TBT4135 - Biopolymerkjemi
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Fakta høst 2009
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Øvingsopplegg høst 200+
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Lab høst 2009
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Innføring i biologiske polymerer (polysakkarider, proteiner), med laboratorieøvinger i anvendte teknikker.
Innhold
Oppsummering
DNA
Deoxyribose (2-deoxy D-ribose), attached to a phosphate group on 3', this is 3' end), and next phosphate group on 3' (3' end). <math>\beta</math>-linked to pyrimidine (cytosin and guanine) or pyrine (adenine or thymin/uracil) at C-1.
PCR
Melt DNA (double -> singlestranded). Add small primers of known sequence near region of interest, bases and DNA polymerase. Rinse and repeat.
Sequencing
Maxam-Gilbert: Base specific cleavage of DNA after marking 5' end by radioactive phosphate, and seperated in gel electrophoresis. Can sequence up to 200 bases. Dideoxy: Synthesize DNA by biological methods, but add small amounts of a type of dideoxy base, which stops synthesis at certain places. Seperate by gel electrophoresis and put together sequence.
Proteins
Amino acids
All proteins formed of L-amino acids.
20 essential amino acids: Non-polar amino acids Alanine (Ala, A): methyl Valine (Val, V): isopropyl Leucine (Leu, L): isobuthyl Isoleucine (Ile, I): 1-methyl propane Proline (Pro, P): Propyl linked to amine in main Phenylalanine (Phe, F): Alanine with phenylfunction Tryptophan (Trp, W): Alanine with indole group Methionine (Met, M): CH3(2)-S-CH3, can be synthesized from cystein.
Polar amino acids Glycine (Gly, G): H Serine (Ser, S): methanol Threonine (Thr, T): tert-propanol Cysteine (Cys, C): methanethiol Tyrosine (Tyr, Y): Phenylalanine with hydroxy in para. Aspargine (Asp, N): Aspartic acid with amino instead of hydroxy Glutamine (Gln, Q): Glutamic acid with amino instead of hydroxy
Acidic amino acids Aspartic acid (Asp, D): Acetic acid Glutamic acid (Glu, E): Propylic acid
Basic amino acids Lysine (Lys, K): amino-buthane Arginine (Arg, R): propyl-guanidinium Histidine (His, H): methyl-imidazole
In general the <math>\alpha</math>-carboxyl group has a pKa of about 2 and the <math>\alpha</math> amino group has a pKa of around 9.5. Asp and Glu have pKa around 4, Cys, Thr and Lys have around 10, Arg has around 12.5 while histidine is special at around 6. Calculate pI by testing what net charge the protein has at a given pH and then try again.
Sequencing
Sanger's method: Attach dinitrofluorobenzene to N-terminal, degrade protein completely and then identify amino acid that is attached to reagent. Can do similar to carboxyl end. This can be used to sequence di- or tripeptides (middle amino acids identified by chromatography). Use mild degradation to obtain mixture, put together puzzle. Not used anymore.
Edman's method: Disconnect only amino-terminal amino acid, identify, and repeat. Can be done automatically in parallell.
Gene coding: Find the first few amino acids (7-10), use this to make a DNA probe (primer in PCR), amplify gene and sequence. Only works on prokaryotes, due to introns in eukaryotes.
Structure
Partial double bond in peptide bond hinders rotation in peptide chain except on each side of the <math>\alpha</math>-carbon (with R-group) although limited to certain angles. R-groups alternating side of chain.
Arnfinsens experiment: Disrupt disulfide bonds with mercaptoethanol and denature with 8M urea, reverse and regain most of activity - folding is native low energy state.
Denaturation can cause changes in acid/base properties and IEP, changes in charge, higher accessibility for proteases, higher reactivity of many side groups, and general conformation changes that influence solution properties.
