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Amino acids share a common structure: <ul><li>20 amino acids found normally in proteins. They share common structural features. </li></ul><ul><li>Except for proline, all amino acids: </li></ul><ul><ul><ul><ul><li>-- Contain a carboxyl group, an amino group and a side chain ‘R’ (except for glycine, which has a hydrogen atom instead of an R group) </li></ul></ul></ul></ul><ul><ul><ul><ul><li>4 different groups surrounding the α -Carbon: makes it a chiral center </li></ul></ul></ul></ul><ul><ul><ul><ul><li>All Molecules with a chiral center are optically active, ie. They rotate plane polarized light. </li></ul></ul></ul></ul>http://www2.glos.ac.uk/GDN/origins/images/amino.gif
Amino Acids can exist in L- or D-forms <ul><li>There are 2 different spatial arrangements around the α -carbon that are non-superimposable mirror images: Enantiomers. </li></ul><ul><li>Stereoisomers related to L-glyceraldehyde are designated ‘L-’ </li></ul><ul><li>Stereoisomers related to D-glyceraldehyde are designated ‘D-’ </li></ul><ul><li>In all proteins (except for some rare cases), amino acids are exclusively in the L-conformation </li></ul>
Amino acids are classified according to the type of R-group they contain <ul><li>Amino Acids are classified into 5 subclasses according to the polarity of their side chains </li></ul><ul><ul><li>Nonpolar (aliphatic): Alanine, Valine, Leucine, Isoleucine, Glycine, Methionine, Proline. Hydrophobic, tend to cluster together on the inside of proteins, lending stability to protein structure. Proline’s cyclic structure makes it moderately polar because of its limited flexibility. </li></ul></ul><ul><ul><li>Aromatic: Phenylalanine, Tyrosine, Tryptophan. Tyrosine and tryptophan are comparatively more polar and absorb UV light. </li></ul></ul><ul><ul><li>Polar uncharged (hydrophilic): Serine, Threonine, Cysteine, Asparagine, Glutamine . These functional groups can form hydrogen bonds with water, found on the outer surfaces of most proteins. </li></ul></ul><ul><ul><li>Positively charged : Lysine, Arginine, Histidine . Strongly hydrophilic </li></ul></ul><ul><ul><li>Negatively charged : Aspartate, Glutamate . Strongly hydrophilic </li></ul></ul>
The 5 categories of amino acids: nonpolar, aromatic, polar, positively charged and negatively charged
The 5 categories of amino acids: nonpolar, aromatic, polar, positively charged and negatively charged
Proline -Has a distinctive ring structure. -Allows for less flexibility in polypeptide regions containing proline as the secondary imino group of proline is held in a more rigid conformation. -Found often in bends of folded protein chains.
Nonstandard amino acids All nonstandard amino acids are derivatives of standard amino acids About 300 nonstandard amino acids have been found. Found in prothrombin Involves 4 lysine residues, Found in elastin Contains selenium instead of sulfur, rare Found in plant cell wall, collagen Derivatives of lysine, found in collagen and myosin, respectively In addition, amino acids can be phosphorylated (thr, tyr, ser) or otherwise modified (cysteinylation. methylation) to modify the function of a protein
Zwitterions <ul><li>From the German “Zwitter”: a hybrid. Amino acids ionize in aqueous solutions. The zwitterionic form dominates at neutral pH. </li></ul><ul><li>Also called ampholytes , as they can act as proton acceptors (bases) and proton donors (acids). </li></ul>
Titration curves of amino acids; prediction of the electric charge of amino acids Titration curve of 0.1M glycine at 25 °C. Blue boxes show the regions of greatest buffering power Inflection point Inflection point Inflection point A quantitative measure of the pKa of the amino acid Note there are 2 regions of buffering Not a good buffer at Physiological pH Isoelectric Point: pI, the Characteristic pH at which The net charge of a Compound is 0.
<ul><li>The pKa of the ionizable groups of glycine are lower than those substituted for methyl substituted –NH2 and –COOH groups due to molecular interactions </li></ul><ul><li>Similar effects can be observed at the active site of an enzyme. </li></ul>
Amino acids with an ionizable R-group have multiple stages corresponding to the multiple possible ionization steps. 3 pKa values The additional stage for the titration of the ionizable R-group merges to some extent with the other two.
