Chem 330 test 1

F=

(q1q2)/ɛr^2

Strong Acid

(small pKa) - weak conjugate base

Protein Isolation and Purification: Some basic techniques

1. Break open cells and extract protein into buffer. Mechanical Forces: Grinding, Ultrasound Detergents or Organic Solvents: Disrupt cell membrane 2. Fractional precipitation: solubility of protein depends on a number of factors. pH: Usually least soluble at pI Concentration of Various Salts: (NH4) 2SO4 often used Concentration of Organic Solvents Strategies: 1. Precipitate other proteins leaving desired protein in solution 2. Precipitate protein of interest leaving others in solution -Often use combination of 1 and 2 3. Column Chromatography

Acid Dissociation Constants (Ka)

1. Calculation of pH of solutions of weak acids 2. Convenient Scale of Acidity Strong Acid -Large Ka (Small pKa) Weak Acid - Small Ka (Large pKa) 3. Also a Scale of Basicity For any conjugate acid-base pair (AH and A-): KaKb=Kw (pKa + pKb=pKw) Ka= acid dissociation constant of AH Kb= base dissociation constant of A

Structures of Biopolymers

1. Structures of monomers units 2. Chemical bonding between monomer units 3. Overall structure of biopolymers and methods for determination of structure -For proper function, biomolecules typical form highly-organized three-dimensional structures

Deoxy Sugars

-2-deoxy-D-ribose -L-Fucose

Cation-Exchange Chromatography

-Column of a sulfonated polystyrene resin is washed with aqueous NaOH to deprotonate sulfonate groups. -Mixture of proteins in a low pH buffer placed on column - positively charged groups displace sodium ions to form ionic interactions with the sulfonate groups of the resin. -Proteins with the highest degree of positive charge bind most tightly to resin. -Elute column with a buffer system that gradually increases in pH and [Na+] - amino acids that are least tightly bound pass through the column at faster rates than more tightly bound species

Hydrophobic Effect - Stabilizing Structures of Proteins

-Cytosolic Proteins:* Most of the nonpolar side-chains lie in the interior regions of the folded protein. -Removing nonpolar groups from aqueous environment provides more freedom of motion of water molecules - makes folding into an ordered structure less unfavorable in terms of entropy. *Proteins tightly associated with membranes often have large numbers of nonpolar groups at their surface

Sugar Acids

-D-Gluconic Acid -D-Glucaric Acid -D-Glucuronic Acid -Abscorbic Acid (Lactone Form) --Vitamin C

Amino Sugars

-D-Glucosamine -N-Acetyl-D-Glucosamine

Sugar Phosphates

-D-Glucose-6-Phosphate -Dihydroxyacetone Phosphate

Sugar Alcohols

-D-Mannitol -Glycerol -Myo-Inositol

van der Waals Attractions

-Dipole-Dipole -Ion-Dipole -Ion-Induced Dipole -Dipole-Induced Dipole -Instantaneous Dipole-Induced Dipole (London Forces) -Short range forces - Individually very weak Close packing of atoms in proteins maximizes strength of interactions Large number provides substantial stability

α-keratins

-Fibrous proteins synthesized by skin cells of vertebrates. Structural proteins in the outer layers of the skin and in various derivatives of the skin (hair, wool, quills, nails, horns, hooves). -Typically contain large amounts of alanine*, leucine*, arginine* and cysteine (*high propensity towards formation of α-helix). -About 310 residues in the central portion of peptides form a α-helix about 450 Å in length. -Two peptides coil about each other to form dimers (below); these dimers associate with others to form strong fibers.

Sequencing Very Large Peptides - Specific Cleavage Methods

-Full sequencing of large peptides requires cleavage of the peptide into fragments, but hydrolysis of acid or base is not very useful since it is non-specific (i.e., cleavage occurs at random points on peptide chain). -Peptides can be cleaved at specific sites with certain reagents. Most of these reagents are proteases, enzymes that catalyze the hydrolysis of peptide bonds

Dipole moment of water

1.87 D

The pH of a buffer solution depends on:

1.the acid dissociation constant of the weak acid 2. the ratio of [base] to [acid] in the mixture -Ka = ([H+][A-])/[HA] -Ka/[H+] = [A-]/[HA] -log(Ka) - log[H+] = log([A-]/[HA]) -pH - pKa =log([A-]/[HA] Henderson-Hasselbach Equation -pH = pKa + log([A-]/[HA])

