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Life characterized by
o High complexity
o Extraction, transformation, systematic use of energy to create and maintain
structures and do work
o Sense and respond to changes in surrounding
o Self-replication, enough change for evolution
o Living organisms have large # of diff compounds
o Macromolecules highly specific interactions
o Internal structures with defined functions
Hierarchy of biomolecular structure
o Supramolecular complexes are held together by noncovalent bonds
o Cell organelle supramolecular complexes macromolecules monomeric
units
o Chromatin DNA nucleotide
o Plasma membrane protein amino acid
o Cell wall cellulose sugars
Biochemistry: atomic composition, role of carbon
o Biomolecules are carbon based (>50% of cellular mass)
o Common elements
H, N, O, P, S
o Metal ions
E.g. K, Na, Ca, Mg, Zn, Fe
Play role in metabolism
o ~30 elements essential for life
o Humans ~80% water
o C (61.7%)
o N (11%)
o O (9.3%)
o H (5.7%)
E. Coli
o Water (70%)
o Protein (15%)
o DNA (1%)
o RNA (6%)
Biomolecules: Amino-Acids and proteins
o Biomolecules are hydrocarbons with Hs replaced by functional groups
o 20 common amino acids, vary by fxn group
Biomolecules contain unique combo of fxn groups
o Fxn of biomolecules depends on 3D structure
Biochemistry is stereospecific
o Stereoisomers (L and D)
o Enantiomers
o
Enzyme and pathways
o Series of related enzymatically catalyzed reactions form a pathway
o E.g pathways
Metabolic pathways produce energy or valuable materials
Signal transduction pathway transmit information
Chapter 2
-
Non-covalent interactions
o Dipole interactions and H-bonds
Electrostatic interactions b/w uncharged polar molecules
o Hydrogen bonds
b/w neutral groups or peptide bonds
o Ionic (Coulombic) interactions
Electrostatic interactions b/w permanent charged species or b/w ion and
permanent dipole
Attraction or repulsion
o Hydrophobic effect
Ordering of water molecules around non-polar substances
o Van der Waals interactions
Weak interactions b/w all atoms
Attractive (dispersion) and repulsive (steric) component
Any 2 atoms in close proximity
Van der Waals interactions
o
o 2 components
Attractive (London force)
Depend on polarizability
Repulsive force (steric repulsion)
Depend on size of atom
o Attraction/repulsion
Attraction dominates longer distance
0.4-0.7nm (4-7 angstroms)
o 1 ANGSTROM = 0.1 nm! Or 10-10 m
Carbohydrates
o Water is poor solvent for nonpolar substances
Nonpolar gases
Aromatic moieties
Aliphatic chains
o Some polar biomolecules
Glucose, Glycine, Aspartate, Lactate, Glycerol
o Some nonpolar biomolecules
Wax, amphipatic phenylalanine, phosphatidylcholine
Water as a solvent: Ionic molecules Salts
o Highly dielectric constant reduces attraction b/w oppositely charged ions in salt
crystal
Almost no attraction at large (>40 nm) distance
o Strong electrostatic interactions b/w solvated ions and water lowers energy of
system
o Entropy increases
Ordered crystal is dissolved
Water as a solvent: Gas molecules
o Polar gases dissolve better than nonpolar gases
E.g.