<math>\alpha</math>-helixes
Left-handed helix, full turn every 3.6 amino acids, with a rise of about 1,5 Å and pitch 5,4 Å. R-groups facing out of helix. Stabilized by uncharged, medium-sized amino acids: Ala, Leu, Phe, Tyr, Trp, Cys, Met, His and Asn. Small or large R-groups, or charged amino acids, de-stabilize the helix: Gly, Ile, Glu, Asp, Lys, Arg, Ser, Thr. Proline and hydroxyproline break the helix, due to hindered rotation in cyclobuthanol-ring. Threonine and serine have intramolecular hydrogen bonds that compete with intermolecular hydrogen bonds. There are other types of <math>\alpha</math>-helixes, such as <math>\alpha_10</math> or pi helixes, which are similar but with less or more amino acid residues per turn. In the standard helix amino acid i and i+3 hydrogen bond. Keratine is rich in <math>\alpha</math>-helixes and <math>\beta</math>-sheets (see below).
Collagen has a triple helical structure that is right-handed with about 20 amino acids per turn, i.e. a much loser structure. Typical sequence is Gly-X-Y where X is often proline and Y is often hydroxyproline. These lock the bond angles to favour this type of helix. Gelatin is denatured collagen that partially reforms the helixes upon gelation. Collagen triple helixes form intermolecular hydrogen bonds to other helixes to make strong filaments, but does not form intramolecular hydrogen bonds.
Collagen is the most abundant protein in mammals, 25-35% of total protein content. Collagen type I, II and III are fibrillar collagen and are found in most connective tissues and bone, cartilage and vitreous humor, and extensible connective tissues respectively. Collagen type IV is part of the basal laminae. Together they account for >90% of the collagen in the body. There are different chain types in the different forms of collagen, type I has two <math>\alpha</math>1 and one <math>\alpha</math>2 chain, type II has three <math>\alpha</math>1 chains, type III has three <math>\alpha</math>3 chains, while type IV has a mixture.
Collagen is built by first forming tropocollagen (three helixes bound together). In the ECM the ends are cleaved and the tropocollagen assembles into fiber bundles. The bundles have a striated appearance. Allysine and lysine residues form Schiff base covalent crosslinks.
Elastin is another ECM protein with a random coil shape, rich in glycine, valine, alanine and proline. Gives flexability to the ECM.
There are many diseases associated with ECM disorders. Marfan syndrome (long arms, legs, extra stretchy) caused by mutation in fibrillin, an important structural protein holding elastin in place. Ehler-Danlos syndrome (stretchy skin, lesions, bruises, bendable limbs) caused by mutation in collagen III.
<math>\beta</math>-sheets
Stretched <math>\alpha</math>-keratins, <math>\beta</math>-keratins and silk fibroin have a common protein structure called a <math>\beta</math>-sheets. Hydrogen bonds are formed between the backbone amide groups, while the R-groups stick up and down in the plane, forming intra-layer bonds in addition.
From the amino acid sequence the secondary structures above can sometimes be estimated. Hydropathy plots can also be made to map regions heavy in hydrophilic or hydrophobic regions, to see where they are most likely to be found in a tertiary structure. Tertiary structures can be assembled into quaternary structures, which are stabilized by weak interactions or disulfide bonds between segments.
Polysaccharides
Polysaccharides are the most abundant biopolymer. The basic building blocks are monosaccharides.
Monosaccharides
They are designated D or L depending on the orientation of the highest numbered chiral carbon atom. If the hydroxy group is pointing right in the Fischer structure it is a D-sugar. This corresponds to the non-ring carbon to be pointing up in the Haworth projection. Opposite for L sugars. L sugars are mirror images of R sugars with the corresponding name, i.e. all groups are mirrored. If only one group is mirrored the sugars are C-X epimers. If the hydroxygroup on C-1 is cis with the non-ring carbon group the sugar is <math>\beta</math>, or <math>\alpha</math> if trans. Pentoses are ribose (RR), arabinose (LR), xylose (RL) and lyxose (LL). Hexoses are allose (RRR), altrose (LRR), glucose (RLR), mannose (LLR), gulose (RRL), idose (LRL), galactose (RLL) and talose (LLL). The hexoses can be in furanose (5-ring) or pyranose (6-ring) forms.
These sugars can be in three forms: Chair, half-chair or boat. Chair is by far most common. The chair form can be 4C1 or 1C4. In general the sugars will be in the form that reduces the amount of bulky axial groups. This glycosidic bonds between monomers can be axial-axial, equatorial-axial, axial-equatorial or equatorial-equatorial, which greatly influences the secondary and tertiary structure of the polysaccharide.
