Proteins, Amino Acids and Peptide Bonds

• Sugars, amino acids, and nucleotides are building blocks for small molecules
• Proteins, polysaccharides, and nucleic acids are macromolecules
• Protein structure and amino acid composition are key

Amino Acid Fundamentals

• There are 20 different amino acids in proteins
• Each amino acid has an amino group, a carboxylate group, and a different side chain
• Proteins have an L-stereoisomer configuration
• Peptide bonds are created through condensation reactions, which involve the removal of H2O between amino acids
• Hydrolysis reactions attack the C=O group and regenerate carboxylic acid and an amino group

Amino Acid Classification

• Amino acids are classified by polarity, charge, and hydrogen bonding ability

Amino Acid Categories

• Non-polar amino acids: Ala, Val, Leu, Ile, Met, Phe
• They consist of only C-C and C-H bonds
• Hydrocarbons are non-polar and hydrophobic
• Moderately non-polar amino acids: Gly, Cys, Pro, Tyr, Trp
• Polar uncharged amino acids: Ser, Thr, Asn, Gln
• Uncharged in solutions with pH 7
• Good HB donors or acceptors
• Positively charged amino acids: His, Lys, Arg
• Contain weak bases that gain H+
• Positively charged in aqueous solutions at neutral/physiological pH
• Negatively charged amino acids: Asp, Glu
• Have carboxylic acid groups that lose H+ at neutral pH, so they are negative

Core Chemical Principles

• Polarity is based on electronegativity differences (O > N > S > C > H)
• Polarity affects hydrogen bonding capabilities
• Atoms with similar electronegativity are non-polar
• Different electronegativity leads to unequal sharing, unbalanced charges, and polar bonds

Acid-Base Properties

• Some groups will protonate (gain) or deprotonate (lose) H+ depending on the availability of H+ in a solution, which functions at physiological pH (7.0-7.4), where normal biochemical processes occur
• The Henderson-Hasselbalch equation is used for ionization states: pH = pKa + log(deprotonated/protonated)
• Find pKa for the study group
• Input into the Henderson-Hasselbalch equation

Hydrogen Bonding Capabilities

• Electrostatic attractions between a H-bond donor and an acceptor are key
• Acceptor: electronegative atom with an available lone pair of electrons
• Donor: Hydrogen atom covalently bonded to another electronegative atom
• Molecules stick closely together, but are not permanently linked and are directional
• Charge states occur at physiological pH

Peptide Bonds

• Charged States at Neutral pH: Individual amino acids are zwitterions at neutral pH
• In a peptide chain, alpha-amino and alpha-carboxylate groups are linked as uncharged amide bonds

Ionizable Side Chains

• Seven amino acids with ionizable side chains can gain or lose H+ based on pH: Aspartate, Glutamate, Cysteine, Tyrosine, Arginine, Lysine, Histidine
• Asp, Glu, Tyr, and Cys: neutral when protonated and negative when deprotonated
• His, Lys, Arg: neutral when deprotonated and positive when protonated

Structures and Charges

• Protonation/Deprotonation: Structures and charges change with pH (e.g., Glutamate, Histidine)
• pKa Values: Specific pKa values for each amino acid's functional groups
• pKa tells you where in the pH scale a group undergoes deprotonation

Charge Calculations

• Calculating the ionization state of a functional group based on pH and pka
• pH is one unit or more below the Pka, the group is fully protonated
• pH is one unit or more higher than pKa, the group is fully deprotonated
• pH is equal to pKa, the group is 50% deprotonated and 50% protonated
• pH is less than one unit away from pKa, use a calculation to determine the exact state
• Charge of an ionized group depends on the functional group
• O or S is neutral when protonated and negative when deprotonated
• N is positive when protonated and neutral when deprotonated

Calculating Partial Charges

• Steps:
• Calculate the ratio of [Dep]/[Pro]
• Calculate [Dep] and [Pro] fractions
• Ratio: [Dep]/[Pro]= a/1-a
• Use the charges associated with each fraction to find out the overall charge

Amino Acid Analysis

• Two processes to determine protein structure:

1. Separation of a mixture
2. Detection of components

• It can be qualitative, quantitative, or preparative

Chromatography Methods

• Partition Chromatography: Separates mixture components.
• Column Chromatography: Identifies by elution volume, concentration measurement, and separation efficiency
• Has stationary and mobile phases
• Polar amino acids spend more time hydrogen bonded to silica and move slowly, non-polar amino acids spend more time in solvent and move almost as fast as solvent

