Compound that exists in a stable form with charges that balance for a total of zero net charge

(ex: any AA w/ uncharged R group)

• covalent bond between carboxyl group and amino group of another amino acid
• formed during translation
• yields rigid planar peptide units
• partial double bond character prevents free rotation
• trans configuration

• Glycine, Alanine, Valine, Leucine, Isoleucine, Proline

(GAVLIP)

• Nonpolar, uncharged
• Hydrophobicity ↑ with size of chain

• cyclic structure introduces a kink into the peptide chain
• typically found as terminator of alpha helices

• common in beta turns

• Serine, Threonine (ST)
• polar, uncharged
• OH can form ester, H-bond, or be attachment site for oligosaccharides in glycoproteins

• Cysteine, Methionine (MC)
• Only cysteine can form disulfide bonds
• Methionine has bulky hydrophobic side chain

• -SH group important component of active sites for many enzymes
• Two cysteines can form cystine with -S-S- bond
• Disulfide bonds important for secondary, tertiary structure (not quaternary)

• Phenylalanine, Tyrosine, Tryptophan (FYW)
• uncharged, only Tyrosene is polar
• Tyrosine and tryptophan absorb uV light at 280nm (useful for protein concentration estimate in acqueous solution)

• Aspartate, Asparagine, Glutamate, Glutamine (DNEQ)
• Polar
• Aspartate and Glutamate negative charge
• Asparagine and Glutamine uncharged, capable of H-bonding

• Histidine, Lysine, Arginine (HKR)
• Net positive charge
• Histidine can be uncharged due to its weak basicity, charge in protein depends on neighboring AA residues

• WaLK To HIVe FaRM

Tryptophan, Leucine, Lysine, Threonine, Histidine*, Isoleucine, Valine, Phenylalanine, Arginine*, Methionine

• Histidine & Arginine nonessential in adults

• Except glycine, all AA's have assymetric α-carbon with D & L configuration
• Only L AA configuration found in human proteins

• pH = pKa + log[A^-]/[HA]
• at half dissociation of the acid, pH = pKa

Buffer definition?

Where does maximal buffering of weak acids and weak bases occur?

• A solution that resists change in pH following addition of acid or base
• Maximal buffering at pH equal to the pKa

pI definition and

EQ's for neutral, acidic, basic AA

• Isolectric point
• pH at which molecule carries no net charge
• neutral - (pK₁+pK₂)/2
• acidic - (pK₁+pK_R)/2
• basic - (pK_R+pK₂)/2

• Histidine
• Cysteine

(FeW MC's have GAVLIP)

Phenylalanine, Tryptophan, Methionine, Cysteine, Glycine, Alanine, Valine, Leucine, Isoleucine, Proline

• Plants and microorganisms can synthesize all AA's
• Mammals can only synthesize the ten nonessential AA's in sufficient quantity, the rest must come from diet

Name the negatively charged AA's

(at physiological pH)

Name the positively charged AA's

(at physiological pH)

Histidine, Lysine, Arginine

(all the basic ones)

Acidic + Basic + uncharged/polar

tHE DaRK is SQuiNTY

 Histidine, Glutamate, Aspartate, Lysine, Arginine, Serine, Glutamine, Asparagine, Threosine, Tyrosene

• Globular
• Fibrous
• Hybrid

• Compact, spherical, tightly folded, H₂O soluble
• Enzyme, transporter, protection, hormone, gene regulatory

• long and H₂O insoluble
• structural support

• Combination of globular and fibrous conformations within the same protein
• Contractile, protection

• α-helix
• β-sheet
• β-turn
• Random coil

• Right-handed, most common 2° structure
• Tightly packed, coiled peptide backbone with side chains directed outside the helix
• Stabilized by H-bonds between -NH and -CO group from every peptide bond
• Proline terminates helix due to structure and inability to H-bond

Coiled-coil:

definition, basic component of, function

• 2+ α-helices intertwine to form coiled-coil
• coiled-coil → protofilament →protofibril → intermediate filament
• intermediate filaments provide strength in hair, myosin, tropomyosin in muscle, fibrin in blood clots