H-O-H bond angle in water

104.5°

Identification of N-Terminal Amino Acid: Sanger Method

2,4-Dinitrofluorobenzene (2,4-DNFB) reacts with amino groups of amino acids under mildly basic conditions to give 2,4-dinitrophenylamino acids (DNP-amino acids) Treat peptide with 2,4-DNFB before hydrolysis and identify DNP-labeled amino acids. As shown above, only the N-terminal amino acid of the peptide will contain a DNP group at the α-position. Note: All lysine residues will be labeled at the side-chain amino group - if lysine is at the N-terminus, it will be labeled with two DNP groups

Most proteins contain at least _______ amino acids with nonpolar side chains; membrane proteins and fibrous proteins often contain larger amounts (up to _____).

30-40%; 90%

Water dielectric constant

78.5

Small Proteins

> 10,000 D (1 Dalton (D) = 1 atomic mass unit).

Large DNA molecules

>10^10 D

Buffer

A solution of a conjugate pair of a weak acid and a weak base. -Buffers are used to maintain the pH of solutions at a relatively constant value.

Chemical properties of acetals

Acetals are unreactive with a numbers of classes of chemical reagents - bases, nucleophiles, oxidizing agents and reducing reagents - but undergo hydrolysis in dilute aqueous acid.

Entropy Change

Increase in entropy is typically thought of as an increase in the "disorder" or "randomness" of a system. Example: Mixing of Ideal Gases Intermolecular attractions between particles of ideal gases are zero (ΔH = 0) Spontaneity of process is due to the increase in entropy of the system - entropy is related to the freedom of motion of molecules

Temperature in denaturing proteins

Increase thermal motion disrupts intramolecular forces.

Proteins containing more than one peptide subunit:

Individual peptide subunits may be identical or different. -Triose Phosphate Isomerase: Two identical peptide chains -Hemoglobin: Four peptide subunits - two a chains and two b chains Peptide subunits may be associated via non-covalent interactions or covalently linked via disulfide bonds

Salt Bridges

Ionic Bonding While ionic bonds are generally considered to be quite strong, the location of a salt bridge is an important factor in determining its contribution to overall stability of the 3°/4° structures of proteins

Weak acid

Large pKa -strong conjugate base

Unfavorable Change in Enthalpy Water

Less hydrogen bonding between water molecules in the liquid

Sequencing via Edman Degradation vs. MS:

Limit of size of peptide to be sequenced is similar for both methods (20-30 amino acids). MS: Impossible to distinguish between Leu and Ile (isomers) and between Gln and Lys (similar mol wt (< 0.1 g/mol)). MS: Much faster than sequencing using Edman degradation

Favorable Change in Entropy

Lower degree of order between water molecules in the liquid

Solvent properties of water: ionic compounds

Many ionic compounds have high solubility in water. Water has a large dielectric constant (below) and solvates ions via ion-dipole and/or hydrogen bonding interactions

Ionic compounds in water

Many ionic compounds have high solubility in water. Water has a large dielectric constant (below) and solvates ions via ion-dipole and/or hydrogen bonding interactions.

Organic Compounds in denaturing proteins

Many organic compounds denature proteins via disruption of hydrogen bonding and/or nonpolar interactions

Change in Enthalpy (ΔH)

Measure of amount of energy exchanged as heat -Relates, among other factors, to strengths of covalent and non-covalent interactions between atoms and molecules

Change in Entropy (ΔS)

Measure of degree of disorder or randomness (most probable distribution of energy)

Dielectric Constant

Measure of the ability of a material to disperse the force between charged particles. Attractive forces between oppositely charged ions decrease with an increase in dielectric constant - the higher the dielectric constant, the lower the tendency of ions to "clump together".

Determination of Amino Acid Composition - Cleave Disulfide Bonds and Protect from Re-forming:

Mecaptoethanol is commonly used to cleave disulfide bonds in peptides; thiol groups can be protected by alkylation (e.g., SN2 reaction with iodoacetate). -Determine if more than one type of peptide chain is present; if there are two or more different peptides, separate and purify each.

Mass Spectrometry

Method to determine the mass-to-charge ratio of ions in the gas phase (species detected by MS must be charged - cationic (most often) or anionic).

Electrophoresis

Method used for the separation and characterization of substances based on their mobility in an electric field.