Thin Layer Chromatography (TLC)

• Overview: Silica gel is spread on a plastic sheet (stationary phase) for TLC. Samples are applied near the lower edge and developed in a non-polar solvent
• Solvent Front & RF Values:
• The solvent front is the highest point reached by the solvent
• RF (Relative Mobility) value = (Distance traveled by amino acid) / (Distance traveled by solvent front)
• Polar amino acids have low RF values; non-polar amino acids have high RF values

Detection of Amino Acids

• Amino acids are colorless
• They can be detected by using ninhydrin (purple for primary/secondary amines, yellow for proline) or fluorescamine (yellow fluorescence under UV light)

Ion Exchange Chromatography

• Separates amino acids/proteins based on net charge using charged resins
• [Na+] concentration influences binding affinity
• The elution volume is the volume of buffer required to elute a specific amino acid
• Standards: Compare elution volumes with standards like Alanine (Ala) or Leucine (Leu)
• Charge Characteristics: Net charge at pH 7 influenced by acidic (Asp, Glu) and basic (His, Lys, Arg) side chains
• relative number of Asp + Glu (negative) versus His + Lys + Arg (positive)

Affinity Chromatography

• Uses ligands covalently attached to beads
• Proteins with a high affinity bind to ligands; eluted by adding salt/competing ligand
• Genetic engineering adds affinity tags (His-tags, GST, Protein A) for purification
• Examples of common tags:
• (His)6 (0.8 kDa, binds Ni2+)
• Glutathione (26 kDa, binds Glutathione)

Immobilized Metal Affinity Chromatography (IMAC)

• Utilizes metal ions (e.g., Ni2+, Co2+) binding to histidine residues in proteins
• Column consists of chelating resin containing Ni2+
• Elution: Adding imidazole releases the protein from the column

Size-Based Separation Techniques

• Gel Filtration Chromatography: Separates proteins by size using polymeric gel beads
• Smaller molecules pass through water-filled pores, so larger molecules are excluded, meaning larger proteins elute first
• Applications: Purify proteins, estimate molar mass using elution volume

Electrophoresis Techniques

• Basics: Movement of charged molecules in an electric field
• Polyacrylamide Gel Electrophoresis (PAGE):
• Separates proteins using polyacrylamide gels
• Visualize proteins using dyes like Coomassie blue
• SDS-PAGE:
• Proteins treated with SDS, partially unfolds them, separated by size, where smaller peptides migrate faster
• All proteins have a uniform charge per unit size

Isoelectric Focusing

• Separation is based on isoelectric point (pI)
• Proteins migrate to pI (no net charge) in a pH gradient

Two-Dimensional Gel Electrophoresis

• Combines isoelectric focusing and SDS-PAGE
• Analyzes complex samples by charge and size

Practical Applications

• Gel filtration is used to purify proteins and measure molar mass
• Electrophoresis is used to analyze protein mixtures and determine size distribution
• Two-dimensional gels in proteomics is used to identify and quantify proteins and study post-translational modifications

Protein Purification

• Methods: Multiple separation methods (Method 1, Method 2, Method 3) Enzyme Activity:
• Measurement of enzyme presence through substrate conversion
• Formula: Enzyme Activity = Total Units of Enzyme
• 1 enzyme unit = amount of enzyme that convert 1 umol of substrate to product per minute
• Specific Activity:
• Enzyme units per milligram of total protein
• Formula: Specific Activity = Enzyme Activity / Total Protein

Purification Steps

• Outcome of Purification:
• Enzyme Activity decreases
• Total Protein decreases
• Specific Activity increases until it reaches a constant value once pure
• Specific Activity Example: Pure enzyme (10 µmol/min/mg) vs. sample (2 µmol/min/mg) = 20% purity

Polypeptides and Proteins

• Structure: Linear chains of amino acids
• Hierarchy includes primary, secondary, tertiary, and quaternary structures
• Primary: linear sequence
• Secondary: regular repetitive patterns, such as helical sections, that run along short sections
• Tertiary: overall pattern of 3D folding of the whole polypeptide
• Quaternary: Assembly of multiple subunits
• Function: Positioning of key amino acids is critical for functionality