There are many modifications that can be done to the monosaccharides. Some of the most common are: D-glucuronic acid (carboxylic acid at C-6), L-rhamnose (6-deoxy-L-mannose). 2-deoxy-D-glucosamine, N-acetyl-D-glucosamine, D-galactose-4-sulphate, D-glucose-6-phosphate and D-mannose-4,6-pyruvate. Some common disaccharides are the glucose dimers maltose (<math>\alpha</math>1-4 ax-eq) and cellobiose (<math>\beta</math>1-4 eq-eq).
Polysaccharides
Cellulose
<math>\beta</math>1-4 linked D-glucopyranose, with eq-eq bonds, unbranched. Insoluble in water. Forms fibrous bundles with high degree of crystallinity, but can also be amorphous. Microcrystalline cellulose is purely crystalline cellulose because the amorphous cellulose has been removed by acid hydrolysis.
There are two main types of cellulose: Cellulose I and II. Cellulose I is the naturally occuring cellulose. The cellulose chains are arranged in a parallel fashion in fully stretched chains, and each glucose is turned 180 degrees compared to the neighbors. C-2 hydrogen bonds with C-6 and the ring oxygen hydrogen bonds with C-3 of the next monomer, and interchain cellulose stabilise the sheets/fibers. Cellulose II is formed when Cellulose I is swelled or dissolved and the precipitated. This form is more thermodynamically stable and has anti-parallel chains arranged in a slightly tilted way.
To functionalize cellulose it is first treated with a strong base so the hydroxyls deprotonate somewhat, then other reagents are introduced to modify the cellulose. Examples of cellulose ethers are carboxymethylcellulose (react with cloroacetic acid), hydroxyethylcellulose (react with ethylene oxide, an epoxide) and methylcellulose (react with methyl chloride). Cellulose esters are cellulose acetate (react with acetic acid anhydride) and cellulose nitrate (react with nitric acid).
Amylose, amylopectin, glycogen
<math>\alpha</math>1-4 linked glucose, i.e. axial-equatorial bonds. Amylose is linear, and may form ordered structures. Amylopectin has a branched structure consisting of amylose linked together with <math>\alpha</math>1-6 at branching points, which are every 12-15 monomers. Each branch keeps on branching, so the whole structure increases in thickness as one moves from the reducing end. Packed together in starch granules. Glycogen is similar to amylopectin, but has a somewhat less regular branching structure and is produced by animals and not plants.
Dextran
<math>\alpha</math>1-6 linked backbone with <math>\alpha</math>1-3 linked branching chains. Commercial dextran used for many experiments as a reference biopolymer is mostly unbranched.
Pullulan
Pullulan is a bacterial polymer produced by A. pullulans. Consists of maltotriose units (three <math>\alpha</math>1-4 linked D-glucose units) linked together with the flexible <math>\alpha</math>1-6 linkage. Easily soluble in water, flexible and available in monodisperse samples.
Chitin and chitosan
Linear <math>\beta</math>1-4 linked N-acetylglucosamine (2-acetamido-2-deoxy-D-glucose). Similar to cellulose, chitin is insoluble in water in unmodified form. Deacetylation of chitin leads to chitosan (<math>F_A</math> less than 80%), which is soluble at low pH when the amine group is protonated. pKa for the amine group is about 6. Chitosan is practically the only positively charged biopolymer at pH 7, and therefore interact with many biological materials. Between 40 and 60% deacetylation the chitosan fibres are so irregular that they can not crystallize, and are therefore soluble at all pH.