Paper Electrophoresis: Anions

Migrate towards anode

Paper Electrophoresis Cations

Migrate towards cathode

Isomerization

Monosaccharides undergo isomerization in base via interconversion of their keto and enol tautomers. -In basic solution monosaccharides undergo isomerization to form complex mixtures; prolonged treatment with base can also result in C-C bond cleavage via base-catalyzed retro-aldol reactions. 5 Isomerization occurs only via the open-chain forms of the monosaccharides - glycosides do not undergo isomerization in aqueous base (acetals are stable under basic conditions)

Acid-Base properties of Carboxylic Acids

Most acidic class of common organic compounds due to resonance stabilization of the carboxylate anion (negative charge delocalized over two oxygen atoms) -Except for formic acid (pKa 3.75), carboxylic acids containing only hydrocarbon groups typically have pKa 4.2-5.2 -Electron-withdrawing groups increase the acidity of carboxylic acids

Conformations of Glycine and Proline

Most peptide bonds (top) prefer trans conformation due to unfavorable steric interactions in the cis conformation (99.9% trans in unstrained peptides). -With glycine (bottom), steric strain in the cis conformation is not as severe. -For peptide bonds to the α-amino group of proline (X-Pro), the cis and trans conformations are of similar energy (both have steric interactions with the adjacent amino acid). Some protein have a considerable percentage of X-Pro peptide bonds in the cis conformation

Polymerization of Acrylamide (pg 7, peptide)

N,N'-Methylenebisacrylamide (above right) is added to cross-link linear chains. Polymerization of acrylamide with N,N'-methylenebisacrylamide produces a continuous gel with reasonably uniform pore size in buffer of choice - pore size can be controlled via amounts of acrylamide and N,N'-methylenebisacrylamide used

Sequences of peptides are always written from the...

N-terminal end to the C-terminal end (left to right)

Typical Strong Bases

NaOH, KOH

Enantiomers

Non-congruent mirror image structures; have opposite configuration at all chiral centers (L-glucose is the enantiomer of D-glucose).

What kind of amino acids are found in proteins?

Only L-amino acids

Oxidation of monosaccharides with Bromine in Water

Oxidizes of the aldehyde group of aldoses to a carboxylic acid group to form an aldonic acid, but the reaction does not change the configuration of the chiral centers of the aldose. Ketoses and glycosides are not oxidized by bromine water.

Sequencing Somewhat Larger Peptides

Partially hydrolyze peptide (e.g., acid hydrolysis) and isolate/purify smaller peptides (2-5 amino acids). Determine composition and sequence of each small peptide by the above methods. Elucidate structure of intact peptide by finding overlapping sequences in the fragments

Non-amide bonds of peptide backbone

Peptide bonds have a rigid, planar conformation favoring the trans conformation, but can have rotation about the non-amide bonds of the peptide backbone

Cysteine Residues

Peptide chains in α-keratins are cross-linked by large numbers of disulfide bonds; hard tissues (horns, nails) contain up to 22% cysteine, while more flexible varieties (hair, skin) possess on the order of 10-14% cysteine

Chymotrypsin

Phe, Trp, Tyr

Acid-Base Properties of Esters of Phosphoric Acid

Phosphoric acid is a triprotic acid - the acid dissociation constants for the three ionizable groups increase substantially for each sequential dissociation -At pH near 7, phosphate diesters (below left) will be virtually 100% ionized while phosphate monoesters (below left) will exist as a mixture of the monoanioic and dianionic forms (typically show as the dianion)

Source of Protein

Plant or animal tissues, bacterial cells, viruses -Protein of interest is usually present in very small amount (< 0.1% dry weight of sample)

Polysaccharides

Polymers of simple sugars linked by acetal groups (glycosidic bonds)

Biopolymers

Polysaccharides Proteins Nucleic Acids

Glucose

Preferred structure is the hemiacetal form with a six-membered ring (glucopyranose). -Formation of the hemiacetal of a sugar creates an additional chiral center in the molecule.

Acid Base Properties of Alcohol

Primary alcohols (pKa ~16) are similar in acidity to water while secondary (pKa ~17) and tertiary (pKa ~19) alcohols are less acidic

Protein Structure

Proteins may contain one or more peptide chain

Fibrous Proteins

Proteins with elongated shapes dominated by long regions of secondary structure; form tough, water-insoluble materials. In some fibrous proteins, peptide chains intertwine to form rope-like structures. Often also have covalent crosslinks between chains. -α-keratins, fibroin (silk protein), collagen,

Basicity of Pyrrole

Pyrrole and related aromatic amines are very weak bases due to the loss of aromaticity upon protonation.

