Biochemistry Final Exam Review PDF

BIOCHEMISTRY FINAL EXAMINATION REVIEW ****  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Ban BIO Câu Lạc Bộ Learning Support xin được gửi tới bạn tài liệu ôn tập môn Biochemistry. Do thời gian thi khá gấp gáp nên chúng mình gửi tài liệu hơi m...

BIOCHEMISTRY FINAL EXAMINATION REVIEW ****  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Ban BIO Câu Lạc Bộ Learning Support xin được gửi tới bạn tài liệu ôn tập môn Biochemistry. Do thời gian thi khá gấp gáp nên chúng mình gửi tài liệu hơi muộn, mong bộ tài liệu có thể phần nào hỗ trợ bạn học ôn tập hiệu quả. File tài liệu tóm tắt sơ lược các lecture, đồng thời bổ sung thêm thông tin ngoài lề cùng lý giải thí nghiệm để bạn đọc tham khảo. Nếu cần bạn có thể tự tìm hiểu thêm thông tin trong sách giáo khoa “Lehninger Principles of Biochemistry” hoặc đọc cuốn “Campbell Biology” (cuốn này viết khá dễ hiểu). Để đạt hiểu quả cao, bạn nên học song hành giữa slide trên moodle, làm quiz, note của riêng bạn và file đề cương này như một cuốn note phụ trợ. Tất nhiên trong quá trình biên soạn, team chúng mình không tránh khỏi có những thiếu xót, mong các bạn thông cảm và nhẹ nhàng góp ý để chúng mình rút kinh nghiệm cho những lần sau (bạn có thể liên hệ chúng mình qua fanpage). Cuối cùng, nếu như bạn học gặp bất kì khó khăn gì, đừng ngần ngại tìm chúng mình trên kênh discord nhé! Chúc bạn ôn tập hiệu quả! 1  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Chapter 3: Amino Acids, Peptides, and Proteins 1. Amino acids: Proteins: ○ Polymers of amino acids, with each amino acid residue joined to its neighbor by specific types of covalent bond. ○ (Amino acid) residue: the loss of elements of water when one amino acid is joined together Common structural features: ○ A carboxyl group and an amino group bonded to the same carbon atom ( the α carbon) ○ Different: R groups (vary in structure, size, electric charge…) By joining the same 20 amino acids in many different combinations and sequences, the cell can produce a protein with strikingly different properties and activities. In addition to these 20 amino acids, there are many less common residues, that can be present in living organisms but not as constituents of proteins. The aa residues in proteins: L stereoisomers ○ Virtually all amino acid residues in proteins are L stereoisomers. Because the active sites of enzymes are asymmetric allowing the reactions they catalyze to be stereospecific, cells are able to specifically synthesize L isomers of amino acids. ○ The α carbon bond to 4 different groups -> a chiral central carbon. Due to the different arrangements, there are 2 possible stereoisomers: L and D enantiomers L-amino acids have alpha-amino groups on the left side D-amino acids have alpha-amino groups on the right side ○ All molecules with a chiral center are optically active: the molecule that rotates plane-polarized light. Classification based on chemical properties of R groups: Nonpolar, aliphatic R group R groups: nonpolar, hydrophobic Side chains of alanine, valine, leucine, isoleucine: tend to cluster together within protein structure by the method of hydrophobic interactions Polar, uncharged R groups R groups: contain functional groups that can form H bonds with water -> more soluble in water, more hydrophilic Negatively charged R groups Hydrophilic, act as a base Positive charged R groups Hydrophobic, act as an acid Aromatic R group R groups: aromatic side chain, nonpolar (hydrophobic) Can participate in hydrophobic interactions 2  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Unusual amino acids found in peptides: Selenocysteine, Pyrrolysine, Desmosine, and its isomer isodesmosine… Classification based on the nutritional requirement: ○ Essential amino acids: Building blocks of protein molecules that bodies cannot synthesize from other amino acids or smaller building blocks, need to get from a food source. There are 9 essential aa divided into 3 groups: Essential aa with hydrocarbon R-groups: isoleucine (ile), valine (val), leucine (leu), phenylalanine (phe) Essential aa with neutral R-groups: tryptophan (trp), threonine (thr), methionine (met) Essential aa with acid or base R-groups arginine (arg), lysine (lys), histidine (his) ○ Non-essential amino acids (body can synthesize) ○ Semi-essential amino acids (histidine and arginine, growing children require them in food but they not essential for the adult individual) Proteins require modification before fully active conformation: 3  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Amino acid derivatives not found in proteins but playing key roles in cells Aa acts as acids and bases: Nonionic and zwitterionic forms of amino acids Ampholytes: amino acid exists in solution as the dipolar ion/ zwitterion, amphoteric (which can act as either an acid or a base) Aa have characteristics titration curves ○ The midpoint: a point of inflection is reached where the pH =pKa ○ pI (Isoelectric point/ electric pH): the characteristic pH at which the net electric charge is 0. 