Biochemistry Lecture Notes PDF

A. SECONDARY STRUCTURE -A HELIX -B BARRELS -LOOPS AND TURNS The alpha helix is a coiled structure stabilized by intrachain hydrogen -Beta sheets are formed by adjacent β-strands. bonds ​-The α-helix is a tightly...

A. SECONDARY STRUCTURE -A HELIX -B BARRELS -LOOPS AND TURNS The alpha helix is a coiled structure stabilized by intrachain hydrogen -Beta sheets are formed by adjacent β-strands. bonds ​-The α-helix is a tightly coiled, rodlike structure, -In contrast to an α-helix, the polypeptide in a with the R groups emanating out from the axis β-strand is fully extended. of the helix. -All of the backbone CO and NH groups form -3 types of b sheets; hydrogen bonds except those at the end of the helix. Β-sheet  in maintaining the structural integrity and function of biological molecules. -disulfide bridges crosslinking These are covalent bonds formed between two cysteine residues in a protein. Disulfide bonds contribute to the stabilization of protein structures, particularly in extracellular proteins like antibodies. Polypeptide chains can change direction by making reverse turns and loops reverse turns are a form of tight turn where the polypeptide chain makes a 180° change in direction. -> Cysteine knots in proteins Cysteine knots, also known as cystine knots or knottins, are structural motifs found in certain proteins. These motifs are characterized by a FIGURE 2.36 Structure of a reverse turn. The CO particular arrangement of disulfide bonds group of residue i of the polypeptide chain is (covalent bonds formed between two cysteine hydrogen bonded to the NH group of residue i + residues) that create a knotted topology within 3 to stabilize the turn. the protein's three-dimensional structure. Cysteine knots provide exceptional stability to Depending of how big the loop is the amino proteins, making them resistant to denaturation acids can be closer or further away and proteolysis. Crosslinking C TERTIARY STRUCTURE Crosslinking in biochemistry refers to the C.1 Structure formation of covalent bonds between different Heptad repeats in a coiled-coil protein macromolecules or within the same macromolecule. This process plays a crucial role The two helices in α-keratin associate with each other by weak interactions such as van der Waals forces and ionic interactions. The left-handed supercoil alters the two right-handed α helices such that there are 3.5 residues per turn instead of 3.6. As a result, the pattern of side-chain interactions can be repeated every seven residues, forming the heptad repeats. Two helices with such repeats are able to interact with one another if the repeats are complementary (Figure 2.44). For example, the repeating residues may be hydrophobic, allowing van der Waals interactions, or have opposite charge, allowing ionic interactions. In addition, the two helices may be linked by disulfide bonds formed by FIGURE 2.44 Heptad repeats in a coiled-coil neighboring cysteine residues. The bonding of protein. Every seventh residue in each helix is the helices accounts for the physical properties leucine. The two helices are held together by of wool, an example of an α-keratin. Wool is van der Waals interactions primarily between extensible and can be stretched to nearly twice the leucine residues. [Drawn from 2ZTA.pdb.] its length because the α helices stretch, breaking the weak interactions between C.2 Protein Domains neighboring helices. However, the covalent -Some proteins have domains, which are disulfide bonds resist breakage and return the independently folding regions within a fiber to its original state once the stretching polypeptide, connected by a short, flexible force is released. The number of disulfide bond linker segment. cross-links further defines the fiber’s properties. Hair and wool, having fewer cross-links, are -The example below has four domains. flexible. Horns, claws, and hooves, having more cross-links, are much harder. FIGURE 2.42 Protein domains. The cell-surface protein CD4 consists of four similar domains. Each domain has a different structure and function [Drawn from 1WIO.pdb.] proper folding of bovine ribonuclease.  