Mechanical Forces and denaturing proteins

Shear forces (e.g., via rapid stirring) can disrupt weak intramolecular forces

Aspartic and Glutamic Acid acidic side chains

Side-chain carboxyl groups will be almost completely ionized at pH 7, but are considerably less acidic than the α-carboxyl group.

"Essential" Carbohydrates

Since mammals can synthesize all required carbohydrates from other precursors, none are truly "essential" for mammals. However, it is energetically favorable for mammals to obtain the majority of the following in the diet.: D-Glucose N-Acetyl-D-glucosamine D-Galactose N-Acetyl-D-galactosamine D-Mannose L-Fucose (6-Deoxy-L-galactose) D-Xylose N-Acetylneuraminic Acid

Solubility of Hydrocarbons - Transfer of Methane from Organic Solvent to Water

Since methane is more soluble in nonpolar solvents than in water, transfer of methane from an organic solvent to water is energetically unfavorable: depending on organic ΔGo = 6-14 kcal/mol. From the general rule that "like dissolves like", one might expect that the reason for the low solubility of methane in water is due to an unfavorable enthalpy change (ΔHo > 0). However, the measured enthalpy change is negative - it is the unfavorable change in entropy that disfavors the process --Since water cannot form strong intermolecular attractions (hydrogen bonding or dipole- dipole attractions) with nonpolar compounds, water forms ice-like cages that encapsulate nonpolar solutes.

Hydrophobic Effect

Since water cannot interact strongly with nonpolar groups, water molecules form ice-like cages about nonpolar regions of solutes. In forming monolayers or micelles, water molecules surrounding the nonpolar portions of amphipathic substances are released from these rigid structures. Increasing the freedom of water molecules makes formation of the self-assembled structures more entropically-favorable; increasing the disorder of water molecules helps drive the increase in the order between the amphipathic molecules. The hydrophobic effect may not make formation of ordered structures completely favorable in terms of entropy (e.g., ΔS > 0), but it will make the process less unfavorable.

Monosaccharides

Single sugar molecules -Polyhydroxy aldehydes (aldoses) and ketones (ketoses).

O-Glycosides

Acetals of carbohydrates, in their cyclic form, and alcohols. The linkage between the sugar unit and the alcohol moiety is often called a glycosidic bond. As with the hemiacetals of monosaccharides, O-glycosides can have α and β anomers. Example: Reaction of D-glucose (which exists as a mixture of α and β anomers interconverted via the open chain structure) with methanol under acidic conditions gives formation of a mixture of the α and β anomers of the O-methyl glycoside of D-glucose (below) O-Glycosides do not undergo mutarotation. Mutarotation requires formation of the open-chain form of the sugar - the cyclic hemiacetal forms of monosaccharides equilibrate with the corresponding aldehyde/ketone structures (open chain structures of sugars), but their acetals do not. As with simple acetals, O-glycosides are stable under neutral and basic conditions, but undergo hydrolysis in the presence of acid - once the hemiacetal structure is formed, mutarotation can again occur.

Examples of Buffers

Acetic acid (weak acid) and sodium acetate (weak base) Ammonium Chloride (NH4+Cl-, weak acid) and ammonia (NH3, weak base) Phosphate Buffer: NaH2PO (weak acid) and Na2HPO4 (weak base) -Not all conjugate acid-base pairs are buffers. For example, a solution of HCl and NaCl is not a buffer because HCl is a strong acid.

Acids and Bases - Bronsted Definition

Acid: Proton Donor (H+, hydrogen cation) Base: Proton Acceptor Acid-Base Reaction: Transfer of a proton from an acid to a base -The products of an acid-base reaction are a second acid-base pair (conjugate acid and base). -By the Bronsted definition, water is both an acid and a base, not only response to added acids or bases, but also in auto-ionization, in which one molecule of water acts as an acid and another as a base to form a hydroxide ion (HO-) and a hydroxonium ion (H3O+)

Determination of Amino Acid Composition: Complete Hydrolysis of Peptide

Acidic Hydrolysis: Heat peptide in 6 M HCl at 100-120 oC for 10-24 hours in a sealed tube. Problems: -Side chains of asparagine and glutamine undergo hydrolysis to yield aspartic and glutamic acid. -Acid hydrolysis causes decomposition of the indole ring of tryptophan -Some loss of serine and threonine (dehydration)