4  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Roles in the living organism: ○ Building blocks of proteins. ○ Precursors for biologically active molecules ( such as neurotransmitters, local mediators, energy-related metabolites, heme, and purines). ○ Serotonin and dopamine (low serotonin levels) ○ Energy sources ○ Regulators gene expressions and cellular signaling (growth, maintenance, reproduction, immunity): Trp operon, high/low tryptophan Peptides are chains of amino acids: ○ Peptide bonds ○ Amino-terminal and carboxy-terminal ends ○ Peptides can be distinguished by their ionization behavior 2. Working with proteins: Proteins can be separated and purified: Protein purification procedure: ○ Crude extract: the solution that protein release after being broken ○ Fractionation: the process that separates the protein into different fractions based on a property such as size or charge ○ Salting out: addition of certain salts in the right amount to selectively precipitate targeted proteins (others remain in solute, usually using ammonium sulfate salt) which are then removed by low-speed centrifugation. Methods for fractionating proteins: ○ Column chromatography: Ion-exchange chromatography 5  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Size exclusion chromatography Affinity chromatography Nitrilotriacetic acid (NTA) Elution with a high concentration of imidazole ○ Protein can be separated and characterized by electrophoresis: ○ Electrophoretic mobility ○ SDS-PAGE (SDS-polyacrylamide gel electrophoresis) SDS (Na-dodecyl sulfate) 2-mercaptoethanol Heat ○ Disulfide bonds formation ○ Breaking disulfide bonds in proteins ○ Absorption of ultraviolet light by aromatic amino acids ○ Isoelectric focusing 3. The structure of proteins: primary structure a. Primary structure: Covalent bonds: peptide and disulfide bonds The sequence of amino acid residues Function of protein depends on its amino acids sequence Amino acid sequence of bovine insulin: Disulfide cross-linkages Steps in sequencing a polypeptide: Step 1: identification of the amino-terminal residue Step 2: the Edman degradation procedure reveals the entire sequence of a peptide Determination of the primary structure of peptides (proteins) Automatic sequencing (sequencer) up to 50 aa Sequencing of large proteins: Breaking disulfide bonds Cleaving the polypeptide chain Sequencing the peptides Ordering the peptide fragments Investigating proteins with mass spectrometry: ○ Measurement of m/z ratio (mass to charge ratio) ○ Matrix-assisted laser desorption/ionization mass spectrometry or MALDI MS Electrospray ionization mass spectrometry (ESI MS) ○ Tandem MS (MS/MS) ○ Small peptides and proteins can be chemically synthesized Amino acid sequences provide important biochemical information Protein sequences can elucidate the history of life on earth 6  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Chap 4: The Three-Dimensional Structure of Proteins 1. Overview of protein structure: Protein structure: is stabilized by multiple weak interactions. ○ Hydrophobic interactions: major contributors to stabilizing the globular form of most solute proteins ○ Hydrogen bonds and ionic interactions: optimized in the thermodynamically most stable structures. Each protein has a unique three-dimensional structure (specific chemical or structural function) Conformation: the spatial arrangement of atoms in the protein. ○ Possible conformation: any structure state without breaking covalent bonds ○ A change in conformation: by rotation about single bonds ○ Native proteins: proteins in any functional, folded conformations A protein's conformation is stabilized largely by weak interactions: ○ The structural patterns reflect 2 rules: Hydrophobic residues are largely buried in the protein interior, away from water. (Hydrophobic residues covered in protein interior (away from water) The number of hydrogen bonds and ionic interactions within the protein is maximized. ○ The peptide bond is rigid and planar: Each peptide bond has some double bond character due to resonance and cannot rotate Carbonyl oxygen: partial negative charge; amide nitrogen: partial positive charge => setting up a small electric dipole Peptide bond: double-bond character due to resonance and cannot rotate ○ The α carbons of adjacent aa residues are separated by 3 covalent bonds: Cα-C-N-Cα. C-N bonds: due to their partial double-bond character, cannot rotate freely (limit the range of conformation possible N-Cα, Cα - C: can rotate Other single bonds in the backbone may also be rotationally hindered, depending on the size and charge of the R groups. ○ 3 dihedral angles/ torsion angles: Ø, 𝛙, Ω reflecting rotation of each of three bonds in the peptide backbone. 7  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Newman projection for determining Phi and Psi angles. 2. Protein secondary structure: Secondary structure: the local spatial arrangement of the segment of a polypeptide chain The regular secondary structure occurs: the dihedral angle remains the same (or nearly) throughout the segment. There are a few common types of secondary structure: α helix, β sheet, β turn, and random coil a. Alpha-helix: α helix structure: ○ Polypeptide backbone is tightly wound around an imaginary axis ○ R groups of amino acid residues protrude outward from the helical backbone (minimize steric repulsive force) The most prevalent arrangement of the polypeptide chain A single turn of helix: ○ 5.4 A ○ Typical; -57 and -47 (slight deviations often found) ○ 3.6 amino acids per turn ○ Right-handed turn 8  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Advantages of α -helix structure: ○ Optimal use of internal H-bond: form between the oxygen of one peptide bond and the amide hydrogen four amino acids away from it along the helix. ○ Participation of every peptide bond in H-binding ○ To provide significant stability on the overall structure => α -heli form more readily than many possible conformations Amino acid sequence affects the stability of the α -helix: Structural integrity of an α -helix depends on correct steric configuration ○ The intrinsic propensity of an amino acid residue to form an α helix ○ The interactions between R groups, mainly those spaced three (or four) residues apart; ○ The bulkiness of adjacent R groups: many adjacent long blocks of negatively charged or positively charged R group prevent the formation of