Initial Steps: -In this example, the exact same sequence (VDLLKN) can be found in one protein’s α helix -The protein was fully unfolded using urea and and in another protein’s β sheet. β-mercaptoethanol. β-mercaptoethanol broke covalent disulfide bonds in the protein. The folding funnel depicts the thermodynamics of protein folding Effect of Denaturation: -Protein folding is often represented as a folding -In 8 M urea with β-mercaptoethanol, the funnel. protein became a randomly coiled into enzymatically inactive polypeptide chain, known -The protein has maximum entropy and minimal as denaturation. structure at the top of the funnel. Renaturation Process: -The folded protein exists at the bottom of the funnel. -Removing urea and keeping a trace of β-mercaptoethanol allowed slow formation and breakage of disulfide bonds. This led to the slow regeneration of the protein's native, stable, and enzymatically active conformation. Role of Disulfide Bonds: -Disulfide bonds are crucial for stabilizing the protein's native structure. The presence of β-mercaptoethanol facilitated the controlled formation and breakage of disulfide bonds during renaturation. Native Conformation and Activity: FIGURE 2.60 The top of the funnel represents all possible denatured conformations—that is, -The native conformation is the most stable and maximal conformational entropy. Depressions enzymatically active form. Regaining enzymatic on the sides of the funnel represent semi-stable activity required removing urea and having a intermediates that can facilitate or hinder the trace of β-mercaptoethanol to allow the protein to rege formation of the native structure, depending on their depth. Secondary structures, such as Alternative conformations of a peptide helices, form and collapse onto one another to sequence initiate folding. Some proteins are inherently unstructured and can exist in multiple Alternative conformations of a peptide conformations sequence refer to different spatial arrangements or structures that the peptide can adopt. The -Intrinsically unstructured proteins (IUP) do not conformation of a peptide is determined by the have a defined structure under physiological rotation about the single bonds in its backbone. conditions until they interact with other molecules. -Metamorphic proteins exist in an ensemble of -Amyloidoses are diseases that result from the structures of approximately equal energies that formation of protein aggregates, called amyloid are in equilibrium. fibrils or plaques. Lymphotactin: an example of a -Alzheimer disease is an example of an metamorphic protein amyloidosis. -An especially clear example of a metamorphic -Normal protein conformations can exist in protein is the chemokine lymphotactin. forms rich in β sheets, which are prone to Chemokines are small signaling proteins in the aggregate. immune system that bind to receptor proteins -An abnormally folded aggregate serves as a on the surface of immune-system cells, nucleus to recruit more proteins. instigating an immunological response. Lymphotactin exists in two very different A model of the human prion protein structures that are in equilibrium (Figure 2.61). amyloid One structure is a characteristic of chemokines, consisting of a three-stranded β sheet and a -Parkinson disease, Huntington disease, and carboxyl- terminal helix. This structure binds to transmissible spongiform encephalopathies its receptor and activates it. The alternative (prion disease), are associated with improperly structure is an identical dimer of all β sheets. folded proteins. All of these diseases result in When in this structure, lymphotactin binds to the deposition of protein aggregates, called glycosaminoglycan, a complex carbohydrate amyloid fibrils or plaques. These diseases are (Chapter 11). The biochemical activities of each consequently referred to as amyloidoses. A structure are mutually exclusive: the chemokine common feature of amyloidoses is that normally structure cannot bind the glycosaminoglycan, soluble proteins are converted into insoluble and the β-sheet structure cannot activate the fibrils rich in β sheets. The correctly folded receptor. Yet, remarkably, both activities are protein is only marginally more stable than the required for full biological activity of the incorrect form. But the incorrect form chemokine. aggregates, pulling more correct forms into the incorrect form. FIGURE 2.61 Lymphotactin exists in two conformations, which are in equilibrium. Protein misfolding and aggregation are associated with some neurological diseases FIGURE 2.62 A model of the human prion protein amyloid. A detailed model of a human prion amyloid fibril deduced from spin labeling and electron paramagnetic resonance (EPR) spectroscopy studies shows that protein aggregation is due to the formation of large parallel β sheets. The black arrow indicates the long axis of the fibril D. QUATERNARY STRUCTURE D.1 Hemoglobin heme group and hemoglobin -Hemoglobin is able to transport oxygen within The heme group is a complex containing iron, the body due to its unique structure. Its crucial for various biological processes. structure consists of four globin subunits: two α Hemoglobin, a protein in red blood cells, utilizes and two β subunits. Each subunit contains a heme to bind and transport oxygen from the heme prosthetic group with an iron-bound. lungs to tissues and facilitates the exchange of Hemoglobin exists in high concentrations in the gases, playing a vital role in oxygenation and cytoplasm of red blood cells, so it needs to be carbon dioxide removal in the body. very soluble in aqueous cytoplasm. This polypeptide chains can assemble into requirement is reflected in the protein's multisubunit structures globular shape, and the fact that it is folded in such a way that hydrophilic residues are found -Many proteins are composed of multiple on the surface of the protein exposed to water, polypeptide chains called subunits. Such while hydrophobic residues are found on the proteins are said to display quaternary interior of the protein. This folding enables structure. hemoglobin to have a stable fold in aqueous -Hemoglobin is a protein with quaternary solution that also allows the protein to interact structure. It has 4 subunits: 2 α subunits and 2 β favorably with water and to be soluble in the subunits. water-filled cytoplasm of the cell. Hemoglobin – globular protein -Each subunit has a heme group which can bind to oxygen Deoxyhaemoglobin Oxygen binding markedly changes the quaternary structure of hemoglobin -The quaternary structure of deoxyhemoglobin tetramer is in the R state increases. is referred to as the T state, while that of Deoxyhemoglobin tetramers are almost oxyhemoglobin is the R state. exclusively in the T state. However, the binding of oxygen to one site in the molecule shifts the -The hemoglobin tetramer can be thought of as equilibrium toward the R state. If a molecule two αβ dimers. The α1β1 dimer rotates 15 assumes the R quaternary structure, the oxygen degrees relative to the α2β2 dimer on oxygen affinity of its sites increases. binding. -This structure alteration, conversion from the T state to the R state, facilitates oxygen binding. Quaternary structural changes on oxygen binding by hemoglobin -hemoglobin tetramer can be thought of as two αβ dimers. FIGURE 7.12 Concerted model. All molecules exist either in the T state or in the R state. At -α1β1 dimer rotates 15 degrees relative to the each level of oxygen loading, an equilibrium α2β2 dimer on oxygen binding. exists between the T and R states. The equilibrium shifts from strongly favoring the T state with no oxygen bound to strongly favoring the R state when the molecule is fully loaded with oxygen. The R state has a greater affinity for oxygen than does the T state. Hemoglobin: sequential model FIGURE 7.11 Quaternary structural changes on oxygen binding by hemoglobin. Notice that, on -A multimeric protein's affinity for a ligand oxygenation, one αβ dimer shifts with respect changes upon binding to a ligand, a process to the other by a rotation of 15 degrees. The known as cooperativity. The concerted model change in shape is called a change in provides a theoretical basis for understanding conformation this phenomenon. The model proposes that multimeric proteins exist in two separate states, Hemoglobin - Concerted Model T and R. Upon ligand binding, equilibrium T state=low affinity for o2 between the two states shifts towards the R R state= high affinity for o2 state, thought to result from protein The binding of ligands simply shifts the conformation changes due to ligand binding. equilibrium between t state and r states (Figure The model is useful in describing hemoglobin's 7.12). Thus, as a hemoglobin tetramer binds sigmoidal binding curve. each oxygen molecule, the probability that the T-to-R Transition -Additional oxygen molecules are now more likely to bind to the three unoccupied sites. Thus, the binding curve for hemoglobin can be seen as a combination of the binding curves that would be observed if all molecules remained in the T state or if all of the molecules were in the R state. As oxygen molecules bind, the hemoglobin tetramers convert from the T state into the R state, yielding the sigmoid binding curve so important for efficient oxygen transport (Figure 7.13). FIGURE 7.8 Oxygen binding by hemoglobin. This curve, obtained for hemoglobin in red blood cells, is shaped somewhat like an “S,” indicating that distinct, but interacting, oxygen-binding sites are present in each hemoglobin molecule. Half-saturation for hemoglobin is 26 torr. For comparison, the binding curve for myoglobin is shown as a dashed black curve. 