Addition of Alcohols to aldehydes and ketones

Alcohols add to the carbonyl group of aldehydes and ketones to form hemiacetals. For most aldehydes and ketones, the position of equilibrium usually favors the carbonyl compound (i.e., the reactants). -Important exception: Cyclic hemiacetals, formed from hydroxy aldehydes or hydroxy ketones, with five-member or six-member rings are relatively stable -Aldoses with four or more carbons and 2-ketoses with five or more carbons can form cyclic hemiacetals:

Acid-Base properties of Thiols

Alkyl thiols (R = alkyl group) are considerably more acidic than the corresponding alcohols -R-OH --pKa 15.5-19.2 -R-SH --pKa 10.3-11.2

Acid-Base properties of amino acids

All amino acids contain at least one acidic and one basic group - the ionic form of the amino acid depends on the pH of the solution.

Benedict's and Tollen's Reducing Sugars

All monosaccharides - ketoses undergo isomerization to aldoses under basic conditions

Diastereomers

All stereoisomers that are not enantiomers (L-idose and D-mannose are both diastereomers of D-glucose).

Basicity of Amides

Amides are usually very weak bases (pKBH 0 to -4) and, although nitrogen is usually a more basic atom than oxygen, undergo protonation preferentially at the oxygen as resonance stabilization would be lost by protonation at nitrogen

Basicity of Amidines and Guanadines

Amidines and guanidines are more basic than alkyl amines due to delocalization of charge via resonance in the protonated species.

Desmosine

Amino acid found in elastin

4-hydroxyproline

Amino acid found in plant cell walls and collagen of connective tissues

Micelle Formation

Amphipathic Molecules with One Hydrocarbon Tail: When small amounts of these compounds are added to water they initially form a monolayer at the surface. Once the surface is coated by a monolayer, they associate to form micelles

Salt

An ionic compound made from the neutralization of an acid with a base.

Basicity of Anilines

Aniline is much less basic than ammonia since resonance stabilization due to interaction between the unshared electron pair on nitrogen and the phenyl group in aniline is lost when nitrogen is protonated

Ramachandran Plot

Areas enclosed by solid lines are predicted to be the most stable combinations of φ and ψ, with areas surrounded by dashed lines somewhat lower stability. Data from measured bond angles in proteins cluster in regions of predicted high stability.

Hybridization state of atoms and acidity

As discussed previously, the hybridization state of an atom influences the acidity of hydrogen attached to that atom, with acidity increasing with the degree of s-character of the hybridization state. For example, ethyne is more acidic than ethene, which in turn is more acidic than ethane. Given the inverse relationship between the acidity of an acid and the basicity of its conjugate base, the basicity of the carbanions of these hydrocarbons increases in the opposite direction. The dependence of the basicity of nitrogen on hybridization state changes in the same manner.

Structure of lipids

Associate to form lipid bilayer of cell membranes

Peptide bond stability

Because of resonance stabilization of the amide C-N bond, peptides are very stable in aqueous solution near room temperature at pH 7 (half-life of ~100 years). -Heating of peptides under strongly acidic or basic conditions for several hours is required for complete hydrolysis of proteins.

Disulfide Bonds

Besides amide bonds, the only other major type of covalent linkage between amino acids in peptides is the disulfide bond, which is formed via oxidation of the thiol groups of two cysteine residues. In some cases, disulfide bonds cross-link two peptide chains (below left). Cysteine residues in a single peptide can also form disulfide bonds, which create loops in the peptide chain (below right)

Determining wolecular weight of Proteins with Multiple Subunits (more than one peptide)

Some methods for determination of molecular weight (e.g., ultracentrifugation) give the total mass of the complete protein (masses of all subunits combined) SDS-PAGE (with mercaptoethanol): Gives molecular weight of individual peptides - separate band for each peptide of different molecular weight SDS-PAGE (without mercaptoethanol): Peptides linked by disulfide bonds give a single band

Salts in denaturing proteins

Some salts destabilize proteins (e.g., LiBr and KSCN) - others have little effect or increase stability

ΔG > 0

Spontaneous in reverse direction

ΔH < 0

Spontaneous processes are favored, stronger interactions between atoms

ΔS > 0

Spontaneous processes are favored; increase in disorder

Paper Electrophoresis Neutral compounds

Stationary

Ion Exchange Chromatography

Stationary phase consists of beads containing either anionic (for cation exchange) or cationic (for anion exchange) groups.

