the top helix forming residues? |
Glu, Met, Ala, Leu, Lys
Cannot have 4 Glu next to each other as Glu is negative and they repel each other unless it becomes protonated which means it cannot only form a perfect alpha helix if it is lower than it's pKa
These are hydrophobic
Negative charge repels each other (cannot form helix with 4Glu in a row) unless you add proton to neutralize it
Lower pH below the pKa of Glu, the negative charge are now neutralize |
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What are the best helix-breaking residues? |
Gly, Pro, Asn, Tyr, Cys
Gly and Pro are most common turners
if Gly is in helix, it has a proton sticking out, it cannot be packed against helical structure
Cys has SH group, Tyr OH group are polar |
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What are the top beta-sheet forming residues? |
Val, Ile, Tyr, Phe, Trp |
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What are the best beta-sheet breaking residues? |
Glu, Asp, Pro, Gly, Lys
Glu has negative charge that can break the structure since it repels |
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What are the top beta-turn-occuring residues? |
Asn, Gly (left handed helix at i+2), Pro (at i+1), Asp, Ser, Cys, Tyr |
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What are the least beta-turning occuring residues? |
Ile, Val, Leu, Phe, Met |
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What is the difference between hemoglobin A vs S? |
Sickle cell anime is a chronic hemolytic disease that is genetically transmitted.
Genetically transmitted means mutation in gene that is passed on from mother/farther to child
Hemoglobin A (HbA) is reversible oxygen carrier
Hemoglobin S (HbS) differs from HbA in one amino acid in the beta chain Glu-6-Val
This is a 'non-conservative' mutation: Glu is polar, charged amino acid, Val is highly non-polar aliphatic amino acid
This creates a hydrophobic patch on the surface of the protein
HbS forms long polymers that change the shape of red blood cells from normal to sickle
HbS tend to aggregate and forms long fibers, which deforms erythrocytes
There is a 'hydrophobic patch' on deoxyhemoglobin S that has a complementary binding site on another deoxyhemoglobin S molecule |
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Why does HbS aggregate? |
Renaturation experiments have shown that information for folding into 2 and 3 structured is coded in the amino acid sequence
Combined knowledge of sequence/structure and prediction can provide insight into why HbS is a disease-causing form
Glu is strong helix former
Val is strong beta-sheet former
Going from Glu to Val, you change the propensity from a helix former to Val which would rather form a beta-sheet which would destabilize it and if this keeps happening, it would be destabilized even more and eventually form a beta sheet
This patch sits on the top of the surface and cannot escape which destabilize the alpha helix which is on the surface of the protein
It forms a helix most of the time but sometimes it might unfold and TRY to form a beta sheet |
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What is Glioblastoma? |
Mutation in membrane protein neu/erbB2 proto-oncogene of mutation Val664 to Glu |
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What is the hormone: glucagon? |
Opposite of insulin
29 residues, 'small' protein = easy to study
Secreted by the pancrease when blood sugar is low
Binds to receptor in liver cells to release glucose into the blood
Has N-terminal beta-strand and C-terminal helical regions
Spectroscopic measurements by circular dichroism have shown that glucagon changes structure as a function of concentration
At low concentrations, glucagon has C-terminal alpha helix (half beta, half alpha)
At high concentrations, glucagon is largely beta-structure (Mostly beta and eventually all beta and precipices out of solution and forms a gel)
Concentrated solutions of glucagon form a gel much like gelatin
CF analysis of glucagon results suggest a biological implication of glucagon segment may change conformation during its biological action
Storm formed is probably beta formed
Released form in blood, changes from all beta to half alpha, half beta
Bound to receptor form which shows helical conformation of peptide when bound to receptor because binding site of peptide is hydrophobic and becomes alpha helices becomes more hydrophobic so it can bind to binding site which has residues with poke out of the helical and fit perfectly to the receptor
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What is gel filtration chromatography? |
Chromatography column separates protein by size (molecular weight)
Column matrix (resin) consists of porous beads with varying pore size
Like a sponge with holes in it which small things can go in and get stuck
Calibrate with protein of known size
smaller proteins are selectively trapped (and slowed)
Large proteins elute first |
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What is affinity chromatography |
Chromatography columns separates proteins by selectively bidning specific proteins
Resin contains covalently bound substrate or ligand that recognizes one protein in the mixture and binds it with high affinity to the colum
You pick the ligand (Ni for His tag)
If you want insulin receptor, insulin would bind to column and if you add free insulin, it knocks it off the column and this would purify the insulin receptor
Selected protein molecules remain bound to the column, when unbound proteins are eluted through the column
Once separated, low molecular weight substrate or ligand is added to the column in excess and "competes" the bound protein off the column |
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What is HPLC? |
High Pressure (Performance) Liquid Chromatography
Not best for proteins because hydrophobic are buried and will not show the hydrophobic patch but there are some proteins that does like HbS