Galactans
Galactans are linear polymers. The basic building blocks are galactans that are alternatily <math>\alpha</math>1-3 and <math>\beta</math>1-4 linked. The galactans are often modified. Carrageenan has 4-linked 3,6-anhydro-<math>\alpha</math>-D-galactose or just <math>\alpha</math>-D-galactose, and 3-linked <math>\beta</math>-D-galactose. <math>\kappa</math>-carrageenan is sulphated at the 4-linked residue, while <math>\iota</math>-carrageenan is sulphated at both repeating residues. Agarose is similar, but the anhydro residue is <math>\alpha</math>-l-galactose instead. Agarose is the ideal unmodified form, while agaropectin is agarose with charged substituents. All linkages in galactans are equatorial, and galactans in nature are structural units in red algae. Carrageenans gel thermoreversibly in the presence of K+ ions, and low sulphate content creates stiffer gels.
Xanthan
Produces by bacteria X. campestris and has a comples penta-saccharide repeating unit. The backbone is a cellulose chain <math>\beta</math>1-4 linked D-gluocse, and every second unit has a chain consisting of: <math>\alpha</math>-D-mannose acetylated at C6, <math>\beta</math>-D-glucuronic acid and 4,6-pyruvated-<math>\beta</math>-D-mannose. Xanthan forms a double helix in solution, making it very stiff with high intrinsic viscosity and shear thinning properties. Has high molecular weight and a persistence length of about 100 nm.
Pectins
Pectins occur in the cell walls of fruits and are rather complex. They have smooth regions consiting of unbranched <math>\alpha</math>-D-galacturonate. They can be partially esterfied. The linkages are diaxial, leading to an eggbox structure that can bind Ca2+ ions and induce gelling. Commercially pectins are used to gel jams, and are then highly esterfied (no charge) and used a low pH. Sucrose content needs to be high so the activity of water is changed.
The hairy regions are highly branched with complex branches, and the backbone can be the same as the smooth regions or alternating rhamnose and galacturonate residues.
Alginate
Alginate is a polysaccharide produced by brown algae. It is linear, and has to components: <math>\beta</math>-D-mannuronate (M) and <math>\alpha</math>-L-guluronate (G). The G-G link is axial-axial, while the M-M link is equatorial-equatorial. Alginate is produced by first making long chains of poly-mannuronic acid, then enzymes (epimerases) epimerize selected C-5, changing the sugar from <math>\beta</math>-D-mannuronic acid to <math>\alpha</math>-L-guluronic acid. Depending on the enzymes it can make long M-blocks, , long G-blocks or alternating MG-blocks, or a mixture. <math>F_G</math> and <math>F_M</math> denotes the fractions of G and M in the alginates, and these fractions can be extended to e.g. <math>F_{GG}</math> for G-dimers, etc. Due to the axial linkages in G-blocks they for eggbox-like structures that bind Ca2+ in the cavities. This associates chains and causes gelation.
The structure of alginates is determined using NMR. NMR can identify the relative fractions of G-blocks, M-blocks, MG-alternating regions, and chain length (of the partially degraded chains used for analysis).
Commercial alginate is often delivered as Na-alginate instead of the acidic form. Natural alginate is very long and therefore behaves almost as a random coil (3-500 kDa), but somewhat stiffer than e.g. pullulan.
Alginate is often used for gelling applications, especially in food products. In medicine it can be used to encapsulate cells. Cells are mixed with an alginate solution, which is dripped into a CaCl2 solution. This causes gelling and cell encapsulation. The beads are then coated with a cationic polymer, and the alginate is dissolved using a chelating agent for Ca2+. Then one has a capsule containing cells. If one wishes homogenous gelation of alginate (not the case if one uses CaCl2) one can mix the alginate with insoluble CaCO3, then add a substance that slowly drops the pH so that the CaCO3 dissolves and starts the gelling.
Alginic acid can also form gels, probably by hydrogen bonds between nearby acidic groups. These show highest gel strength at low and high fractions of G (or M), and gelling increases with increasing molecular weight.
Adding free G-blocks can do one of two things, depending on the Ca2+ concentration: At low Ca2+ the G-blocks bind the calcium and "gel" with the alginate, reducing crosslinking between the alginate chains and thus reducing gel strength/viscosity. At high Ca2+ concentration the G-blocks seem to help crosslink the alginate chains and therefore increase the gel strength.
Properties of biopolymers in solution
Biopolymers can adopt three idealized shapes in solution: The stiff rod, the compact sphere and the random coil.