the α helix If a polypeptide chain has a long block of Glu residues, the (-) charged carboxyl group of adjacent Glu residues repel each other -> prevent forming α helix (similar to Lys or Arg: positive charged R groups repel each other) ○ The occurrence of Pro and Gly residues (rarely Steric hindrance in proline: nitrogen atom 9  Câu lạc bộ USTH Learning Support trân trọng giới thiệu of pro residue in peptide linkage has no H atom to participate in H bond with other residues, also the N atom in a part of rigid side chain ring so it cannot rotate. Interactions between amino acid residues at the ends of the helical segment and the electric dipole inherent to the α helix b. The β conformation: β- sheet: ○ The backbone of the polypeptide chain is extended into a zigzag structure which can be arranged side by side, forming a sheet-like structure. ○ The H bonds form between carbonyl and amino groups of the backbone, while R groups extend above and below the plane of the sheet ○ The adjacent polypeptide chains in β-sheet: parallel or antiparallel Parallel: having the same amino-to-carboxyl orientations Repeat period: 7A Antiparallel: having the opposite amino-to-carboxyl orientations ○ Repeat period: 6,5A ○ 10  Câu lạc bộ USTH Learning Support trân trọng giới thiệu ○ Amino acid propensities Large aromatic residues (Tyr, Phe, Trp) β - branched amino acids (Thr, Val, Ile) c. β turns: β turns: connect the ends of two adjacent segments of an antiparallel β sheet Gly and Pro residue often occur in β turns Type I and type II β turns are most common, type I occur more than twice as frequently as type II Note: Common secondary structure have characteristic dihedral angles: ○ Secondary structure of a polypeptide segment can be defined if the dihedral angles are known for all amino acid residues in that segment. Circular dichroism spectroscopy: ○ Measures the difference in absorption of left-handed versus right-handed plane-polarized light due to the characteristic CD spectra of each conformation. 3. Protein tertiary and quaternary structures: Tertiary structure: overall three-dimensional arrangement of all atoms in a protein Quaternary structure: the arrangement of two or more separate polypeptide chains, or subunits, which may be identical or different. Classify protein into 2 major groups: fibrous proteins and globular proteins Fibrous proteins Globular proteins - Polypeptide chains arranged in long strands - Polypeptide chains folded into a spherical or sheets or globular shape - Consist largely of a single type of secondary - Contain several types of secondary structure structure and their tertiary structure is - Made up most of enzymes and regulatory relatively simple proteins - Provide support, shape, and external protection to vertebrate a. Fibrous proteins: Properties that give strength and/or flexibility to the structures A simple repeating element of secondary structure 11  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Insoluble in water Due to high concentration of hydrophobic amino acid residues both in the interior of the protein and on its surface. These hydrophobic surfaces are largely buried similar polypeptide chains are packed together to form elaborate supramolecular complexes. Three common fibrous protein structures: α- Keratin; β-strands/sheets (fibroin and elastin), and triple helices (collagen) b. Globular proteins: Shared properties of globular proteins ○ Compactly folded ○ Interior: hydrophobic amino acids ○ Exterior: hydrophilic amino acids ○ Stabilized by H-bond and ionic bonds ○ Assemblage of a-helixes, b-sheets, and connecting segments Motif (super secondary structure, fold) ○ A folding pattern involving two or more elements of 2° structures and the connection between them ○ Example: helix turn helix motif (DNA binding) ○ Independently unstable ○ Protein motifs are the basis for protein structural classification Domain ○ A part of a polypeptide chain that is independently stable ○ Potentially a single entity concerning the entire protein Protein quaternary structures range from simple dimers to large complexes: ○ Rotational symmetry ○ Cyclic symmetry ○ Dihedral symmetry ○ Icosahedral symmetry ○ Helical symmetry 4. Protein denaturation and folding: Loss of protein structure results in loss of function. Denaturation: ○ A loss of three-dimensional structure to cause loss of function ○ Not necessarily complete unfolding or randomization of conformation ○ Partially unfolded state, in most cases Denaturation by heat ○ To exert complex effects on the weak interactions 12  Câu lạc bộ USTH Learning Support trân trọng giới thiệu ○ The abrupt conformational change at a narrow range of temperature ○ Protein specific denaturation temperature ○ Cooperative process ○ The heat-stable proteins of thermophilic bacteria and archaea have evolved to function at the temperature of hot springs. Denaturing agents ○ Extremes of pH -> alter the net charge on the protein, causing electrostatic repulsion and disruption of some H bond ○ Miscible organic solvents: alcohol or acetone ○ Solutes: Urea, guanidine hydrochloride (GdnHCl) ○ Detergent: SDS,... => Miscible solvents, urea, and detergents: disrupting the hydrophobic interaction ○ Heavy metals: Pb, Hg, Au -> disrupt S-S bonds Renaturation ○ Regaining of native conformation (structure) ○ Example: denaturation and renaturation of ribonuclease A ○ Denaturation: urea solution in presence of reducing agent Reducing agent cleaves the 4 S-S bonds to yield 8 Cys residues Urea: disrupts the stabilizing hydrophobic interactions ○ Renaturation: removed reducing agent and urea -> spontaneously refolds into its correct tertiary structure with full restoration of its catalytic activity Molecular chaperones: ○ Proteins that interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways, providing microenvironments on which folding pathways can occur and protect degradation and denaturation of proteins during folding process ○ 2 class of molecular chaperones; ○ Hsp70: More abundant in cell stressed by elevated temperatures Protect proteins subject to denaturation by heat and new peptide molecules being synthesized (not yet folded) Block the folding certain proteins that must remain unfolded until translocated across a membrane ATP-dependent process Hsp70 proteins bind to and release polypeptides in a cycle that uses energy from ATP hydrolysis, involve other proteins (include Hsp40) 13  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Chaperonins: GroEL/GroES: in ecoli 10%-15% of cellular proteins require the resident chaperonin system for folding under normal conditions (30% require when the cells are heat stressed) 14  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Enzymes: catalyze isomerization reactions ○ PDI (Protein disulfide isomerase): Catalyzes the interchange or shuffling of disulfide bonds of the native conformation are formed Catalyzes the elimination of folding intermediates with inappropriate disulfide cross-links ○ PPI (peptide prolyl cis-trans isomerase) Catalyzes the interconversion of cis and trans isomers of Pro residue peptide bond C. Defects in protein folding may be the molecular basis for a wide range of human genetic disorders. Misfolding proteins which not goes through refolding or denaturation can develop some serious disease: type 2 diabetes, Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease ○ A soluble protein is secreted in a misfolded state, converted into an insoluble extracellular myeloid fiber. ○ Formation of disease-causing amyloid fibrils: The fibers are highly ordered and unbranched, with a high degree of β-sheet structure. Before folding is complete, the β-sheet regions of one polypeptide associate with the same region in another polypeptide, forming the nucleus of amyloid. ○ Alzheimer’s disease Amyloid fiber formation by misfolded b-amyloid peptide (36-43 aa) A kind of amyloidoses Alzheimer's disease - plaques, tangles, causes, symptoms & pathology Prion: ○ Prions: ○ An infectious agent that consists of aggregates protein which is normally in our body but misfolding ○ Cannot be broken down or denatured by typical method ○ Transmissible ○ Respond for mad cow disease in cattle, scrapie in sheep, Cretzfeldt-Jakob disease in human ○ PrP (or PrPc): normal protein in brain (which consist predominantly of alpha helices) ○ PrPsc: under certain condition, the protein misfolding into this contagious infectious form (which consist predominantly of β-pleated sheets, they tend to bind with other β-pleated sheets -> forming aggregates) ○ Interacting PrP with PrPsc can convert the latter into PrPsc, initiating a domino effect in which more and more of the brain protein convert to the disease-causing form. 15  Câu lạc bộ USTH Learning Support trân trọng giới thiệu CHAPTER 8: NUCLEOTIDES AND NUCLEIC ACID 8.1 Some Basics 8.2 Nucleic Acid Structure 8.3 Nucleic Acid Chemistry 8.4 Other Functions of Nucleotides —--------------------------------------------------------------------------------------------- 8.1 Some basics + Nucleotides and Nucleic Acids Have Characteristic Bases and Pentoses. - Nucleotides characteristic components: (1) a nitrogenous (nitrogen-containing) base (2) A pentose (3) a phosphate - The molecule without the phosphate group is called a nucleoside. - The formula above shows the structure of ribonucleotide. - In deoxyribonucleotides, the -OH group on 2’C is replaced by -H. 16  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - The picture above shows the rule of numbering. - A and G are the purine bases in DNA and RNA. - C and T are the pyrimidines in DNA, U and C are the pyrimidines in RNA. + In solution: 17  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - In solution, the straight-chain (aldehyde) and ring (β-furanose) forms of free ribose are in equilibrium. - RNA contains only the ring form, β-D-ribofuranose. - Deoxyribose undergoes a similar interconversion in solution, but in DNA exists solely as β-2′-deoxy-D-ribofuranose. - Ribofuranose rings in nucleotides can exist in four different puckered conformations. In all cases, four of the five atoms are in a single plane and the fifth atom (C-2′ or 18  Câu lạc bộ USTH Learning Support trân trọng giới thiệu C-3′) is on either the same (endo) or the opposite (exo) side of the plane relative to the C-5′ atom. + Deoxyribonucleotides and ribonucleotides: + Some minor purines and pyrimidines bases are shown as nucleosides: - Minor bases of DNA: 5-Methylcytosine: Occurs in the DNA of animals and higher plants. N6-methyladenosine in bacterial DNA. 19  Câu lạc bộ USTH Learning Support trân trọng giới thiệu N2-Methylguanosine. 5-hydroxymethyl cytidine in the DNA of animals and of bacteria infected with certain bacteriophages. * Roles: - Altered or unusual bases in DNA molecules often have roles in regulating or protecting genetic information. + Some adenosine monophosphates: - Cells also contain nucleotides with PO4- group in a position other than on 5’C. + Phosphodiester Bonds Link Successive Nucleotides in Nucleic Acids. - The 5’ phosphate group of one nucleotide unit is joined to the 3’ hydroxyl group of the next nucleotide, creating a phosphodiester linkage. - RNA is hydrolyzed rapidly under alkaline conditions, but DNA is not; the 2’- hydroxyl groups in RNA (absent in DNA) are directly involved in the process. 