Contains hydropbic resin
Separates peptides/proteins primarily by 'hydrophobicity'
More hydropbic compounds interact strong with the column, have longer 'retention times' on the colum |
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What is Ion Exchange Chromatography? |
Separates amino acids or peptides mainly by positive or negative characters
Very useful to separate protein because charges are on surface on protein to combine as resin and get them off by adding a salt like KCl
Used in amino acid analysis to determine protein composition
Cation exchange resin has negative sites: attracts and tightly binds positively charged peptides/proteins
Anion exchange resin has positive sites: attracts and tightly binds negatively charged peptides/proteins |
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What is Edman degradation? |
Chromophoric reagent combines with N-terminal amino acid, cleaves next peptide bond in the process
Creating a derivative and do this again
Limited by length of sequence |
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What is carboxypeptidase? |
Enzyme that cleaves amino acids one at a time from C(carboxyl)-terminus
Limited by length of sequence |
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What is Trypsin? |
Enzyme cleaves to carboxyl side of basic residues (Lys, Arg) |
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What is Chymotrypsin? |
Enzyme cleaves to carboxyl side of aromatic residues (Trp, Tyr, Phe)
Binding site in active site that recognizes aromatic residues of Trp, Tyr and Phe |
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What is Cyanogen bromide (CNBr)? |
Chemically cleaves to the carboxyl side of Met residues |
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How can you use Mass Spectrometry (MS) to identify peptide fragments? |
Protein (peptides) bombarded by high energy electron beams or laser beams
Ionized molecules created, which impact upon a charged plate: exact molecular weight detected
Charged = lose protons and become negatively charged and mess up the protein and ionizing them into gas phase |
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What is X-ray crystallography? |
(Some) proteins can form ordered crystals
Crystals placed in X-ray diffractometer produce diffraction patterns that can be interpreted in terms of atomic positions
Three-dimensional structures of proteins can be reconstructed at high resolution (2A), including backbone and side chains
Protein have hydrophilic surface that interacts with water, we want to use reagents that interacts with water in order to remove the water (ammonium sulphate)
Or force proteins in an ordered array to remove water
Membrane proteins are hard to crystallize because you must extract the proteins away from the lipid bilayer
The folding of the protein backbone can be specified preceisely using crystallographic data
The phi/psi angels of each residue on the alpha-carbon
Position (coordinates: x,y,z) of every atom is 3-dimensional space |
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What is NMR spectropscopy? |
NMR = Nuclear magnetic resonance
In the presence of external amgnetic field, the nucleus (H proton) can exhibit more than one spin state (up/down)
Can move between these states by the absorption of electromagnetic radiation of specific frequency (energy)
The energy absorbed can be detected and from this information about the environment of the nucleus can be deduced
Very high field superconducting magnets (up to 800 MHz) provide spectra with great resolution and sensitivity
Protein structures can be solved with NMr |
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What are the advantages and disadvantages of x-ray and NMR? |
Advantage: These are two most established high resolution techniques for deduction of protein structure
Disadvantages: X-ray proteins must be crystallized and for NMr proteins must be small
Large complexes can be visualized using electron microscopy (EM) |
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What is Infra-red spectropscopy? |
Proteins contain vibrating, stretching and bending groups
In an infra-red beam, these motions lead to absorption of IR light as function of wavelength
Proteins have numerous C-H, N-H and C=O groups so these are most prominent in the Ir spectrum
Ir has the ability to detect and evaluate quantitatively H-bonding interactions
Band positions in the carbonyl amide region can distinguish helix and beta-sheet structures
Band positions correlate with "strength" of hydrogen bond |
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What are protein chromophores? |
Operational definition: a chemical group that absorbs ultraviolet (UV) and/or visible light at characteristics wavelengths
Chromophore groups are usually contain conjugated double bonds
Amide carbonyls and aromatic rings are important chromophores in proteins |
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What is circular dichroism spectroscopy? |
CD spectra record absorption of UV light, and also take advantage of asymmetry of proteins
Each alpha-carbon in each amino acid (except Gly) is asymmetric -L- vs D- configuation
Secondary structures are asymmetric - the twist of a helix can be left or right handed
Proteins with asymmetric features can polarize light
Dissolve protein sample in water or suitable solvent
Place in small cuvette in UV beam
Polarize the UV beam
Spectrum records composite content of alpha-helix, beta-sheet and non-ordered structure in the protein |
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Which of the following represents an amide linkage? |
A |
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What is the corn rule? |
With hydrogen in back:
If you spell corn clockwise with hydrogen back, that is D
If you spell corn ccw with H back, that is L |