Polar: Ammonia, Hydrogen sulfide
Nonpolar: N, O, CO2
o Nonpolar gases chemically converted, or bound to other molecules for biological
transport
E.g. myoglobin
Water as a solvent: Hydrophobic molecules
o Hydrophobic molecules poorly dissolved in water explained by entropy
o Bulky water has little order
High entropy
o Water near hydrophobic solute highly ordered
Low entropy
o Low entropy thermodynamically unfavorable
Hydrophobic solutes have low solubility
o Hydrophobic effect minimizes loss of water entropy
Water as solvent hydrophobic effect
o Association or folding of nonpolar molecules in aq solution
o Is one of the main factors behind
Protein folding
Protein-protein association
Formation of lipid micelles and membranes
Binding of steroid hormones to their receptors
o Does NOT arise b/c of direct attractive force b/w 2 non-polar molecules
In fact, H > 0 for transfer of many nonpolars into benzene from water;
while G < 0 (so dominated by TS)
o Origins of hydrophobic effect
Consider amphipathic lipids in water
Lipid molecules disperse in solution
Nonpolar tail is surrounded by ordered water molecules
Entropy of system decreases
System is now unfavorable
o Nonpolar chains of the amphipathic molecule aggregate
Fewer water molecules are ordered
Released water molecules will be more random, entropy increases
o Aggregation continues
Only polar head groups of ampipathic molecules are exposed
Make energetically favorable H-bonds with water molecules
Water and protein-ligand interactions: H-bonding
o Water is a ligand
o Most water molecules remain unionized, thus pure water has low electrical
conductivity
o Equilibrium strongly to left
o Extent of dissociation depend on temperature
Proton hydration
o Protons do not exist free in solution
They are hydrated forming hydronium H3O+ ions
o Hydronium ions are solvated by nearby water molecules
o Covalent and hydrogen bonds are interchangeable
Allows for fast mobility of protons in water via proton hopping
Water ionization: quantitative treatment
o Concentrations of species involved in chemical equation described by the
equilibrium constant
Keq = [H+]*[OH-]/[H2O]
o Keq can be experimentally determined = 1.8*10-16 M at 25C
o [] = concentration Molarity, M, mol/Liter
o [H2O] = 55.5M determined from water density @ 25C
(1000 g/L) / 18 (g/mol) = 55.5 M
o
The pH scale
o Way to express [H+]
o In neutral solution [H+] = [OH-] and pH = 7
o pH and pOH always add to 14
o pH can be negative ([H+] > 1 M)
o What is pH?
pH = -log [H+]
log[10-7] = 7
Kw = [H+][OH-] = 1*10-14 M2
log[H+] log[OH-] = +14
pH + pOH = 14
o pH depends little on H+ from water
o pH altered by presence of other acids and bases that inc or decr [H+]
Acid release proton [H+] in water
Bases accept proton [H+]
o Acid dissociation in water
HA H+ + A Conjugate pairs
o
o
o
o
o
o
o
o
o
o
o The HH equation
Quantitatively describes the shape of the titration curve
Determine pKa, given pH and molar ratio of HA/A Determine pH, given pKa and molar ratio of HA/A Determine ratio of HA/A- given pH and pKa
Proteins and other Biomolecules
o Amino acids are weak acid/bases
Useful to know their protonation state
Henderson-Hasselbalch Equation
o
o Determine the fraction of histidine group that is protonated at pH = 7.3
pK1 = 1.8 (carboxyl group)
pK2 = 6.0 (imidazole group)
pK3 = 9.2 (amino group)
Ratio vs Percentage
o
HH Equation
o
o The cell cytoplasm is maintained at pH~7.4
o Under normal conditions, lactic acid is at a concentration of 1mM
What are the concentrations of the acid and base forms?
o Condensation/Hydrolysis Rxns
o
o ATP made from energy from proton gradients across membranes of chloroplasts
and mitochondria
o Other condensation reactions
Polymerization of amino acids, nucleic acids, carbohydrates
ALL THESE ARE ENDERGONIC REQUIRE HYDROLYSIS
OF ATP
o Oxidation/Reduction Rxns
Aromatic (3)
Polar, uncharged (5)
(+) charged (3)
(-) charged (2)
Nonpolar aliphatic R-groups
o G, A, P, V, L, I, M
Aromatic R-groups
o F, Y, W
These AA side chains absorb UV light at 270-280 nm
Polar, uncharged R groups
o S, T, C, N, Q
Cysteines can form disulfide bonds covalent bonds
o Acetylation
o Methylation
o Adenylation
o Amino acids with uncharged side chains, e.g. GLYCINE, have 2 pKa values
o For glycine
pKa of -carboxyl = 2.34
pka of -amino = 9.6
It can act as a buffer in two pH regimes
o
o Zwitterions predominate at pH values b/w the pKa values of amino and carboxyl
group
for amino acid without ionizable side chains, the ISOELECTRIC POINT
(equivalence point, pI) is
pI = (pK1 + pK2) / 2
At the pI, net charge = 0
Least soluble point in water
Does not migrate in electric field
o The chemical environment affects pKa values:
-carboxy group is much more acidic than in carboxylic acids
-amino group is slightly less basic than in amines
o Some Amino acids with ionizable side chains, such as the charged amino acids, have
3 pKa values
Take the average of these two pKa values (For Glu = 3.22)
o Example
What is the pI of histidine?