The Hendersson-Hasselbach equation also applies to the amino acid acid-base reactions: 2. What is the ratio of forms of glycine when the pH = 10? 1. What is the pH at which the ratio of is 0.25? to to
The Lambert-Beer Law <ul><li>Biomolecules absorb light at characteristic wavelengths. This property is widely exploited to detect, identify and measure their concentration in solution. </li></ul><ul><li>The fraction of incident light absorbed is related to the thickness of the absorbing layer and the concentration of the absorbing media. </li></ul>Absorbance is directly proportional to the concentration of the solution and the Intensity of transmitted light. Log I 0 /I = ε cl Log I 0 /I = Absorbance , where I 0 = intensity of the incident light, I = intensity of transmitted light, ε = the molar extinction coefficient (liters/mole-cm) c = concentration of the absorbing species, and l = the path length
The absorbance of UV light by tryptophan (W) and tyrosine (Y) is exploited in the measurement of amino acids and peptides.
Reversible disulfide bond formation by cysteine residues <ul><li>Some proteins contain disulfide bonds that act to stabilize their structure. </li></ul><ul><li>Formed via the oxidation of 2 cysteine residues: the resulting amino acid is called Cystine </li></ul>Very hydrophobic Very insoluble molecule
Objectives <ul><li>Description of peptide bond formation </li></ul><ul><li>Sequencing a protein </li></ul><ul><li>Identification and purification of proteins </li></ul>
Polypeptides and proteins <ul><li>Amino acids form polymers - long chains of amino acids connected via peptide bonds : a substituted amide linkage. </li></ul><ul><li>Peptide bonds are formed via condensation </li></ul><ul><li>The 2 amino acid residues are covalently joined by the removal of a water molecule from the α -carboxyl group of one amino acid and the α -amino group of another </li></ul>Peptide bond Reaction is thermodynamically unfavorable under physiological conditions Requires modification and activation of carboxyl group.
Acid-Base properties of peptides 1. Isoelectric points of peptides - Peptides have characteristic titration curves and a characteristic isoelectric point (pI) at which their overall charge is neutral and at which they do not move in an electric field 2. peptides can be distinguished by their ionization behavior 3. Acid-Base properties of peptides - The α -amino and carboxyl groups of all non-terminal amino acids are covalently bound in peptide bonds, which do not ionize and therefore do not contribute to the total acid-base behavior of peptides -The R groups of some amino acids can ionize, and in a peptide these R groups contribute to the overall acid/base properties. -The pKa value for an ionizable R group can change when an amino acid becomes a residue in a peptide, due to a change in its surroundings
Peptide bonds connect amino acids to form polypeptides and proteins. -The peptide bond is rigid and planar -One exception is the cis-configuration Of peptide bonds involving proline.
-3 bonds separate the sequential α -carbons in a peptide chain. The N - C α and C – C α bonds are able to rotate around bond angles designated Φ and Ψ respectively. -The C – N bond is not free to rotate, and other bonds may be sterically hindered depending on the R-group.
The conformations of peptides are defined by the values of phi( Φ, degree of rotation at the N- α C bond) and psi( Ψ, degree of rotation at the α -C bond). Conformations deemed possible are those that involve little or no steric interference, based on calculations using known van der waals radii and bond angles. Allowed values for Φ and Ψ are graphically revealed in a Ramachandran plot.
Sanger’s sequencing method and the (Pehr) Edman degradation method The Edman degradation removes only the amino-terminal amino acid from a polyeptide, leaving all other bonds intact. This process has been automated using a machine called a sequenator .
Homologs, Paralogs, Orthologs <ul><li>Evolutionarily, protein sequence is important. </li></ul><ul><li>Homologous proteins: Members of the same protein “family” For example, hemoglobin, cytochrome C </li></ul><ul><li>Paralogs: Two homologs found in the same species eg. Hemoglobin and myoglobin </li></ul><ul><li>Orthologs: homologs found in different species, eg. Whale vs. human hemoglobins. </li></ul>
Objectives <ul><li>Different Structures of proteins (primary secondary or tertiary) </li></ul><ul><li>How amino acid sequence of a protein determines its secondary or tertiary structures </li></ul><ul><li>How structure of the proteins determines the </li></ul><ul><li>properties of proteins such as solubility, stability and thermodynamic properties. </li></ul><ul><li>How the structure of proteins determine their function. </li></ul>
Some proteins have chemical groups other than amino acids -The conjugated group is called the prosthetic group .