Chemical Reactivity

• Principles: Unbalanced distribution of valence electrons, reactivity from nucleophiles (has a lone pair) and electrophiles
• Reactions:
• Nucleophilic Substitution: Incoming nucleophile displaces a leaving group
• Nucleophilic Addition: Nucleophile adds to a double bond

Amino Acid Sequencing

• Tagging N-Terminus: Using fluorodinitrobenzene for identifying the first amino acid
• Hydrolysis Methods: Acid and base hydrolysis; digestive enzymes (proteases)

Structural Hierarchy in Proteins

• Question: Describe the four levels of protein structure and their significance
• Answer:
• Primary: Linear amino acid sequence
• Secondary: Regular patterns (e.g., helices)
• Tertiary: Overall 3D folding
• Quaternary: Assembly of multiple polypeptides

Nucleophilic Reactions

• Question: Explain the difference between nucleophilic substitution and addition
• Answer:
• Substitution: Nucleophile replaces a leaving group
• Addition: Nucleophile adds to a double bond without displacing a group

Edman Degradation

• Developed by Pehr Edman in 1956
• Determines the amino acid sequence of proteins
• Sequentially removes and identifies N-terminal amino acids
• Improves upon Sanger's method
• Sanger only identifies the N-terminal amino acid once because hydrolysis destroys the remaining polypeptide chain
• Allows for repeated identification of amino acids
• Suitable for sequencing up to 50 amino acids using automated sequencers

Mechanism of Edman Degradation

• The process involves two main steps: Coupling and Cyclization
• Coupling: The N-terminal amino acid is reacted with phenylisothiocyanate (PITC) in the presence of a base, forming a thiourea derivative
• Cyclization: The thiourea derivative is then treated with an acid. This leads to the release of the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative, which can be identified
• The cycle can be repeated to identify subsequent amino acids, allowing for the determination of the entire sequence

Limitations of Edman Degradation

• Limited to short sequences (up to 50 amino acids)
• Not effective for proteins with blocked N-termini or those that contain many similar amino acids
• The efficiency of the reaction decreases with longer peptides
• Due to incomplete reactions and side reactions that can occur
• Automated sequencers have improved the speed and efficiency of Edman Degradation, but the fundamental limitations remain

Trypsin and Chymotrypsin

• Trypsin: Cleaves polypeptides at the carboxyl side of lysine (Lys) and arginine (Arg) residues, producing smaller peptide fragments
• Chymotrypsin: Cleaves at the carboxyl side of aromatic amino acids such as phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr)
• Both enzymes are used to generate oligopeptides of defined sizes for further analysis, such as mass spectrometry

Selective Hydrolysis

• A technique used cut polypeptides at specific locations, yielding a limited number of oligopeptides
• The enzyme trypsin recognizes Arg or Lys side chains, proline (Pro) residues can hinder hydrolysis due to their unique structure
• Chymotrypsin generates characteristic patterns of fragments Analyzed for protein identification

Cyanogen Bromide (CNBr)

• Chemical reagent that cleaves polypeptide chains at methionine (Met) residues, converting Met to homoserine (Hse)
• The reaction occurs on the carboxyl side of Met, breaking the peptide bond and yielding fragments that can be analyzed
• This method is useful for generating specific peptide fragments for sequencing and analysis
• Trypsin: Arg and Lys
• Chymotrypsin: Phe, Trp, and Tyr
• CNBr: Met to Hse

Comparison of Enzymatic and Chemical Cleavage

• Enzymatic cleavage (trypsin, chymotrypsin)
• Specific and can yield predictable patterns of fragments
• Chemical cleavage (CNBr)
• Less specific but can target specific residues
• Both methods can be used in combination for a comprehensive analysis of protein sequences
• The choice of method depends on the specific protein and the desired outcome the sequencing process

Concept of Overlap Method

• The overlap method
• cuts a protein with different enzymes to generate overlapping peptide fragments that can be sequenced individually
• By comparing the sequences of overlapping peptides, the complete amino acid sequence of the original protein can be reconstructed
• Useful for complex proteins with multiple potential cut sites

Overview of the Overlap Method

• Cuts a polypeptide into smaller peptides with different hydrolysis methods, such as trypsin and chymotrypsin
• Trypsin cleaves at the carboxyl side of lysine (K) and arginine (R) residues, chymotrypsin cleaves at the carboxyl side of aromatic amino acids like phenylalanine (F), tryptophan (W), and tyrosine (Y)
• This method allows for the identification of overlapping sequences, which can be aligned to deduce the original polypeptide sequence
• Overlapping sequences help in reconstructing the full polypeptide chain by identifying common amino acids

