2024 Medicinal Biochemistry I Lecture Notes PDF

Medicinal Biochemistry I Protein structure and function Žarko Bošković [email protected] KU MDCM...

Medicinal Biochemistry I Protein structure and function Žarko Bošković [email protected] KU MDCM October 2 – October 8, 2024 Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 1 / 41  Goals and objectives Goals To understand that proteins act as the workhorses of the cell. The shape, functions, and response of the cell to its changing environment are defined by the combination of the proteins present within the cell. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 2 / 41  Review of some basics Review A group’s pKa is the pH at which it is 50% protonated. A group is primarily protonated when the ambient pH is less than its pKa. (Think of this as when there are more than enough protons around to keep the group half protonated.) We call the group acidic (e.g. carboxylic acid) if they have a low pKa (i.e. they are deprotonated at neutral pH) and basic if they have high pKa. Elements are most electronegative towards the upper right of the periodic table. Peptide bonds are formed via a dehydration reaction. Peptide bonds have a resonance structure whereby about 40% of the time they are double bonds, which is why they stay fairly planar. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 3 / 41  Review of some basics Review Cysteines can be oxidized to form disulfide bonds. The cytosol is reducing; disulfide bonds exist in oxidizing environments (e.g. extracellular). Air in the laboratory can oxidize disulfide bonds, which is why some protocols require addition of reducing agents. A hydrogen bond has a length of 1.6 - 2.0 Ångstrom. Van der Waals forces between nonpolar groups are very weak; the collapsing of hydrophobic domains of proteins is driven primarily by entropy of the surrounding water molecules rather than by Van der Waals forces. Chiral amino acids (all except G) are found in their levorotary (L) form in all life on Earth. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 4 / 41  Review of some basics Levels of structural organization There are 4 levels of protein structural organization: Primary: Amino acid sequence Secondary: Helices, sheets, loops Tertiary: Entire chains, bundles of helices, loops, domains Quaternary: Multiple subunits folded into a complex Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 5 / 41  Amino acids Amino acids structures Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 6 / 41  Amino acids Amino acid polarity Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 7 / 41  Amino acids Isoelectric point Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 8 / 41  Amino acids Isoelectric point Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 9 / 41  Amino acids Dihedral angle Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 10 / 41  Peptide bond Dihedral angles in a polypeptide Ψ is the angle along the Cα-C bond. Φ is the angle along the N-Cα bond. Ω is the angle along the C-N bond Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 11 / 41  Peptide bond Dihedral angles in a polypeptide Ω is always 0 or +180. If you plot Φ against Ψ, there are only a few clusters are well-represented: a range of α-helix combinations, a β-sheet area, and a third rarer area (called Lαand populated by left-handed α-helices). Ω is usually found in the trans conformation due to steric hindrance of the consecutive side chains, however, proline because it is anchored to the backbone has a unique twist that enables a cis conformation. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 12 / 41  Peptide bond Dihedral angles in a polypeptide Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 13 / 41  Peptide bond Chirality in amino acids Figure: Mirror image relationship between configurational isomers. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 14 / 41  Peptide bond Chirality in helices Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 15 / 41  Peptide bond Levels of structural organization There are 4 levels of protein structural organization: Primary: Amino acid sequence Secondary: Helices, sheets, loops Tertiary: Entire chains, bundles of helices, loops, domains Quaternary: Multiple subunits folded into a complex Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 16 / 41  Peptide bond Secondary structure α-helices and β-sheets are two ways of allowing the NH and C=O groups on the backbone to form hydrogen bonds. α-helices contain 3.6 residues per rotation, or in other words, each residue spans 100 degrees of rotation. Consecutive rungs of an α-helix turns are separated by 5.4Å. α-helices are almost exclusively right-handed. If you plot out where each residue falls on the helix based on the 3.6 residues/turn rule, you find that amphipathic, half-buried helices have all the hydrophobic residues on one side and the hydrophilic ones on the other side. A fully buried helix will be all hydrophobic residues and a fully exposed helix will be all hydrophilic residues. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 17 / 41  Secondary structure Secondary structure In β-sheets, all potential H-bonds are satisfied except for the “flanking” strands at either end of the sheet. About 20% of β-sheets found in nature are mixed parallel and anti-parallel, the other 80% are pure one or the other. β-sheets are not flat, but pleated. