Biochem Ch 4, 5, & 6

• Amine (pKa ~9.5)
• Carboxylic Acid (pKa ~2)

• A molecule that cannot be superimposed on its mirror image
• Are optically active and rotate plane polarized light
• Amino acids (except glycine) are chiral bc bond to the central carbon is different

L-stereoisomers

• Based on the structure of L-glyceraldehyde
• L: (-) Left
• D: (+) Right

• Presents three-dimensional chemical structures in two-dimensions

• Positively charged amine
• Negatively charged carboxyl group

Zero net charge- Zwitterion

• Glycine (G)
• Alanine (A)

• Valine (V)
• Leucine (L)
• Isoleucine (I)
• Methionine (M)

• Met's side chain is very flexible and can adjust to geometrical constrains of other residues
• Often found in amphiphilic helices involved in protein-protein interactions
• Easily (and reversibly) oxidized, so can be used to "protect"

• Phenylalanine (F)
• Tyrosine (Y)
• Tryptophan (W)

• Genetic Disorder characterized by a mutation in the gene for the hepatic enzyme phenylalanine hydroxylase (PAH)
• Can lead to intellectual disability
• Treatment: strict Phe-restricted diet supplemented by other amino acids

• Precursor of serotonin "happiness hormone"
• Serotonin is increased by several antidepressant drugs and sunlight
• Some psychedelics are agonists of this molecule

• Tyr and Trp absorbs UV light at 280 nm and most proteins contain either or both
• Therefore, the protein can be determined based on detection of these
• Both residues are also fluorescent. This can be used to probe conformational changes of unmodified proteins

• Cysteine (C)
• Serine (S)
• Threonine (T)

• Curly vs. Straight Hair
• Keratin contains cysteines and the cross linking provide hair stability and define straight or curly hairs

• Asparagine (N)
• Glutamine (Q)

• Asparagine (N)
• Aspartate (D)
• Glutamate (E)

• Important neurotransmitter (memory)
• MSG is commonly used as taste enhancer

• pH at which a particular molecule carries no net electrical charge
• Determined as an average value

• Lysine (K)
• Arginine (R)

• Neighboring charges
• Nonpolar environments
• Hydrogen bonding
• In enzyme active sites: pKa is often drastically different

• Valine
• Isoleucine
• Threonine

• Selenocysteine has a lower pka and reduction potential than cysteine which makes it suitable in proteins that are involved in antioxidant activity.
• More active than S, present in several metabolic enzymes, mainly reductases and dehydrogenases

Reprogramming the UGA "stop" codon- translational recoding

• Pyrrolysine is encoded in Archaea by another "stop" codon-UGA
• Helped the

• Formed by the condensation of two amino acids accompanied by the release of a water molecule 
• Bond specific to amino acids between the carboxylic acid and amine groups

• Always written N to C
• If one-letter codes, all capital letters
• If three-letter codes, first letter capitalized
• Termini are sometimes specified

• Partial double bond character maintains planarity of peptide (amide) bonds
• Protein backbone rotations are similar to sheets of paper
• Planar because of the orbitals that form
• In linear form side chains are on alternating sides but not extensively found in folded proteins

• Free rotation is limited to the NH-C_alpha (Phi) and C_alpha-CO (Psi)
• Angles around these bonds are known as "dihedral" or "torsion" angles

• If both C_alpha atoms on the same side of the peptide bond- the bonds are Cis
• Too many interactions in the Cis position so not commonly found in nature

• If both the Calpha atoms are on the opposite sides of the peptide bond

• X-Pro
• Due to the ring structure it is ~30% cis

• Side chains extend on opposite sides of the backbone
• Full extended conformation is rare in protein structures
• Easiest semi-realistic geometry on the plane of the paper

• Phi, Psi dihedral coordinates
• Amino acid side chain determines allowed conformations
• The allowed or favored (green) regions also correspond to the main types of secondary structure

Glycine has more surface covered on the graph because there are no side chains

• Translation proceeds N to C
• Carried out by ribosomes

• Forms complex structures incorporating standard and modified amino acids (including d-amino acids)
• Utilizes modular enzyme architecture