Pk1 = 1.82
pKR = 6.0
pK2 = 9.17
(9.17 + 6)/2 = pI
pI = 7.59
MUST KNOW HOW TO DRAW TITRATION CURVE FOR GIVEN
AMINO ACID AND LABEL THE PKs and pI!
o
o Ribosome can synthesize 100aa in 5 seconds (error rate 1/10,000)
o Amino acid activated by ATP and ribosome catalysis
Polypeptides (covalently linked a-amino acids and possibly:
o Cofactors
general term for functional non-amino acid component
Metal ions, organic molecules
o Coenzymes
Used to designate organic cofactors of enzymes
NAD+ in lactate hydrogenase
o Prosthetic groups
Covalently attached cofactors
Heme in myoglobin
o Other modifications
Proteins size and composition
o Simple proteins contain only amino acids
o Conjugated proteins contain prosthetic groups:
Lipids
Sugars
Metals
Cofactors
o
o Peptide vs Protein (latter are typicaly ~10,000 Da)
o
o Proteins vary in their amino acid composition
o The molecular weight of a simple protein estimated by # of AA it contains
(# of AA) x 110 Da = ~Molecular weight
Note:
Average amino acid mass is 138 Da
However, small amino acids are more common
The average Mw of amino acids used by proteins is = 128 Da
In a polypeptide, H2O is removed due to peptide bond, so average mw
is reduced to 128-18 = 110 Da per amino acid
Peptides and proteins, What we trying to learn?
Sequence and composition?
o How vary b/w individuals and species?
o How related to other proteins?
What is 3d structure?
o How related to fxn?
o How did it find native fold
How achieve its biochemical role?
o How is its biochem role disrupted in disease?
For non-enzymes require a different assay
o General assay to assess protein purity
Gel Electrophoresis
Electric field pulls proteins according to their charge
Gel matrix hinders mobility of proteins according to their size and
shape
o Smaller proteins migrate faster than larger ones
o
red one is cathode (+)
o Polyacrylamide Gel Electrophoresis (PAGE)
Separates biological macromolecules, usually proteins or nucleic acids,
according to their electrophoretic mobility.
o
Protein sequencing
o Primary AA sequence provides
Protein ID
Insight into physico-chemical properties (MW + pI)
Insight into 2, 3 structure (sometimes 4 structure)
Insight into biological function, disease and evolutionary history
o
o Benefits
Only require 10-100 pico-mole of protein
o Drawbacks
Can only sequence ~30 AA
o How to overcome drawback?
Cleave larger proteins into smaller pieces first
E.g. using proteases, or chemical cleavage
Protein Sequencing Protease Enzymes
o
o Electrospray Ionization Mass Spectroscopy (ESI-MS)
o
DNA Sequence analysis
Chapter 4
3D structure of proteins
o Major themes
3D structure determined by 1 sequence
Protein fxn depend on structure Form & Function
Most proteins exist in one or a few # of stable structures
Protein structure is primarily stabilized by non-covalent interactions
Proteins may share common structural features
Protein structures are not static Dynamics is important to function, and some
are completely unstructured
Overview of Protein Structure
o Unlike most organic polymers, protein molecules adopt a specific 3D conformation
o This structure is able to fulfill a specific biological function
o
Overview of Protein Structure
o Native fold is stabilized by a large # of favorable interactions w/I protein
Typically, this is the THERMODYNAMICALLY MOST STABLE
There is a COST IN CONFORMATIONAL ENTROPY of folding the protein
into one specific native fold
Proteins MAY HAVE MORE THAN ONE STABLE CONFORMATION
Important to catalysis (e.g. binding and release of substrate)
Non-covalent interactions are maximized
o Hydrophobic effect (Major component)
Release of water molecules from the structured solvation layer around the
molecule as protein folds increase the net ENTROPY
o Hydrogen bonds
Interactions of N-H and C=O of the peptide bond leads to local regular
structures such as a-helices and B-sheets
o London dispersion
Medium-range weak attraction b/w all atoms contribute significantly to the
stability in interior of protein
o Electrostatic interactions
Long-range strong interactions b/w permanently charged groups
Salt-bridges, esp. buried in hydrophobic environment strongly stabilize the
protein
Structure of the peptide
o Protein structure partially dictated by properties of the peptide bond
o Peptide bond is a resonance hybrid of 2 canonical structures
o Resonance causes peptide bonds
To be less reactive (compared to esters for example)
To be rigid and nearly planar
To exhibit large dipole moment in favored trans configuration
o
The Helix Dipole
o Recall that the peptide bond has a strong dipole moment