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 18 / 41  Secondary structure B. mori silk β sheets fetch 3ua0 Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 19 / 41  Secondary structure Keratin α helices fetch 4zry Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 20 / 41  Secondary structure Disulfide bonds Ribonuclease fetch 1fs3 Anfinsen’s experiment: primary sequence determines structure. (This notion is now somewhat revised.) Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 21 / 41  Secondary structure Ribonuclease Ramachandran plot Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 22 / 41  Secondary structure Disulfide bonds Insulin fetch 3i40 Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 23 / 41  Disulfide bond Disulfide bonds Immunoglobulin chains are held by disulfide bonds fetch 1igt Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 24 / 41  Disulfide bond Protein carriers Living organisms can’t let simple diffusion and physicochemical properties of molecules dictate where they go. There are specialized proteins that transport molecules. For example: Cholesterol is carried by HDL and LDL particles. More precisely apolipoproteins within these particles. Solute Lipid Carriers (SLC family of proteins) carries carboxylic acids and other small molecules Myoglobin (Mb) and Hemoglobin (Hb) carry oxygen Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 25 / 41  Hemoglobin Oxygen-carrying team Hemoglobin carries O2 (transport function), while its “cousin” myoglobin stores it (repository function). There are 5 billion red blood cells per mL of our blood, and each one has 280 million molecules of Hb (MW = 64,500 g/mol) with 4 heme groups per each molecule. Mb is similar, but monomeric (it consists of only one unit) and has one heme. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 26 / 41  Hemoglobin Heme is central to these proteins Heme = Iron + porphyrin ring around it Porphyrin is aromatic (like benzene); it has delocalized pi electrons in a circle, the number of which fit the 4n+2 formula. It has 4-fold symmetry and it’s largely planar. This is one of the nature’s solutions to the oxygen-carrying problem; Others exist: hemerythrin, erythrocurorines, clorocruorines. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 27 / 41  Hemoglobin Myoglobin and hemoglobin Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 28 / 41  Hemoglobin Myoglobin and hemoglobin Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 29 / 41  Hemoglobin Properties of oxygen-transporting molecule Carrier (Hb) should have high affinity for O2 when it is abundant, but lowered affinity when it is low – it should release O2 when there’s not enough of it. Repository (Mb) should have high affinity when Hb is giving it up Carrier should carry CO2 away from the muscles into lungs. This prevents acidification of the tissues and pH from falling too low. Carrier should have low affinity for O2 in low pH (it should release it into the tissue that requires O2 ) Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 30 / 41  Hemoglobin Saturation curves Myoglobin Mb + O2 ↽ −−−⇀ − MbO2 Keq pO2 y = Keq pO +1 2 Parabolic curve Hemoglobin Hb + n O2 ↽ −− ⇀ −− Hb(O2 )n n Keq ∗pO y = Keq ∗pn +1 2 O2 Sigmoidal (S-shape) curve indicating that hemes are not independent in the tetrameric structure. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 31 / 41  Hemoglobin Saturation curves Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 32 / 41  Hemoglobin Hemoglobin and oxygen Hemoglobin binds oxygen in such a way that at low oxygen pressure it doesn’t bind it very much but it does when the pressure rises. Oxyhemoglobin is the name of species when O2 is bound, and deoxyhemoglobin when there’s no O2 HbO2 is a stronger acid than Hb. Le Chatelier’s principle requires that at low pH equlibrium is shifted towards weaker acid – Hb; this means tha O2 is released in acidic environments, exactly what’s needed for tissue that’s rapidly consuming O2 such as muscles during exercise. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 33 / 41  Hemoglobin Crystal structures of Mb and Hb X-ray diffraction crystal structures of Mb and Hb were the first to be solved (Perutz and Kendrew, 1961 Nobel prize) Mb is “a box” for heme Amino acid sequences for Mb and Hb are very similar Mb contains 7 helical chains. Heme is in the middle with histidines holding on to it. Prolines make bends from one helix to another. Why can proline do that? These chains prevent oxidation of Fe(II) to Fe(III); ferric ions do not bind oxygen at all. Heme is not covalently attached to protein (this is different in cytochromes) CO (carbon monoxide) binds to iron better than oxygen (lethal) Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 34 / 41  Hemoglobin Hemoglobin fetch 1buw Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 35 / 41  Hemoglobin Hemoglobin A single chain of hemoglobin. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 36 / 41  Hemoglobin Hemoglobin Whenever prolines (Pro, P) are found, polypeptide changes its secondary structure; there’s a bend. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 37 / 41  Hemoglobin Myoglobin fetch 1mbo Myoglobin with O2 bound (MbO2 ). Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 38 / 41  Hemoglobin Myoglobin Myoglobin with O2 bound (MbO2 ). Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 39 / 41  Hemoglobin Metal cofactors fetch 1ca2 Metals are often held by histidines (His, H) and cysteins (Cys, C) in the active sites of enzymes. Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 40 / 41  Hemoglobin Leucine zipper fetch 1nkp Transcription factors are held together through VdW interactions between complementary leucines (Leu, L). Žarko Bošković [email protected] (KU MDCM) MDCM601 October 2 – October 8, 2024 41 / 41