2,3-bisphosphoglycerate is crucial in determining the oxygen affinity of FIGURE 7.13 T-to-R transition. The binding curve hemoglobin for hemoglobin can be seen as a combination of -2, 3-Bisphosphoglycerate (2,3-BPG) stabilizes the binding curves that would be observed if all the T state of hemoglobin and thus facilitates molecules remained in the T state or if all of the the release of oxygen. molecules were in the R state. The sigmoidal curve is observed because molecules convert -2, 3-BPG binds to a pocket in the hemoglobin from the T state into the R state as oxygen tetramer that exists only when hemoglobin is in molecules bind. the T state. Oxygen Binding by Hemoglobin FIGURE 7.17. 2,3-Bisphosphoglycerate binds to the central cavity of deoxyhemoglobin (left). There, it interacts with three positively charged groups on each β chain (right).  Oxygen binding by pure hemoglobin compared with hemoglobin in red blood cells FIGURE 7.18 Oxygen affinity of fetal red blood cells. Fetal red blood cells have a higher oxygen affinity than do maternal red blood cells because fetal hemoglobin does not bind FIGURE 7.16 Oxygen binding by pure 2,3-BPG as well as maternal hemoglobin does. hemoglobin compared with hemoglobin in red blood cells. Pure hemoglobin binds oxygen Mutations in genes encoding more tightly than does hemoglobin in red blood hemoglobin subunits can result in cells. This difference is due to the presence of disease 2,3-bisphosphoglycerate (2,3-BPG) in red blood cells. -Sickle-cell anemia is a genetic disease caused by a mutation resulting in the substitution of Fetal Hemoglobin native glutamic acid for valine at position 6 of the β chains. -Fetal hemoglobin must bind oxygen at the same pO2 at which the mother’s hemoglobin is releasing oxygen. -In fetal hemoglobin, the β chain is replaced with a γ chain. -The fetal α2γ2 hemoglobin does not bind 2,3-BPG as well as adult hemoglobin does. The reduced affinity for 2,3-BPG results in fetal hemoglobin having a higher affinity for oxygen, binding oxygen when the mother’s hemoglobin is releasing oxygen Oxygen Affinity of Fetal Red Blood Cells The formation of HbS aggregates  although it does not markedly alter the properties of oxyhemoglobin. Examination of the structure of hemoglobin S reveals that the new valine residue lies on the surface of the T-state molecule (Figure 7.26). This new hydrophobic patch interacts with another hydrophobic patch formed by Phe 85 and Leu 88 of the β chain of a neighboring molecule to initiate the aggregation process. More-detailed analysis reveals that a single hemoglobin S fiber FIGURE 7.27 The formation of HbS aggregates. is formed from 14 chains of multiple interlinked The mutation to Val 6 in hemoglobin S is hemoglobin molecules. Why do these represented by the red triangles, while the aggregates not form when hemoglobin S is hydrophobic patch formed by Phe 85 and Leu oxygenated? When oxyhemoglobin S is in the R state, residues Phe 85 and Leu 88 on the β chain 88 in deoxyhemoglobin is represented by the are largely buried inside the hemoglobin blue indentations. When HbS is in its deoxy assembly. In the absence of a partner with form, it exhibits the complementary features which to interact, the surface Val residue in necessary for aggregation position 6 is benign (Figure 7.27). D.2 Collagen BOHR effect Collagen: fibrous protein The Bohr effect describes hemoglobin's lower affinity for oxygen secondary to increases in the -Collagen consists of three polypeptide chains partial pressure of carbon dioxide and/or that form a superhelical cable. decreased blood pH. This lower affinity, in turn, enhances the unloading of oxygen into tissues -2.9 Å per turn, 3.3 residues per turn. Different to meet the oxygen demand of the tissue. from α-helix qn; Is there a Bohr effect for myoglobin? Why or why not? No, myoglobin does not exhibit a significant Bohr effect. Unlike hemoglobin, myoglobin is primarily found in muscle tissues where pH remains relatively stable during normal physiological conditions. Myoglobin's main role is in oxygen storage and release in tissues with consistent pH, reducing the need for a Conformation of a single strand of a pronounced Bohr effect. collagen triple helix cause of sickle cell disease -It contains three helical polypeptide chains, each nearly 1000 residues long. Glycine appears In people with sickle-cell