20  Câu lạc bộ USTH Learning Support trân trọng giới thiệu + The Properties of Nucleotide Bases Affect the Three Dimensional Structure of Nucleic Acids - Electron delocalization among atoms in the ring gives most of the bonds partial double-bond character. - Tautomeric form of U: Lactam prodominate at pH = 7.0 pH decrease=> Other forms become more popular. Other free pyrimidines and free purines also have tautomeric forms, but they are rarely encountered. - All nucleotide bases absorb UV light, and nucleic acids are characterized by a strong absorption at wavelengths near 260 nm. - The spectra of corresponding ribonucleotides and deoxyribonucleotides are identical. - Functional groups of purines and pyrimidines are: Ring nitrogen, carbonyl group, and exocyclic amino. 8.2. Nucleic Acid Structure + DNA Is a Double Helix That Stores Genetic Information - Watson-Crick model for the structure of DNA. The original model proposed by Watson and Crick had 10 base pairs, or 34 Å (3.4 nm), per turn of the helix. - In aqueous solution the structure differs slightly from that in fibers, having 10.5 base pairs, or 36 Å (3.6 nm), per helical turn. - The pairing of the two strands creates a major groove and a minor groove on the surface of the duplex. 21  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - Two antiparallel strands are not identical but complementary. - Purines have two stable conformations with respect to deoxyribose called sync and anti. - Pyrimidines are generally restricted to the anti-conformation because of steric interference between the sugar and the carbonyl oxygen at C-2 of the pyrimidine. + DNA can occur in different 3-D structures. 22  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - Whether A-DNA occurs in cells is uncertain, but the evidence for some short stretches (tracts) of Z-DNA in both bacteria and eukaryotes. - Z-DNA may play a role ( not yet identified) in regulating the expression of some gene or genetics recombinant. + Certain DNA sequences adopt unusual structures: - Palindrome: - Minor repeat: 23  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - Palindromic DNA (or RNA) sequences can form alternative structures with intrastrand base pairing. When only a single DNA (or RNA) strand is involved, the structure is called a hairpin. - When both strands of a duplex DNA are involved, it is called a cruciform - Roles: Site recognized by many sequence-specific DNA binding proteins arranged as palindromes. 24  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - DNA structures contain three or four DNA strands. Base-pairing patterns in one well-characterized form of triplex DNA. Polypirimidines and polypurines that can form triple helices are found within the regions involved in the regulation of expression in some eukaryotic genes. In principle, triple DNA synthesized can disrupt gene expression => Controlling cellular metabolism, applied in medical and agricultural sectors. + Messenger RNAs Code for Polypeptide Chains - Monocistronic: - Polycistronic: + Many RNAs Have More Complex Three-Dimensional Structures: - Secondary structure of RNAs. The paired regions generally have an A-form right-handed helix, as shown for a hairpin. 25  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - Base-paired helical structures in an RNA. The blue dots indicate non-WatsonCrick G=U base pairs that G=U base pairs are allowed only when presynthesized strands of RNA fold up or anneal with each other, no RNA polymerases that insert a U opposite a template G, or vice versa, during RNA synthesis. 8.3 Nucleic Acid Chemistry + Double-Helical DNA and RNA Can Be Denatured - Reversible denaturation and annealing (renaturation) of DNA. 26  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - t(m): The temperature at the midpoint of the transition, is the melting point. 27  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - Heat denaturation of DNA: t(m) and %G+C in total nucleotides are in a proportional relationship, higher t(m), larger %G+C and vice versa. 28  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - Partially denatured DNA: The regions that denature are highly reproducible and are rich in A=T base pairs. + Nucleic Acids from Different Species Can Form Hybrids - Two DNA samples to be compared are completely denatured by heating. - When the two solutions are mixed and slowly cooled, DNA strands of each sample associate with their normal complementary partner and anneal to form duplexes. - If the two DNAs have significant sequence similarity, they also tend to form partial duplexes or hybrids with each other + Nucleotides and Nucleic Acids Undergo Nonenzymatic Transformations 29 Câu lạc bộ USTH Learning Support trân trọng giới thiệu 30  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - Formation of pyrimidine dimers induced by UV light The formation of a cyclobutane pyrimidine dimer introduces a bend or kink into the DNA. Chemical agents that cause DNA damage: Precursors of nitrous acid, which promotes deamination reactions. Alkylating agents can alter certain bases of DNA. - Some Bases of DNA Are Methylated A defense mechanism that helps the cell to distinguish its DNA from foreign DNA. Repairs mismatched base pairs formed during DNA replication. + The Sequences of Long DNA Strands Can Be Determined (in a separate document) 8.4. Other Functions of Nucleotides + Nucleotides Carry Chemical Energy in Cells (NMPs, NDPs, and NTPs) + Adenine Nucleotides Are Components of Many Enzyme Cofactors (CoenzymeA, NAD+, FAD) + Some Nucleotides Are Regulatory Molecules (cAMP, cGMP,ppGpp) 31  Câu lạc bộ USTH Learning Support trân trọng giới thiệu CARBOHYDRATE 1. Key concept: - Chiral carbon (C*/ 𝐶α) atom is one contains four different attachments. A carbon atom with two or more of the same attachment, such as two or three hydrogen atoms, is not