Protein separation, purification, and characterization <ul><li>Purification Steps </li></ul><ul><ul><li>Preparation of crude extract: may involve subcellular fractionation </li></ul></ul><ul><ul><li>Utilizing protein solubility: Salting out </li></ul></ul><ul><ul><li>Dialysis </li></ul></ul><ul><ul><li>Column chromatography </li></ul></ul>
Types of column chromatography Ion exchange chromatography: Cation or anion exchange The column matrix is a synthetic polymer containing bound charged groups. The affinity of each protein for the charged groups on the column is affected by -pH (ionization state) -Concentration of salt Size exclusion: separates by size. -Matrix is a cross linked polymer of defined size -Larger protein migrates faster as they are too large to enter pores
Types of column chromatography (cont.) Affinity: Separates by specifically binding a ligand (such as an antibody) cross-linked to the column
Electrophoresis (used for detection and analysis) : -Separates proteins by size, charge and shape. -SDS allows estimation of molecular weight. CH 3 -(CH 2 ) 11 -O-SO 3 - Na +
Estimating the molecular weight of a protein Most common is the use of marker proteins of known molecular weight A plot of the log M r of the marker proteins, which allows the estimation of the molecular weight of the unknown protein from the graph.
Overview of polyacrylamide gel electrophoresis (PAGE)
Isoelectric Focusing 2D Electrophoresis <ul><li>Electrophoresis in 2 dimensions: </li></ul><ul><ul><li>1. Separation by isoelectric point using a pH gradient </li></ul></ul>
Isoelectric Focusing 2D Electrophoresis (cont.) -2. PAGE is then performed on the focused samples. Picture of a 2-dimensional gel
Levels of Structure in Proteins Sequence -peptide and disulfide bonds A stable arrangement Folding Arrangement of multimers -Spatial arrangement of atoms in a protein is called conformation -The conformation of a protein under a given condition is thermodynamically stable -Proteins in their functional folded conformation are called native proteins - Stability of a protein is defined as their tendency to maintain a native conformation
Secondary structure refers to the arrangement of adjacent amino acids in regular, recurring patterns; a few types of secondary structure are particularly stable and occur widely in proteins: The α helix, β conformation, and β turns The peptide bond is rigid but other bonds are free to rotate. α helix makes optimal use of H-bonds and forms readily. In α helices, the properties of the side chains place five types of constraints on the stability of the helix a. The electrostatic repulsion (or attraction) between successive amino acid residues with charged R groups. For example long block of Glu residue will not form helix as the COO- groups will repel each other and overcome influence of hydrogen bond. The positive charge on Lys and Arg residue would do the same. b. The bulkiness of adjacent R groups. Such as Asn, Ser, Thr and Leu c. The interactions between amino acid side chains spaced three (or four) residues apart. d. The occurrence of Pro and Gly residues. The N atom of Pro is rigid and cannot rotate. Also, it cannot form hydrogen bond as it does not have H. Gly has more conformational flexibility and tends to form a coil.
e. The interaction between amino acid residues at the ends of the helical segment and the electric dipole inherent to the a helix. Often the negatively charged amino acids reside near the N-terminus where they stabilize the interaction with positive charge. Positively charged residue near the N-terminus would destabilize the helix. *Right-handed alpha helix
The β Conformation of Polypeptide Chains 1. The backbone of the polypeptide chain is extended into a zigzag rather than a helical structure 2. The zigzag chains can be arranged side by side to form a structure resembling a series of pleats, called a β-sheet 3. H-bonds are formed between adjacent segments of polypeptide chain 4. the adjacent polypeptide chains can be either parallel or antiparallel
β turns are common in proteins 1. In globular proteins, which have a compact folded structure, nearly one third of the amino acid residues are in turns or loops where the polypeptide chain reverses direction 2. These are the connecting elements that link successive runs of a helix or β conformation 3. Particularly common are β turns that connect the ends of two adjacent segments of an antiparallel β sheet
Tertiary Structure <ul><li>The overall 3-D arrangement of all atoms in a protein is referred to as the protein's tertiary structure. </li></ul><ul><li>Most structural patterns reflect two simple rules: </li></ul><ul><li>Hydrophobic residues are largely buried in the protein interior </li></ul><ul><li>The number of H bonds within the protein is maximized </li></ul>
In a multi-subunit protein, the arrangement of the subunits in 3-D complexes constitutes quaternary structure Quaternary structure of deoxyhemoglobin: 2 α subunits and 2 β subunits pack together to make a multimer of 4 subunits.