Practical Application of the Overlap Method

• The overlap method is crucial in protein sequencing, especially when direct sequencing is not feasible

Mass Spectrometry (MS)

• A powerful analytical technique used to identify and quantify proteins by measuring the mass-to-charge ratio of ionized particles
• Tandem mass spectrometry (MS/MS)
• Allows for the fragmentation of peptides
• Provides detailed information about their structure

Steps in Mass Spectrometry

1. Sample hydrolysis: Proteins are digested into peptides using proteases like trypsin
2. First MS (MS-1): Peptides are sorted and selected for further analysis
3. Collision cell: Selected peptides are fragmented at peptide bonds, producing y-type and b-type fragments
4. Second MS (MS-2): The masses of the fragments are measured to deduce the original peptide sequence

Fragmentation Patterns and Sequence Deduction

• Trypsin digestion results in y-type fragments
• Carry a positive charge at the C-terminus due to the presence of K or R residues
• The mass differences between peaks in the mass spectrum correspond to the masses of the amino acids lost during fragmentation, allowing for sequence reconstruction
• For the peptide GASSISYPAR, the mass spectrum peaks can be analyzed to identify the sequence by calculating the mass differences

Levels of Protein Structure

• Proteins have four levels : Primary, secondary, tertiary, and quaternary
• Primary structure : The linear sequence of amino acids in a polypeptide chain
• Secondary structure : Regular patterns such as alpha-helices and beta-strands formed by hydrogen bonding between backbone atoms

Secondary Structure Formation

• Alpha-helices
• Formed by hydrogen bonds between the carbonyl oxygen of one amino acid and the amide hydrogen of another, creating a coiled structure
• Beta-strands
• Formed by hydrogen bonds between adjacent strands, resulting in a sheet-like structure
• The flexibility of the polypeptide chain allows for various conformations due to bond rotation

Conformations vs. Configurations

• Conformations
• Can change without breaking covalent bonds
• Configurations Require bond breaking to change

NCBI and BLAST

• The National Center for Biotechnology Information (NCBI)
• Provides a vast database of biological information, including protein sequences and genomic data
• BLAST (Basic Local Alignment Search Tool)
• Used to compare an input sequence against a database of known sequences to find similarities
• Applications of BLAST
• Can identify homologous proteins, providing insights into evolutionary relationships and functional similarities
• Helps in discovering new proteins by comparing unknown sequences to known sequences in the database

X-ray Diffraction in Protein Structures

• Concept: Measures regular repeating patterns on a molecular scale
• Application: Identifying structural parameters from diffraction patterns in fibrous proteins like α-keratin and β-keratin
• Has major and minor patterns
• The ångstrom unit, 1 Å = 1′10 -10 meter
• Key Contributions:
• Linus Pauling's work on peptide chains and bond lengths

Protein Secondary Structures

• Peptide bond has two resonance forms one with a double bond
• Alpha-amino and -carboxylate are locked in rigid planar peptide bonds
• a helical shape, alpha-c bonds turn in same direction
• in extended shape alpha-c bonds turn in alternate directions
• ɑ-helix: 3.6 amino acids per turn, stabilized by hydrogen bonds
• Distance between each turn of helix is 5.4Å, distance along the helix per amino acid is 1.5Å
• #1 C=O lines up with H-N #5 to form HB
• β-sheet: Alternating orientation of amino acids forming antiparallel and parallel sheets
• Distance between each turn of helix is 7.0A, distance along the helix per amino acid is 3.5Å

Formation of Secondary Structure

• Factors Influencing Structures:
• Amino acids like Ala, Arg, Gln, Glu, His, Leu, Lys, Met, and (Phe) tend to form ɑ-helices
• Trp, Tyr, (Phe), Val, Ile, Thr, Cys prefer β-sheet structures
• Secondary Structure Breakers
• Amino Acids:
• Gly, Pro, Asn, Asp, Ser often disrupt secondary structures
• Have side chains that interfere with secondary structure H-bonds
• 2 breakers in a group of 4 amino acids interrupts the secondary structure