a chiral carbon atom. If they have 2 attachments symmetric – similar 🡪 This is achiral carbon, not chiral carbon. - Carbonyl (C=O) at C1 🡪 Aldehyde (R-CHO) 🡪 Called Aldose Carbonyl (C=O) at C2 🡪 Ketone (R-O-R’) 🡪 Called Ketose ⇨ Fehling’s test (CuSO4.5H2O + NaOH): Detect what kind of sugar: Aldehyde: Red α -hydroxyl – ketone: Red Aldehyde and α -hydroxyl – ketone are absent: Blue (CuSO4) - α: Hydroxyl (-OH) group below the surface of the ring (anomers) β: Hydroxyl (-OH) group above the surface of the ring (anomers) - L - configuration: hydroxyl (-OH) group of the last 𝐶α is left side the linear structure (Fischer projection) D - configuration: hydroxyl (-OH) group of the last 𝐶α is right side the linear structure (Fischer projection) 32  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - - 2. Carbohydrate: Classification Member Monosaccharides 1 sugar containing 3,4,5,6,7 carbon Glucose (Glc) atoms. Fructose (Fru) Disaccharides 2 monosaccharides linked by Sucrose 🡪 Glc + Fru (sugar cane/ glycosidic bonds sugar beets) Latose 🡪 Gal + Glc (milk) Maltose 🡪 Glc + Glc (Starch, glycogen) Oligosaccharides 3 – 20 monosaccharides 🡺 A saccharide polymer but containing a small number of monosaccharides Polysaccharides > 20 monosaccharides 33  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - Enzyme: Sucrose Sucrase Lactose Lactase Maltose Maltase Glycogen, Strach (Amylose, Amylpectin) α – amylose Cellulose Cellulase Chitin Chitinase 3. Monosaccharide: a) Properties: - Colorless - Crystalline solids - Freely soluble in water but insoluble in nonpolar solvents - Sweet taste b) Function: - Building blocks for bigger molecules - Energy source - Regulation - Signaling - Monosacchairde derivatives: Amino sugar formation Phosphate ester formation Oxidation to acidic sugars Glycoside formation Reduction to sugar alcohols 34  Câu lạc bộ USTH Learning Support trân trọng giới thiệu c) Fischer projection and Haworth projection Fischer projection Haworth projection - Vertical lines are away from the - The cyclic form of cacbohydrate viewers – Dashes in the normal - Furanose – cyclic hemiketal: 5C + 1O formula - Horizontal lines are towards the viewers – Wedges in the normal formula - Pyranose – cyclic hemicetal: 6C + 1O - Ex: - From Fischer projection to Haworth projection: Up – left Down – right Anomeric carbon become C=O at C1 35  Câu lạc bộ USTH Learning Support trân trọng giới thiệu 4. Disaccharide: - In sugar cane, sugar beets (sucrose); milk (lactose); starch and glycogen (maltose) a) Glycosidic bond: - Two monosaccharide units can be joined together by glycosidic bond (ring strucrure – anomers) If not ring structure, linkage is similar but different name O -linked and N - linked - There are 2 type: Linkage by Oxygen 🡪 O – glycosidic bond / Glycosidic bond Linkage by –NH– 🡪 N – glycosidic bond b) Condensation reaction or a dehydration synthesis: - Disaccharides are made join via a condensation reaction and a dehyfration synthesis - Sucrose (sugar cane/ sugar beets) 🡪 α – D – Glc + β – D – Fru (1-2 glycosidic bond) - Latose 🡪 β – D – Gal + α – D – Glc (1-4 glycosidic bond) - Maltose (Starch and glycogen) 🡪 α – D – Glc + α – D – Glc (1-4 glycosidic bond) ⇨ Most monosaccharides join by 1-4 glycosidic bond, but some may join by 1-2 glycosidic bond (Ex: Sucrose) c) Relative sweetness in human taste tests: - Increasing sweetness: Gal 🡪 Glc 🡪 Sucrose 🡪 Fru 🡪 Aspartame 🡪 Sucralose 36  Câu lạc bộ USTH Learning Support trân trọng giới thiệu 5. Oligosaccharides: - In cabbage, brussels sprouts, broccoli, sweet potato, and asparagus - Function: Play important role in cell recognition Energy storage in plants Enzyme catalyzes the first reaction in N-glycosylation pathway 37  Câu lạc bộ USTH Learning Support trân trọng giới thiệu 6. Polysaccharide: a) Homopolysaccharides: - Polymers contain a single type of monosaccharide Starch Glycogen Cellulose Chitin Plants cell Animal cells Plant cell wall Exoskeleton (tissues, muscle, liver) Monosaccharides Glucose N-acetylglucoamine (NAG or GlcNAc) Linkage - Amylose (20%) ~ 100 - Linear chains: link - Unbranched: - Unbranched: glucose units - by α − thousands of glucose thousands of GlcNAc unbranched: link by 1,4-glycosidic bonds unit: link by β unit: link by β α −1,4-glycosidic -1,4-glycosidic -1,4-glycosidic bonds bonds - Branched: link by bonds α −1,6-glycosidic - Amylopectin (80%) ~ bonds - Fibrils 🡪 Parallel 100,000 units - chains by hydrogen branched: link by bonds linkages α −1,6-glycosidic ⇨ Glycogen a highly bonds branched molecule (1/10 unit) ⇨ Straight chains by β linkage 🡪 High ⇨ Strach a lower ⇨ Having spiral form, tensibe strength 🡪 branched molecule heavily branched 🡪 β is allow very (1/30 unit) Easy to breakdown long form, straight for glycosis chains ⇨ Glycogen released more Glucose energy than starch Enzyme - α – amylose - Cellulase Chitinase Function - Energy strorage in - Most abundant - Cell wall plants - Cell wall in plants energy storage in insects, fungi, animals and some crustaceans b) Heteropolysaccharides: - Polymers containing more than one type of monosaccharide (Ex: Peptidoglycan in bacterial cell wall) 38  Câu lạc bộ USTH Learning Support trân trọng giới thiệu 7. Glycoconjugate: - Consists of a small number of carbohydrate units (oligosaccharides) covalently attached to a core protein. Peptidoglycans Proteoglycans Glycoprotein Found in Gram-Positive bacteria cell wall (thick peptidoglycan – Purple) and Gram-Negative cell wall (thin peptidoglycans – Pink) Units N-acetylglucosamine + Glycosaminoglycans N-acetylmuramic acid (GAG) Linkage β (1🡪4) - Sulfate-free: - The hydroxyl N-acetylglucosamine and Hyaluronic acid oxygen atom in N-acetylmuramic acid 🡪 GlcA + GlcNAc (link by the side chain of