Random coil
There are many models for the random coil, ranging from simple to relatively complex. The worm-like chain model is the most accurate and can account for the transition from random coil to stiff chain. Starting simple we have a chain without restrictions. The end-to-end distance is the sum over all the monomer vectors: <math>\vec{r}=\sum_{i=1}^n \vec{l_i}</math>, while the square length is of course <math>r^2=\vec{r} \cdot \vec{r}=\sum_{i=1}^n \sum_{j=1}^n \vec{l_i} \vec{l_j}</math>. If we assume all lengths are identical, we can take the average over the square length: <math>\langle \vec{r}^2 \rangle=nl^2</math>, because all other <math>l_i\not=l_j</math> will null each other out. This gives <math>\langle r^2 \rangle = n^{0.5}l</math> for a random coil. For a stiff rod we naturally have <math>r=nl</math>.
If we extend the model so that we have hindered rotation around bonds, we get a parameter <math>\langle r^2 \rangle=n\beta^2</math>, where <math>\beta</math> is a fictitious bond length that includes hindered rotation, and will always be larger than 1. This parameter is related to the stiffness parameter <math>C_\infty=\beta^2/l^2</math>, so we can write <math>\langle r^2 \rangle=n C_\infty/l^2</math>. The last modification we can do to this simple model is adding a monomer excluded volume, which accounts for the volume of the monomers in the chain. This parameter is called <math>\alpha</math>, and <math>\alpha < 1</math> if we are in a bad solvent, because bad solvents cause contraction of the polymer, <math>\alpha=1</math> is a special case called <math>\theta</math>-conditions, which is where a slightly bad solvent exactly balances the effect of the monomer volume. <math>\alpha > 1</math> happens if we are in a good solvent. In good solvents <math>\alpha</math> has a small molecular weight dependence, such that <math>\alpha \propto n^{0-0.1} \Rightarrow r \propto n^{0.5-0.6} \propto M^{0.5-0.6}</math>
There are other models for the random coil as well. Kuhn used equivalent statistical segments called Kuhn segments, which kind of average a little over the segments. We introduce the parameters <math>l_k = C_\infty l</math>, the equivalent Kuhn length, and <math>N_K=\frac{n}{C_\infty}</math>, the equivalent number of Kuhn segments. Another much used parameter is the persistence length <math>a=\frac{1}{2}(C_\infty +1)l \Rightarrow l_K \approx 2a</math>. The persistence length is defined as the projection of the average end-to-end distance onto the first vector, but can also be interpreted as the distance one must travel along the chain until the direction of the first bond is independent of the bond direction. The persistence length is valid for all chains, while <math>C_\infty</math> only is valid for flexible coils.
Random coils in nature are typically mostly water, often 90-95% of the hydrodynamic volume is really water. Thus they are rather open structures.
Worm-like chain model
This model is used for a common type of conformation: somewhere between a random coil and a stiff rod (called a stiff coil). It models the chain as a randomly, continuously curving chain of uniform thickness, and has the stiff rod and random coil as limiting values. The persistence length (above) a, the mass per unit length, the contour length and the diameter. <math>Sett inn formel her</math>
Ionic strength
The ionic strength will affect the shape of the biopolymer. The ionic strength is given as <math>I=\frac{1}{2} \sum_i C_i z_i^2</math>, so it has a large dependance on the valency of the salt. Increasing ionic strength causes contraction of polyelectrolytes, due to charge screening between repulsive groups.
Radius of gyration
The radius of gyration is the radius at which one could concentrate all the mass into a spherical shell and maintain the same moment of inertia. Other definitions are: the moment of inertia is the mass times the radius of gyration square. This can e.g. be defined as <math>R_G^2=\frac{\sum_i m_i r_i^2}{\sum_i m_i}</math>, i.e. the radius of gyration is the root mean square of the inertia divided by the total mass.
Spheres
Integrating the moment of inertia of a sphere and dividing by the total mass gives <math>R_G^2=3/5 R^2</math>. Since <math>R \propto R</math> and <math>M \propto R^3</math>, this means <math>R_G(sphere) \propto M^{1/3}</math>.