Tertiary and Quaternary Structures

• Tertiary structure
• Overall folding of the polypeptide chain
• Definition Quaternary structure: Assembly of multiple polypeptide subunits
• Examples
• α-keratin: Fully α-helix
• Fibroin: Fully antiparallel β-sheet
• Collagen: Unique triple-helix structure

Denaturation of Proteins

• Causes
• Heat, disruptive solvents, harsh detergents
• Effects
• Loss of function, often irreversible

Protein Folding

• Breaks in secondary structure: Gly, Pro, Asn, Asp, Ser (GPNDS)
• Hydrophobic effect Drives protein folding by enclosing non-polar amino acids in the core

Stability and Interactions

• Van der Waals Forces
• Role in packing amino acids together
• Impact of weak interactions and close contacts

Amino Acid Distribution

• Hydrophobic vs. Hydrophilic:
• Hydrophobic effect Non-polar AAs group together
• Hydrophilic effect Polar AAs form the outer layer and interact with water or ions

3.tertiary Structures

• Protein Folding Patterns:
• Groups with mostly groups of ɑ-helix-forming AAs will fold into an ɑ-helix bundle
  • Non-polar patch or stripe
  • Myoglobin
• β-sheet: 3.6 amino acids per turn, stabilized by hydrogen bonds -Anti-parallel more stable because H-bonds are in straight line
• Importance of amino acid sequence

Protein Stability

• Non-covalent Interactions dictate folding pattern and stability
• Backbone H-bonds stabilize the secondary structure
• Hydrophobic effect, van der Waals interactions
• Dipole-dipole interactions, salt bridges, and H-bonding

Disulfide Bonds

• When pairs of Cys-SH groups react with O2, releasing H20
• Role in Tertiary Structure:
  • Formation and importance outside cells
  • Impact on protein stability

Binding and Catalysis

• Substrate Binding:
  • Enzymes binding to specific target molecules
  • Non-covalent interactions: Hydrophobic patches van der Waals forces and H-bonds
• • Example: Chymotrypsin binding to aromatic amino acids (Phe, Tyr, Trp)

Enzyme Catalysis

• Mechanisms and Speed:
• Factors influencing uncatalyzed reactions
• Specificity of enzyme-substrate interactions

Arrhenius Equation

• Z Collision frequency
• P Probability factor (related to reactant orientation)
• Ea Activation energy
• Fraction of molecules at temperature T that have energy Ea

Enzyme Mechanisms

• Proximity Effect:
  • Enzyme binds substrates in the active sight, increasing likelihood of reaction by putting molecules closer together/Increases Z
• Orientation Effect: -Enzymes holds substrates in correct orientation, aligning reactive groups for reaction/Increases P

Lower Activation Energy (Ea)

Enzymes speed up reactions by:

• Proximity and Orientation Effects: physically bringing reactants closer and aligning
• Chemical Catalysis: Providing an alternative pathway with a lower Ea

Types of Chemical Catalysis

• Nucleophilic Catalysis: Enzyme provides a better nucleophile (Cys-SH,His-N2,Asp-COO)
• Electrophilic Catalysis: Enzyme contains an electrophilic group or prosthetic groups (pyridoxal

Stabilizing TransitonState Enzymes can stabilize the transionstate by

Binding substates in an ideal shape lowering Fa by distorting leading to the transition stat

• Chymotrpisin Mechanism* -Binging Chymontrypsin bins weakly u steam of target amine
• Tartet amine acid (Phey. The, Tip) into binding pocket
• Cadytic Trod:* ASpo22 Statilbes the positive changes on H. 51
• Alp 57:Acys as general base -Seli995: Better mudeophite due to +Chymotrpisin Mechanism* +Oxuaniole Stabilizes transsition state by forming favorable interactions with the transit

Chymotrypsin Catalytic Mechanism

• His-57 Role Acts as a general acid or base or stabilizes the charge on reactive groups

Properties

• Pka close to physiological pH (able to act as acid/base)

Stabilizing TransitonState Enzymes can stabilize the transion state by binging substates in an ideal shape by leading Fa to distorting leading to the transion state.

Mutation Studies

• Asp to Ala 100% normal rate
• Mechansum cycle can repeat ~10 times
• Identity mechanism int Ser toala

Definition

Enzyme assay reaction rate kinetics is mathematical analys of subtrate concentration and enzyme conatration by measuring of produced -