anemia, both alleles of the hemoglobin β-chain gene (HbB) are at every third residue in the amino acid mutated. The HbS substitution substantially sequence, and the sequence decreases the solubility of deoxyhemoglobin, glycine-proline-hydroxyproline recurs frequently (Figure 2.45). Hydroxyproline is a derivative of c)glycosylation I of OH groups with galactose proline that has a hydroxyl group in place of one and glucose of the hydrogen atoms on the pyrrolidine ring. 3 chains assemble to form pro collagen -The collagen helix has properties different from Golgi; glycosylation II and packed into excretory those of the α helix. Hydrogen bonds within a vesicles strand are absent. Instead, the helix is stabilized by steric repulsion of the pyrrolidine rings of the Extracellular; collagen peptidase enzyme cuts proline and hydroxyproline residues (Figure ends and forms tropocollagen 2.46). The pyrrolidine rings keep out of each role of lysyl and prolyl hyroxylase other’s way when the polypeptide chain assumes its helical form, which has about three copper is essential for the activity of lysyl residues per turn. Three strands wind around oxidase, which, in turn, is crucial for the proper one another to form a superhelical cable that is cross-linking of collagen fibrils. Hydroxylysine, stabilized by hydrogen bonds between strands. through its hydroxylation and subsequent glycosylation, contributes to the stability and The hydrogen bonds form between the peptide correct assembly of collagen molecules. These NH groups of glycine residues and the CO processes are essential for the formation of groups of residues on the other chains. The functional collagen structures that provide hydroxyl groups of hydroxyproline residues also strength and support to various tissues in the participate in hydrogen bonding. body. role of vitamin c vitamin C is required for the hydroxylation of proline and lysine residues in the synthesis of collagen. The hydroxylation of proline and lysine is essential for the stability and structure of FIGURE 2.46 Conformation of a single strand of collagen. Hydroxyproline and hydroxylysine a collagen triple helix. residues allow collagen molecules to form strong and stable triple helical structures. synthesis of collagen Without adequate vitamin C, these hydroxylation reactions cannot proceed nucleus;mRNA transcription efficiently, leading to the synthesis of structurally weaker collagen. Cytosol; ribosome translation ->produces precursor pre-pro-a peptide Er; post translational modification a)cleavage of signal peptide from N-terminal ->produces pro-a peptide b)hydroxylation of lysine and proline (OH) vitamin C cofactor  –Lack of vitamin K prevents carboxylation of clotting proteins, which can lead to hemorrhaging. Posttranslational hydroxylation of proline and lysine -Hydroxyproline stabilizes the triple helix by forming interchain H-bonds -Hydroxylysine allows interchain covalent bonds Protein analysis and purification -Biophysical properties of proteins and their amino acids allow a multitude of techniques to be used for analysis 1.Size Exclusion Chromatography: separates E. MODIFICATIONS biomolecules based on their size, with larger molecules eluting faster through a porous Post-translational modifications stationary phase than smaller ones. -Post-translational modifications confer 2.Ion-Exchange Chromatography: separates additional functionalities to amino acid molecules based on their net charge, utilizing a charged stationary phase to attract or repel -Phosphorylation, acetylation and ubiquitylation analytes according to their charge most common and cover >90% of all PTMs characteristics. Some common and important covalent 3.Affinity Chromatography:exploits specific modifications of amino acid side chains interactions between a biomolecule of interest and a ligand on the stationary phase, allowing selective purification based on binding affinity. 4. High-Performance Liquid Chromatography (HPLC): uses high pressure to separate, identify, and quantify components in a mixture with high resolution and efficiency. 5.Gel Electrophoresis: separates biomolecules Posttranslational modifications confer based on their size and charge by migrating new capabilities to proteins through a gel matrix in response to an electric field, allowing the visualization and analysis of -Lack of appropriate protein modification can nucleic acids or proteins. result in pathological conditions; for example: –Lack of vitamin C prevents hydroxylation of proline in collagen, which can lead to scurvy.