Linear sequence of amino acids linked by peptide bonds

• alpha helices, beta sheets, loops & turns
• Defined by local interactions of primary sequence

Protein domains- defined by long range interactions between secondary structural units

Multi-subunit protein complexes- associations between two or more proteins

• Protein primary sequence is determined by DNA and encodes all required information for protein to fold and function properly 

• Sequence of amino acids from N- to C- terminus 
• May include disulfide bonds

Few amino acids linked

Many amino acids linked

Large polypeptide, typically with MW>10,000 Da (g/mole) cutoff

• Common repeating patterns of relative orientation of amino acid residues in a peptide in 3D space stabilized by hydrogen bonds
• The stabilizing hydrogen bonds connect backbone elements of the polypeptide chain

• Alpha helix
• Beta sheet

Stable secondary structural elements

• Predicted and described structures thaat contained planar peptide bonds and were: 
• 1) Sterically allowed and favorable
• 2) Maximized the hydrogen bonding capacity of backbone amines and carbonyl groups

• 3.6 residues per turn
• 5.4 Pitch
• i to i+4 hydrogen bonding pattern: C=O of residue and i and amide N-H of residue i+4
• Phi= -57degrees; Psi= -47degrees
• Hydrogen bond distance= 2.8

• 3.6 residues per turn, encloses 13 atoms in a ring by formation
• 3.6₁₃-helix is more tightly and has 13 residues

• 3 is the residue with 10 turns
• Is more tightly wound helix
• Stabilized by i, i+3 bonds

• Side chains point "out" and "back"
• Very few steric clashes
• Maximized hydrogen bonding of backbone amides

• Electrostatic interaction across a turn of the helix
• Side chains a and d (i and i+3) or a and e (i and i+4) are close in space
• These interactions can stabilize or destabilize the helix

• Are common
• Occur when an amphipathic helix is primarily hydrophilic, but has hydrophobic residues in the i and i+3 positions which allow two helices to interact with each other
• Can be further stabilized by interstrand salt bridges
• Can be parallel or antiparallel; homo- or hetero-dimers

Antiparallel bonds are straight and stronger than parallel

• By large hydrophobic residues 
• They keep large and branched side chains far apart, minimizing steric clash

Alpha helix are more likely to form since they are closer together and form faster

In 3D these are twisted and arrows indicate N to C directionality

• Green florescence protein which allow us to see other proteins in the cell
• Tracking individual neurites (axons and dendrites) labeled by a combination of flurescent proteins

High conformational flexibility and no detectable structure

Any structured region that does not fit standard secondary definitions

• Short secondary structural units that connect more standard units
• Short beta hairpin turns

Longer connecting segments (any number and sequence of amino acids)

• Generally connect antiparallel beta strands 
• Can occur in isolation
• Typically 4-5 amino acids stabilized by a hydrogen bond between residues i and i+3
• Glycines and prolines are often found in beta turns

Stabilized by a hydrogen bond between residues i and i+3

Hydrogen bonds between residues i and i+2

• Separate structural clusters within one protein chain 
• One protein will have multiple domains
• Some are very clearly separated and others are not as distinct

Separate protein chain

Has multiple chains

Viruses: polio and tobacco mosaic virus

Vault a large cellular particle (organelle)- multiple copies of three proteins and RNA

When you have multiple synthesis site on mRNA

Enthalpy and entropy

Reduced water cages (hydrophobic effect)

• The chemical bonds between: 
• H bonds betwene polar groups
• Van-der-waals bond between hydrophobic sidechains (plentiful, but weak bonds)
• Salt bridges (strong but few)

• Reduced configurational entropy
• Just sheer folding is not favorable but the hydrophobic effect is greater than this so folding still occurs

Desolvation cost: to form hydrogen bonds or salt bridges between polar groups within a protein in a folded state, the same groups should break bonds with water molecules in the unfolded state