β-sheet:

structure, stability, function, pathology

• Extended conformation of laterally packed β-strands in parallel or anti-parallel
• Stabilized by H-bonds with side chains of adjacent AA's pointing in opposite directions
• Multiple β-sheets provide strength and rigidity in structural proteins
• principal 2° structure in amyloidosis patients

β-turn

• Consists of 4 AA's (often proline, glycine)
• Stabilized by H-bonds, ionic bonds
• Allow polypeptide chains to fold into more compact shapes (ex: globular)

• 2° structure found in collagen
• Peptide chains twisted into 3-stranded helix which is twisted into a superhelix

Protein tertiary structure:

components, stability

• dicated by AA sequence
• Secondary structures combine to form domains
• maintained by ionic conds, H-bonds, -S-S bonds, van der Waals forces, hydrophobic interactions

• organization of multiple polypeptide chains into functional multimeric protein
• dimer (2 subunits), tetramer (4 subunits)
• NOT stabilized by -S-S bonds between subunits

• Denaturing agents (such as urea and β-mercaptoethanol) can fully unfold a protein
• Upon removal of denaturing agents, protein returns to catalytically active structure

Prion Disease:

What is prion?

What causes prion diseases?

Examples of prion diseases

• prions are soluble cellular protein made of mostly α-helices present on neurons (PrPc)
• with prion disease, α-helices are replaced with β-sheets to form an insoluble aggregate protein  resistant to proteolysis (PrPsc)
• leads to neurodegenerative diseases (scrapie, "mad cow", Creutzfeldt-Jacob)

• immunoglobulin chains form insoluble protein aggregate in organs and tissues
• leads to Alzheimer's

• molecular chaperone proteins assist in folding
• when misfolding occurs, fxn is lost and protein is either degraded to recycle products or can sometimes form damaging aggregates that lead to disease

• Uses porous beads packed in a column to separate protein based on size and shape
• Large prtoeins elute first, while smaller ones get caught in pores and elute more slowly

• protein passes through column containing charged beads
• proteins with opposite charge will bind to beads while same charge and then neutral pass through

• column of immobilized ligand separates protein of interest bc it binds to the ligand while other proteins pass through (ex: inhibitor to purify enzyme, hormone to purify receptor)
• to release protein can add excess soluble ligand or alter buffer properties

• protein sample denatured with β-mercaptoethanol and sodium dodecyl sulfate (SDS)
• SDS gives overall negative charge to proteins
• smallest proteins will move farthest away from the cathode (-) toward the anode (+)
• can determine degree of purity of a sample, mass of protein, number of subunits in heteromeric protein

• proteins subjected to PAGE in a pH gradient
• proteins separate on basis of net charge until they reach their pI (pH where net charge = 0)
• Can be further separated by SDS-PAGE to obtain higher resolution

• protein + HCL+heat+24hrs → AA's
• (Trp destroyed, Ser/Thr partially destroyed, Asn→Asp, Gln→Glu)

• Perform ion exchange chromatography
• Quantify AA amounts by measuring light absorption

• uncharged N-terminal residue labeled with FDNB (Sanger's reagent) or dansyl chloride

• Hydrolyze w HCL and separate using chromatography

C-Terminal identification of AA sequence

(2 methods)

treat with hydrazine (reacts w all AA except C-terminus), then Hydrolyze with HCL

OR treat protein w carboxypeptidase to cleave C-terminal AA

Determine unreacted or cleaved C-terminal residue w chromatography

• phenyl isothiocyanate reacts w N-terminus to form derivative which is released from peptide under mildly acidic conditions
• remainder of peptide is intact, ready for another round of labeling and release
• automated to analyze up to 50AA at a time

• Certain enzymes cleave between specific AA's
• Cleave w enzyme and determine AA sequence of fragments
• Then cleave w another enzyme and determine AA sequence of these fragments
• Now you can determine the entire protein sequence