The light and heavy chains of an antibody molecule.
Higher order structured proteins can be classified into 2 groups: fibrous proteins and globular proteins <ul><li>- Fibrous Proteins have polypeptide chains arranged in long strands or sheets, usually have a structural function. </li></ul><ul><li>- usually consist largely of a single type of secondary structure </li></ul><ul><li>- provide support, shape, and external protection to vertebrates </li></ul><ul><li>- Insoluble (hydrophobic amino acid residues) </li></ul><ul><li>- examples include "-keratin, collagen, silk fibroin </li></ul>
α -Keratin -Alpha keratins belong to the intermediate filament (IF) protein family. -An all α -helix protein. -Rich in hydrophobic amino acids: Ala, Val, Leu, Ile, Met, Phe
Collagen -A repeating tripeptide: Gly-X-Pro or Gly-X-Hyp -Left-handed helical structure: 3 residues per turn -3 helices wrap around each other in a right- handed twist -Great tensile strength!
Fibroin (silk protein) <ul><li>Anti-parallel β-sheet rich in Ala and Gly, small side chains allow close packing. </li></ul>
Globular proteins <ul><li>have polypeptide chains folded into a spherical or globular shape </li></ul><ul><li>folding provides structural diversity necessary for biological functions </li></ul><ul><li>often contain several types of secondary structure </li></ul><ul><li>include most enzymes and regulatory proteins </li></ul><ul><li>examples include human serum albumin, sperm whale myoglobin </li></ul>-A. A ribbon representation of the backbone -B. A “mesh” image – surface -C. surface contour image: pockets -D. Ribbon + hydrophobic residues -E. Space-filling model
The heme group <ul><li>Present in hemoglobin, myoglobin, cytochrome C. </li></ul>Porphyrin ring structure -Contains 1 iron molecule. -2 open coordination Bonds.
<ul><li>Stable folding patterns are common in globular proteins </li></ul><ul><li>Hydrophobic interactions make a large contribution to the stability of protein structures. </li></ul><ul><li>Burial of hydrophobic R groups to exclude water requires at least two layers of secondary structure β-α-β loop and α- α corner. </li></ul><ul><li>Where they occur together in proteins, α helices and β sheets generally are found in different structural layers. </li></ul>Tertiary Structure of Globular Proteins
- Polypeptide segments adjacent to each other in the primary sequence are usually stacked adjacent to each other in the folded structure. - Connections between elements of secondary structure cannot cross or form knots - β conformations are more stable when the segments are slightly right handed twist Other common motifs (mostly functional) found in many proteins. -Supersecndary structures (motifs): particularly stable arrangements of several Elements of 2° structure and the connections between them.
Protein denaturation and folding <ul><li>A loss of 3-D structure sufficient to cause loss of function is called denaturation. </li></ul><ul><li>- Proteins can be denatured by heat, </li></ul><ul><li>extremes of pH, or by chemical denaturing reagents. </li></ul><ul><li>- During denaturation, no covalent bonds are broken; rather, the weak interactions in the protein are affected. </li></ul><ul><li>- Some denatured proteins can renature spontaneously, showing that amino acid sequence determines tertiary structure. </li></ul><ul><li>- Protein folding in cells involves multiple pathways. </li></ul>
Methods to determine the 3-D structure of a protein <ul><li>1. X-ray diffraction </li></ul><ul><li>the spacing of atoms is located by intensities of spots on a photographic film by a beam of X ray of given wavelength after the beam has been diffracted by the electrons of the atom. </li></ul><ul><li>2. Nuclear magnetic resonance (NMR). </li></ul>
X-ray diffraction patterns from whale myoglobin are used to extract diffraction patterns to determine the 3-D structure of the protein Three dimensional electron density map calculated from the diffraction patterns
The use of 2D nuclear magnetic resonance (NMR) to determine the 3D structure of whale myoglobin