β - 1,3 – glycosidic bonds) Ser or Thr 🡪 O – linkage - Sulfate: Chondroitin sulfate 🡪 - The amide GlcA + nitrogen atom in GalNAc-4-Sulfate ((link the side chain of by β - 1,3 – glycosidic Asn 🡪 N - bonds) linkage Dermatan sulfate 🡪 IdoA + GalNAc-4-Sulfate ((link by β - 1,3 – glycosidic bonds) Keratan sulfate 🡪 D-Gal + GalNAc-4-Sulfate (link by β -1,4 – glycosidic bonds) Heparan sulfate/ Heparin 🡪 L-IdoA-2-sulfate + N-sulfo-GlcNAc-6-sulfat e (link by β -1,4 – glycosidic bonds) 39  Câu lạc bộ USTH Learning Support trân trọng giới thiệu LIPIDS 1. Functions: - Energy storage - Major structural elements of biological membranes - Electron carriers - Light-absorbing pigments - Hydrophobic anchors for proteins - Emulsifying agents - Hormones - Intracellular messengers - Enzyme cofactors - “Chaperones” 2. Classifications: - Saponification: Triglyceride + 3KOH or 3 NaOH 🡪 Glycerol + Carboxylate salts of fatty acids (soaps) 40  Câu lạc bộ USTH Learning Support trân trọng giới thiệu 3. Fatty acids/ FA; a) Stucture of fatty acid: - Polar head: Carboxylic acid (COO-) 🡪 Hydrophilic - Nonpolar tail: Hydrocarbon 🡪 Hydrophobic 🡺 Fatty acid micelles or bilayer (phospholipids /cell membrane): Always tail inside and head outside. 🡺 Micelles are important in the transport of insoluble lipids in the blood, ans in the actions of soaps. b) Typical saturates and unsaturated fatty acids: 𝐶,𝐷 - Name numerical symbol: A: B (∆ ) A: The number of Carbon atom B: The number of double bond ∆: Position of double bond C and D 9,12 Ex: 18:2 (∆ ): Have 18 C and 2 double at (C9 = C10 and C12 = C13) 41  Câu lạc bộ USTH Learning Support trân trọng giới thiệu C2 (From Head - COOH 🡪 Tail ): α Last C (From Head - COOH 🡪 Tail ): ω - Special saturated and unsaturated fatty acid: Myristic acid 14:0 Saturated (only single bond in Palmitic acid 16:0 hydrocarbon tail) Stearic acid 18:0 Arachidic 20:0 Monounsaturated Palmitoleic acid 7 16:1 (∆ ) (only 1 double bond in hydrocarbon tail) Oleic acid 9 18:1 (∆ ) Linoleic acid 9,12 18: 2 (∆ ) Polyunsaturated (many double bond in α – Linolenic acid 9,12, 15 18: 3 (∆ ) hydrocarbon tail) (ALA) – 1 of 3 main omega-3 fatty acids Arachidonic acid 5,8, 11, 14 20: 4 (∆ ) Eicosapentaenoic acid 3,6, 9, 12,15 20: 5 (∆ ) (EPA) – 1 of 3 main omega-3 fatty acids Docosahexaenoic acid 4,7, 10, 13,16,19 22: 6 (∆ ) (DHA) –1 of 3 main omega-3 fatty acids c) Melting points: ▪ Saturated fatty acid > Unsaturated fatty acid 🡺 The more suface area, the more melting point 🡪 (C13 > C12) ▪ Fatty acid > Methyl fatty acid Ex: Melting point: Lauric acid (12:0): 44oC > Methyl laurate (12:0): 5 oC d) Properties of fats and oils: - Fats: Form of solid because it contains a high proportions of saturated fatty acids - Oils: Form of liquid because it contains a high proportions of unsaturated fatty acids 42  Câu lạc bộ USTH Learning Support trân trọng giới thiệu 4. Triacylglycerols/ Triglycerides/ fats/ TG: a) Classification of trigacyglycerols: - A simple triacylglycerol – same fatty acids - A mixed triacylglycerol – different fatty acids b) Function: - Energy storage (storage lipids – neutral) c) Properties: Insoluble and water repellent 43  Câu lạc bộ USTH Learning Support trân trọng giới thiệu d) Reaction: - Hydrolysis: Triglycerides can be broken apart by digestive enzyme called lipases → + TG + 3H2O 𝐻 𝑜𝑟 𝑙𝑖𝑝𝑎𝑠𝑒 Glycerol + FA1 + FA2 + FA3 - Hydrogenation and Partial Hydrogenation: → TG with unsaturate FA + zH2 𝑁𝑖 TG with saturated FA (with z = the number of the double bond) 5. Glycerophospholipids/ Phosphoglycerides/ Phospholipids: a) Classification of phospholipid: - Ester lipids: only ester linkage between glycerol and fatty acids ▪ In cephalins, lecithins, cephalins, mitochondria, heart 44  Câu lạc bộ USTH Learning Support trân trọng giới thiệu - Ether lipids: one of the two acyl chains is attached to glycerol in ether linkage ▪ In brains, heart, muscle, blood flow, basophil ⇨ Ester more reactive than ether 🡪 Ester less stable than ether ⇨ Ether lipids 🡪 Cell membranes of archaea 🡪 Hyperthermophilles Otherwise 🡪 Ester lipids 🡪 Cell membranes of bacteria/ eukaraya b) Function: - Signaling messengers 45  Câu lạc bộ USTH Learning Support trân trọng giới thiệu 6. Spingolipids: - Spingolipids are complex lipids that contain sphingosine instead of glycerol a) Classification of sphingolipids: - Spingomyelin (Phosphoric acid + Choline) ▪ In myelin sheath (neuron) - Gangliosides (Monosaccharide/ Disacchide/ Oligosaccharide) ▪ In gray matter, white matter (brain); Blood antigen 7. Steroids: a) Structure of steroids: - Ring system b) Cholesterol: - The most abundant steroid in the body ▪ An essential component of cell membranes; ▪ A precursor for other steroids, such as the bile salts, sex hormones, vitamin D, and the adrenocortical hormones 46  Câu lạc bộ USTH Learning Support trân trọng giới thiệu c) Steroid: 🡺 Plant sterol - Fungi, yeast sterol, and Cholesterol – Vitamin D are almost similar except B ring - Fat-soluble vitamins: D, E, A, K d) Hormones: - Adrenocorticoid hormones (in Adrenal glands and Kidneys) - Sex hormones: Estrogen (Woman): C18H24O2 Testosterone (Man): C19H28O2 e) Bile salts: (CONNECT WITH DIGESTION AND METABOLISM) - A yellowish-brown or green fluid produced in the liver and stored in the ball bladder - For emulsification, digestion and absorption of dietary lipids 47  Câu lạc bộ USTH Learning Support trân trọng giới thiệu 8. Membrane structure and function: a) Cell membrane structure: ⇨ Majority of the cell membrane is phosphatidylcholine. b) Function of cell membrane: - Protection - Transportation - Signal transduction - Energy storage c) Properties of cell membrane: - Impermeable to most polar or charged solutes, but permeable to nonpolar compounds; - Unsaturated