Carbonyl O = negative
Amide H = positive
o All peptide bonds in the a-helix have a similar orientation
o Result in a large macroscopic dipole moment
Negatively charged residues often occur near the positive end of the helix
dipole
Beta-sheet
o The backbone is in a more extended conformation
o
o Parallel or Antiparallel orientation of two chains w/I a sheet are possible
o Parallel B-sheets
H-bonded strands run in the SAME DIRECTION
Resulting in bent H-bonds (weaker)
o Antiparallel B-sheets
H-bonded strands run in the OPPOSITE DIRECTIONS
Resulting in linear H-bonds (stronger)
Beta-turns
o B-turns occur frequently whenever strands in B-sheets change the direction
The 180 turn is accomplished over 4 amino acids
o The turn is stabilized by H-bond from carbonyl oxygen to amide proton at the n+3
position
o Proline in position 2 or Glycine in position 3 are common in B-turns
o
Proline Isomers (Cis and Trans)
o Most peptide bonds not involving proline are in the trans configuration (<99.95(
o For peptide bonds involving proline, about 6% are in the cis configuration. Most of
this 6% involves B-turns
o Proline isomerization is catalyzed by proline isomerases
o
Analysis of Secondary Structure Circular Dichroism (CD)
o Spectroscopic technique to quantitatively assess secondary structure content
o
Protein Tertiary Structure
o Tertiary structure
Overall spatial arrangement of atoms in a polypeptide chain or in a protein
o 3 major classes
Globular proteins
Water-soluble proteins
Lipid-soluble membranous proteins
Fibrous proteins
Typically insoluble; made from a single secondary structure
Intrinsically disordered proteins
Lack stabile/ordered structure
o Found with a huge variety of shapes and sizes but some general features are shared
Compact fold
Hydrophobics concentrated at the interior of hydrophilics at surface
Stabilized by many H-bonds and ion-pairs
o While overall structures are often very complex, most proteins are assembled by
linking together common structural features motifs or folds
Protein Motifs (Folds)
o Motif/Fold recognizable folding pattern of 2 or more connected 2ndary structural
elements
o All alpha, All beta, or Mixed alpa/beta
o Motifs can be found as reoccurring structures in numerous proteins
o Most proteins are made of different motifs folded together
o
o Specific connectivity b/w sequential 2ndary elements are favored
o
o Larger and more complex folds may be constructed from smaller simpler motifs
o
o Hierarchy
Class: all , all , /, and +
Each class has many folds
Some are very common, some are unique to only 1 known protein
Protein Domains
o Domain part of a protein that is independently stable
Multiple domains may have independent functions within the entire protein
May be separated by a linker (independent), or closely packed with eachother
o
Fibrous Protein Structure
o Collagen
Gly and Pro-rich left handed helix
Three chains form a right handed triple helix
Higher tensile strength than steel wire
Gly residues facilitate fibril packing
Many triple helices assemble into a collagen fibril
Stabilized by inter-strand H-bonding and crosslinking b/w modified lysines
(unusual)
o 4-Hydroxyproline in Collagen
Forces the proline into a favorable pucker conformation
Offer more hydrogen bonds b/w 3 strands of collagen
The post-translational processing is catalyzed by prolyl-hydroxylase and
requires: a-ketoglutarate, oxygen and ascorbate (vitamin C)
Lack of vitamin C can cause scurvy (degeneration of connective tissue)
o
o Example of B-sheet fibrous protein: Silk Fibroin
Main protein in silk from moths and spiders
Anti-parallel B-sheet structure
Small side chains (Ala- and Gly) allow the close packing
Structure is stabilized by H-bonding w/i sheets
London dispersion interactions b/w sheets
o Protein Kinase A
Tethering of Functionally Coupled Proteins/Domains
o
o Pros
o Cons
No size limits
Well-established
High resolution
o Required steps
Isotope labeling
Purify protein (mg quantities)
Collect NMR data
Assign NMR spectra
Calculate structure
o Pros
Does not require crystals
Information on structure and dynamics
o Cons
Difficult for insoluble membrane proteins
Difficult for larger proteins (>50 kDa)
Determining Protein Structure (Biomolecular CryoEM)
o Electron Cryo-Microscopy (cryoEM)
Imaging technique using electron beam as light source
o Required steps
Purifying the protein (ug quantities)
Collect image data
Calculate 3d reconstruction
Build model into density map
o Pros
Does not require crystals
No size limit (except for small proteins)
o Cons
Technically difficult
Best for large proteins (>200 kDa)