Rods
Integrating the moment of inertia of a cylinder and dividing by the total mass gives <math>R_G^2=1/12 L^2</math>. Since <math>L \propto M</math> for a rod, this means <math>R_G \propto M</math> for a rod.
Random coils
Here we can only use averages. Calculations give <math>R_G = \sqrt{1/6 \langle r^2 \rangle}=\sqrt{1/6 n \beta^2 \alpha^2}=\sqrt{1/6 \beta^2} n^{0.5} \alpha</math>. Since <math>n \propto M</math> and <math>\alpha \propto n^{0-0.1} \propto M^{0-0.1}</math>, <math>R_G \propto M^{0.5-0.6}</math>
Most biopolymers have intermediate forms, and these intermediate forms can be estimated from the radius of gyration, which gives if they are in the collapsed coils (between random coil and sphere) or stiff coils (between random coil and rods).
Disorder and order
Bond angles
As mentioned above there can be hindered rotation around certain bonds. A Ramachandran plot can be used to gain an overview over allowed angles.
=Types of bonds
The main types of bonds are: covalent bonds (200-1000 kJ/mol), ion-ion bonds (40-400 kJ/mol), ion - induced dipole and hydrogen bonds (4-40 kJ/mol) and other types of bonds (0-4 kJ/mol). In comparison the thermal energy is 2.4 kJ/mol at 25C, the bond strength must be greater than this to be stable. A hydrogen bond can when a hydrogen atom is trapped between two highly electronegative atoms, such as oxygen or nitrogen. It is covalently bound to one of them at a distance of about 1 Å, and electrostatically bound to the other at a distance of 1.8 Å. The angle also plays a role in the bond strength.
Thermodynamics
The shape of the biopolymer in solution will depend on the thermodynamics of the system. In general, a random coil is a state of high entropy for the biopolymer, while an ordered conformation such as an insoluble crystal or a specific protein fold leads to loss of entropy for the polymer. For water, the entropy depends on the solute. If the solute is small, there is favorable entropy in dissolution. The larger the solute is, the less entropy gain one gets from dissolution, because water molecules must form ordered structures encapsulating the biopolymer. Thus solubility increases for smaller solutes. Enthalpy is also sometimes a driving force. However, if the enthalpic contribution to a specific type of conformation comes from hydrogen bonds, this change is almost 0 since water can form the same hydrogen bonds. Thus the thing that causes e.g. certain folds in proteins is often the entropy gain in water from "releasing" the protein into a folded shape.
The contributions from entropy and enthalpy lead to these possible scenarios for dissolution of biopolymers or denaturation of proteins, for the total change in entropy and enthalpy for the system:
<math>\Delta H > 0, \Delta S > 0</math>: Increasing temperature will make the entropy contribution dominate, dissolution or denaturation at increasing temperature.
<math>\Delta H < 0, \Delta S > 0</math>: Always soluble.
<math>\Delta H > 0, \Delta S < 0</math>: Never soluble.
<math>\Delta H < 0, \Delta S < 0</math>: Can be soluble at low temperatures where the entropy contribution is low compared to the enthalpy contribution.
For polyelectrolytes the entropy gain from the dissolution of counter-ions is large. Since electroneutrality must be maintained, polyelectrolytes are mostly soluble, but this solubility decreases with increasing ionic strength.
Denaturation/solubility
In light of the above the following factors can influence the solubility/denaturation of biopolymers:
- Chaotropic agents: Bind to water, decreasing the entropy gain of water leaving the biopolymer, denatures proteins.
- Temperature: Denaturation/solvation according to the balance of enthalpy and entropy, stated above.
- Acid/base equilibrium: Can cause charge changes, repulsion between charged groups, etc. Similar as ionic strength, and is a contribution to ionic strength.
- Organic solvents: Changing the solvent for something other than water can cause denaturation or solvation.
- Mechanical treatment: Stress on the system can cause changes in conformation.
- Pressure: Denaturation/solvation often causes a decrease in net volume. A higher pressure will favor this transition.
Thermodynamics of dilute solutions
In a dilute solution the concentration must be lower than a given critical overlap concentration. For spheres this concentration is <math>c^*=\frac{2.5}{[\eta]}</math>