Size-Exclusion Chromatography (gel chromatography)

Stationary phase consists of beads containing pores with a particular size range. -Larger Proteins: Pass rapidly through column -Smaller Proteins: Entry into pores slows rate of elution

Affinity Chromatography

Stationary phase contains beads covalently bound to compound that reversibly binds to protein of interest 1. Load mixture of proteins to stationary phase - only proteins that bind the specific substrate adhere to beads 2. Wash column to rinse away unbound proteins 3. Elute column with solvent that dissociates protein from substrate bound to bead

Anomers

Sugar hemiacetals (or acetals) with opposite configuration at the hemiacetal/acetal carbon (anomeric carbon) of the monosaccharide (e.g., α-D-glucopyranose and β-D-glucopyranose). Some sugars form stable hemiacetals with significant amounts of both the pyranose and furanose structures

Furanose

Sugar in which the hemiacetal (or acetal) form contains a five-membered ring.

Pyranose

Sugar in which the hemiacetal (or acetal) form contains a six-membered ring.

Melting Temperature of Proteins (Tm)

Temperature at which sample of protein is 50% denatured; for most proteins, Tm<< 100°C

Quaternary Structure

The arrangement of peptide chains in multi-subunit proteins.

Basicity of Heterocyclic Amines

The basicities of heterocyclic alkyl amines are similar to their acyclic counterparts.

Basic Side chains and other nitrogen-containing groups

The basicity of nitrogen atoms in the various amino acids spans a large range

Oxidation of monosaccharides with Benedict's Reagent and Tollen's Reagents:

The carbohydrate must be in the open-chain form for oxidation with Benedict's or Tollen's reagent to occur. Benedict's and Tollen's reactions are used to classify carbohydrates as "reducing sugars" or "non-reducing sugars"

What has important consequences as to the structure and reactivity of peptides?

The high degree of resonance stabilization/double bond character of peptide bonds (amide bonds)

isoelectric point (Isoelectric pH, pI)

The pH at which the amino acid (or peptide) has no net charge - pH at which the concentration of the zwitterionic form is at a maximum

Tryptophan side chain

The side chain nitrogen of the indole ring is not basic since its protonation would result in the loss of some resonance stabilization - protonated form is still aromatic (benzene ring) but aromaticity no longer extends over the nitrogen containing ring.

Dielectric Constants (ɛ) and Ionic Bonding

The strength of electrostatic forces between charged particles, whether attractive or repulsive, decreases with increasing dielectric constant of the medium.

Tertiary Structure of Proteins

The three-dimensional structure of the peptide chain in the native conformation (i.e., functional form) of the protein

G.N. Ramachandran

Using computer models of small polypeptides, examined changes in degree of steric strain associated with rotations of the dihedral angles φ and ψ gave the most stable peptide bonds.

Strengths of Bases

Usually uses Ka (pKa) of the conjugate acid as a measure of basicity

Sequencing Small Organic Molecules

Vaporize molecule by heating. Ionize molecule via collision with a beam of high energy electrons - the parent ion usually undergoes subsequent fragmentation to yield smaller ions. Separate and detect ions on basis of mass-to-charge ratio

Solvation involves two competing effects:

Water molecules next to a cation (anion) are arranged such that the negative (positive) end of their dipoles are oriented towards the ion - favorable formation of favorable attractions between solvent and solute molecules (ΔH<0) -Formation of the solvation shell increases the local structure of the solvent - unfavorable restriction of the motion of water molecules (ΔS < 0)

Water and Polar Compounds

Water solvates polar molecules via dipole-dipole and hydrogen bonding interactions. Small organic compounds (1-4 carbons) containing one oxygen or nitrogen are very soluble in water (> ca. 8 g/100 mL H2O) due to hydrogen bonding, but solubility drops off rapidly with increasing number of carbons

Polar compounds in water

Water solvates polar molecules via dipole-dipole and hydrogen bonding interactions. Small organic compounds (1-4 carbons) containing one oxygen or nitrogen are very soluble in water (> ca. 8 g/100 mL H2O) due to hydrogen bonding, but solubility drops off rapidly with increasing number of carbons.