fatty acid chains that increase the flexibility or fluidity of the membrane - Asymmetric; polar 48  Câu lạc bộ USTH Learning Support trân trọng giới thiệu d) Cell membrane: 49  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Chapter 5 – Metabolism (2) 1. Citric acid cycle: Convert acetyl group derived from the carb, fatty acids, and amino acids à CO2, NADH, FADH2, GTP Glycolysis happens in the cytoplasm, leading to the formation of two molecules of pyruvate (per one molecule of glucose). In mitochondria matrix: pyruvates are converted into acetyl-coA (coenzyme A) -> The link between glycolysis and citric cycle (Kerb’s cycle or tricarboxylic acid cycle - TCA) Acetyl-coA = acetate (2 carbon atoms) + enzyme portion. A carrier of the acyl group, an energy-rich compound 50  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Acetyl-coA formation: pyruvate (2C) thoát 1 CO2, gắn thêm coA à Acetyl coA (2C + enzyme) Chú ý vào 2 phản ứng thoát CO2 liên tiếp, có sự tham gia của NAD+ và NADH ○ NAD+ (Nicotinamide Adenine Dinucleotide) ○ NADH (Nicotinamide Adenine Dinucleotide Phosphate) Net effect: ○ 1 pyruvate 3CO2 ○ 1 NAD -> 4NADH + H (12ATP) ○ 1 FAD -> 1FADH2 (2ATP) ○ 1 GDP + P -> GTP (= 1ATP) ○ Tổng = 15 ATP 2. Regulation of Citric acid cycle The ability to regulate the cycle keeps the cell in a stable state, and this function is maintained by three mechanisms: ○ The availability of substrates. ○ Inhibition of the products formed. ○ Inhibition of enzymes through allosteric feedback. -> Through Pyruvate dehydrogenase complex (PHD complex) PHD complex: the mitochondrial matrix multienzyme complex, plays an important role in energy homeostasis in the heart by providing the link between glycolysis and the tricarboxylic 51  Câu lạc bộ USTH Learning Support trân trọng giới thiệu acid (TCA) cycle. In TCA cycle, PDH complex catalyzes the conversion of pyruvate into acetyl-CoA. * In anaerobic bacteria: Incomplete TCA cycle. 3. Electron-transport chain (respiration chain) Mitochondria are double-membraned organelles that harness most of the energy that cells need. Nearly all of this energy comes from reactions that take place at the inner mitochondria membrane. ATP synthase (complex V - Chemiosmosis is the movement of ions across a semipermeable membrane-bound structure, down their electrochemical gradient). Example: formation of ATP by the movement of hydrogen ions (H+) across a membrane during cellular respiration or photosynthesis. -> Use proton gradient to the synthesis of ATP: ADP + Pi -> ATP Electron transport chain: 4 complexes, establishing this gradient and maintaining it. 52  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Electron transport chain gồm có: ○ Complex I, III, IV directly pump protons from the matrix to the inner membrane space (Ngược chiều pump của ATP synthase) ○ Complex II not directly pump protons, but it promotes proton pumping in complex III and IV. Complex I – NADH-coenzyme Q Oxidoreductase Complex II – Succinate-coenzyme Q Oxidoreductase Succinate + CoQ -> Fumarate + CoQH2 Complex III – Coenzyme Q – cytochrome c Oxidoreductase QH2 + 2 Cyt Cox + 2H+ (matrix) -> Q + 2 Cyt red + 4H+ inner membrane space Complex IV – Cytochrome c Oxidase 4 Cyt c red + 8H+ (matrix) + O2 -> 4 Cyt c ox + 2H2O +4H+ (inner membrane space) 53  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Chapter 6 – Basic methods I. Safety rules & basic tools 1. Micropipette Volume range First stop: aspirate the liquid into the pipette tip in the set volume; Second stop: liquid dispensed 2. Serial dilution: * Serial dilutions: Usually the dilution factor at each step is constant. 54  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Để làm loãng 10 lần (giảm nồng độ đi 10): Lấy x chất tan + 9x dung môi (thường là H2O) Các bước sau làm tương tự. 3. Centrifugation: Separate particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed. Phải đặt các mẫu cân bằng (đối xứng với nhau trong máy quay ly tâm) 4. pH Buffer: a solution that can resist pH change upon the addition of an acidic or basic. Đo pH: Calibrating: using distilled water 5. Spectrophometer – Máy quang phổ 55  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Máy quang phổ đo độ hấp thụ (Absorbance) của sample. Sample được đặt trong cuvette có Opaque sides và transparent sides, phải đặt transparent sides trên đường đi của tia sáng. II. Working with DNA 1. Agarose gel Electrophoresis: DNA is negatively charged, moving from wells towards the positive pole. Separate DNA samples based on mass 2. PCR: to amplify a fragment of DNA Denaturing: xấp xỉ nhiệt độ sôi của nước (khoảng 95 độ C -> tách mạch DNA) Annealing: primers bind to template Extension. 3. DNA isolation Organic extraction 56  Câu lạc bộ USTH Learning Support trân trọng giới thiệu Magnetic separation 4. RNA isolation: 5. Quantitative determination of nucleic acids Principle: Beer Lamberts’ Law which states that the concentration of the sample is directly proportional to the absorbance of light done by the sample III. Working with proteins 1. Measure concentration Bradford Assay: ○ Principle: the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. ○ After adding protein, samples are taken to spectrophometer to determine the absorbance of UV light => concentration. BCA protein determination: ○ Sodium salt of bicinchoninic acid reacts with the cuprous ion generated by the biuret reaction under alkaline conditions. The bicinchoninic acid cuprous complex forms a deep blue color that is read at 562 nm. 2. Dialysis – lọc The process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, such as dialysis tubing Nguyên lý: Solvent moves from lower concentration -> higher 57 Câu lạc bộ USTH Learning Support trân trọng giới thiệu 58