• Buried hydrophobic groups
• Shielded from water

• Unfavorable water cages
• Entropic cost

• Protein backbones are polar
• Polarity of side chains vary from very hydrophilic to very hydrophobic
• Proteins fold to minimize contacts between hydrophobic residues and water (maximize intramolecular hydrophobic interactions)

Hydrophobicity of sequential regions can predict location

• Are specific interactions between hydrophobic groups that stabilize a well-folded protein interior
• Weak enthalpic effect

• Hydrogen bonds and are necessary to remain stable
• It is also one of the major forces in protein stability

Hydrogen bonds are often stronger between protein groups that between protein and water

• Electrostatic interactions in proteins
• Specify combined electrostatic and hydrogen bondings
• Recovers approx. the same energy as the cost of taking charges out of water

• Interior: contribute to stability and conformational specificity
• Exterior: charged residues contribute specificity for interaction with other proteins and ligands

• May stabilize protein structures when not in a reducing environment (cytoplasm is reducing)
• Stability of small proteins may depend on these
• Larger proteins may retain fold in absence of disulfides

• Unfolded proteins, or proteins in which the secondary and tertiary structure has been disrupted to create random coil, are termed "denatured"
• Can be caused by chemical or thermal means

• Small molecules commonly used to denature proteins
• Used in high concentrations
• Bind water and reduce hydrophobic effect (can form many H bonds with water and decreases water cage)
• Typically reversible 

Cooperative unfolding; when part of the structure is disrupted, it destabilizes the remaining structure

• Do proteins fold through a random, sequential search pattern?
• It takes approx 10-13 secs to rotate around a bond i.e to sample a possible protein backbone conformation
• To sample all of the possible conformations of all the residues in a fairly small, 100-amino acid protein, it would take: 
• Time to fold randomly 10n * 10-13s= 1087s
• which is over 5 billion years
• Proteins fold in milliseconds to seconds, sometimes minutes
• Proteins do not fold by sequential random search

• Structural
• Local structures
• Stabilization of secondary structure

• Rapid formation of local interactions (i.e. secondary structure), which then determine the further folding
• These local interactions serve as nucleation points in the folding process

Undergo hydrophobic collapse to molten globule state

Internal side chains pack together, water is expelled from protein core

• Folding funnel
• Proteins fold via a series of conformational changes that reduce their free energy and entropy until the native state is reached
• there are many paths to the bottom of the funnel

• Simplified smooth folding funnel
• "single step" folding

• More realistic folding funnel
• "multi-step" folding

• Synthesis
• Denatured (unfolded) 
• Intermediate
• Native
• Fiber

• A misfolding that is tightly packed and therefore hard to degrade
• Also does not take more energy to fold an amyloid than amorphous aggregate and will spontaneously form

• Beta sheets which are very stable due to a large number of hydrogen bonds (often hydrophobic clusters)
• Folding beta sheets is kinetically slower as the stabilizing H-bonds are separated by longer distances as compared to alpha helices (i-i+4)

Amyloid beta or "Abeta" peptide

alpha-Synuclein

Prion protein

Huntingtin with polyQ tracts

Large family of proteins that function to "inhibit inappropriate interactions between potentially complementary surfaces and disrupt unsuitable liasons so as to facilitate more favorable associations"

Help to minimize aggregation and provide a framework for proper protein folding

• A particular class of chaperones
• Aid in the proper folding of misfolded proteins with exposed hydrophobic patches
• Some proteins cannot get folded without these

• Initially exposed hydrophobic patches interact with an unfolded protein
• ATP hydrolysis hides hydrophobic patches, exposing polar residues allowing folding and expelling of the bound protein

• Some proteins are natively unfolded i.e present completely or partially in a random coil form
• The fraction of these increase from prokaryotes -> unicellular -> multicellular

• In "interaction hubs" and play a critical role in signaling and other processes when interaction with numerous partners is essential
• Are highly interactive and can form complexes with a large variety of different proteins

• An IDP that is a transcription factor and controls gene expression
• Arrests cell cycle progression if the DNA is damaged