Common elements in biomolecules

Carbon, Hydrogen, Oxygen, Nitrogen, Sulfur and Phosphorous

α-Amino Acids

Carboxylic acids with an amino group at the α-position (carbon adjacent to carbonyl group); in their predominant ionic form at pH 7, the α-amino group is protonated and the α-carboxylate group is deprotonated.

Proteins Prolyl Isomerase (PPI)

Catalyzes cis-trans isomerization of X-Pro bonds

Protein Disulfide Isomerase (PDI)

Catalyzes reversible cleavage/formation of disulfide bonds

Peptides

Chains of α-amino acids linked by amide bonds (peptide bonds). In proteins, peptide bonds between amino acids involve the α-carboxyl and α-amino groups. The peptide chain is directional -one end of the peptide has an unbound α-amino group, the other end has an unbound α-carboxylate group.

Mutarotation

Change in the specific rotation (i.e., optical activity) of a sugar due to changes in the relative concentration of two anomeric forms of the compound, for example, conversion of a single anomer of a sugar to a mixture of anomers. --can only occur via the open-chain form of the sugar. -Although many monosaccharides exist primarily in the hemiacetal form (furanose and/or pyranose), many of their reactions rely upon their equilibrium with the open chain form (i.e., require the carbonyl group of the open chain sugar as the site of reaction).

pH in denaturing proteins

Changes in pH will alter the protonation states of acidic/basic groups in peptides -can disrupt salt bridges and hydrogen bonding interactions

Identification of C-Terminal Amino Acid(s): Carboxypeptidases

Class of enzymes that sequentially cleave amino acid residues from the C-terminal ends the peptides. Once cleaved from the peptide, the amino acid can be identified using chromatography. Depending upon the selectivity of the enzyme for the amino acids at and adjacent to the C-terminus, it may be possible to sequence the first 2-3 amino acids at the C-terminal end of peptide by this method.

Globular Proteins

Compact folding of peptide chain; the overall shape is spherical to elipisoidal. In many cases, much of the tertiary structure of a protein consists of combinations of various secondary structures (super-secondary structures or structural motifs).

Fibroin (Silk Protein)

Composed largely of anti-parallel β-sheets; large percentage of amino acids with small side chains (Gly, Ala, Ser) allow tight association of peptides.

Amphipathic Molecules

Compounds that contain both hydrophilic (polar or ionic) and hydrophobic regions

β-Sheet

Conformation of the peptide bonds gives an extended chain structure. Side-by-side arrangement of two or more peptide chains leads to association C=O---H-N hydrogen bonding between α-carboxyl and α-amino groups.

Acid-Base properties of Phenols

Considerably more acidic than alcohols due to resonance stabilization of the conjugate base.

Lipoproteins

Contain lipid groups

Metalloproteins

Contain metal ions

Proteins

Contain one or more chains of amino acids linked by amide bonds (peptides) Twenty amino acids commonly found in proteins - different R groups

Conjugated Protein

Contain permanently-associated non-peptide components

Simple Protein

Contains only peptide chains

Other monosaccharides are designated as D or L as follows:

D-Monosaccharides: Same configuration as D-glyceraldehyde at the chiral center furthest from the carbonyl group. L-Monosaccharides: Same configuration as L-glyceraldehyde at the chiral center furthest from the carbonyl group.

Nucleic Acids

DNA and RNA -Polymers of nucleotides linked by phosphodiester bonds

R-S Stereochemistry

Defines the absolute configuration of groups about a chiral center.

D-L System

Defines the relative configuration of a chiral center via comparison with the configurations of the two enantiomers of glyceraldehyde (glyceraldehyde is the reference compound for definition of D and L). The enantiomer of glyceraldehyde that rotates plane-polarized light in the clockwise (dextrorotator, d) direction is defined as D-glyceraldehyde. Its enantiomer, L-glyceraldehyde, rotates plane-polarized light in the counterclockwise (levororotatory, l) direction

Salt Bridges on Interior of Proteins

Depending on types of amino acid side-chains in the vicinity (polar vs. nonpolar), the dielectric constant in the interior of a protein can be fairly small (on the order of 3-4 near nonpolar groups) - salt bridges in these regions can play a large role in the stabilization of tertiary/quaternary structure.

Hayworth Projection

Depict the stereochemistry of all chiral centers in cyclic forms for monosaccharides (as with Fischer projections) without showing the overall conformation of the compound (as with structures of the chair conformations of pyranoses).

What is water important in?

Determining the structures and properties of biomolecules

Epimers

Diastereomers that differ in configuration at a single chiral center. D-Mannose is the C-2 epimer of D-glucose and L-idose is the C-5 epimer of D-glucose.

Sequencing Short Peptides

Dipeptides: Amino Acid Composition and N-Terminal Analysis Tripeptides: Amino Acid Composition, N-Terminal Analysis and C-Terminal Analysis -May be able to sequence peptides with 4-5 residues via C-terminal analysis using carboxypeptidase.

Lithium Chloride: LiCl(s) → Li+(aq) + Cl-(aq) ΔHosoln = -37.0 kJ/mo

Dissolution of LiCl is enthalpically favorable.

Sodium Chloride: NaCl(s) → Na+(aq) + Cl-(aq) ΔHosoln = 3.9 kJ/mol

Dissolution of NaCl is has an unfavorable enthalpy change - but NaCl is very soluble in water.

Confirmation of Peptide Bonds

Due to resonance, the C-N bond of peptides (amide bond) has considerable double bond character. The geometry about the peptide bond is planar, and the trans confinguration is usually much more favorable than cis due to steric effects. -While rotation about peptide (C-N) bonds is highly restricted, it is possible to have rotation about the non-amide bonds of the peptide backbone.

Basicity of Alkyl Amines

Due to the electron donating effect of alkyl groups, aliphatic amines are somewhat more basic than ammonia; the variation in basicity with alkyl substitution is due to a solvation effect

Solid Water (ice)

Each water molecule is hydrogen bonded to four others in a tetrahedral arrangement - water molecules are not packed closely together -Unlike most substances, water in the liquid state (d = 1.00 g/mL) is denser than the solid (d = 0.92 g/mL)

ΔG = 0

Equilibrium

Paper Electrophoresis for compounds with ionizable groups

For compounds with ionizable groups (i.e., acids and bases), mobility depends on the pH of the buffer solution

Enthalpy Change (ΔH) in Water as a Solvent

For dissolution of solids and liquids in a solvent, interactions between particles of solute and between solvent particles must be disrupted (endothermic process) and replaced by solvent-solute attractions (exothermic process).

ΔG =ΔH - T Δ S

Free Energy Change (ΔG) -Determines spontaneity of a process

Assisted Protein Folding

While information required for proper folding (3° structure) contained in the amino acid sequence (1° structure), some proteins do not achieve proper conformation via unassisted folding - there are proteins whose function is to assist in the proper folding of other proteins

Polyacylamide Gel Electrophoresis (PAGE)

Gel Electrophoresis: Method of separation of large molecules (Proteins, DNA) Gel contains regularly-sized pores - similar to size-exclusion chromatography Analytes placed in a buffer to give them a net electrical charge - in the diagram shown below, proteins would be placed in a high pH buffer to give them a net negative charge. -Polymerization of acrylamide

Forces Stabilizing the Tertiary and Quaternary Structures of Proteins

In their native state (i.e., physiologically active conformation(s)), atoms in proteins are tightly packed (packing density - fraction of space in a solid occupied by atoms). 1. Disulfide Bridges 2. Salt Bridges - Ionic Bonding 3. Hydrogen Bonding 4. van der Waals Attractions 5. Hydrophobic Effect

Basicity of Amines and Other Nitrogen-Containing Compounds

While it is possible to compare strengths of bases using base dissociation constants (Kb or pKb), it is more common to discuss basicity by referring to the acid dissociation constants (Ka or pKa) of the conjugate acids of the bases using the inverse relationship between the acidity of an acid and the basicity of its conjugate base (i.e., strong acid - weak conjugate base and vice versa). In is also common to use pKBH to represent the pKa of the conjugate acid of a base - the larger the value of pKBH, the stronger the base

Zwitterion

a molecule or ion having separate positively and negatively charged groups.

Dipole-Induced Dipole

a weak attraction that results when a polar molecule induces a dipole in an atom or in a nonpolar molecule by disturbing the arrangement of electrons in the nonpolar species

pH>pI

amino acid migrates towards anode (net negative charge on amino acid)

pH<pI

amino acid migrates towards cathode (net positive charge on amino acid)

pH=pI

amino acid remains stationary (no net charge on amino acid)

N-Terminus of peptide

amino acid with an alpha-amino group but not bonded together

Basic Side Chains

arginine, lysine -Lysine contains a primary amino group in the side chain while arginine contains a guanidino group, both of which are sufficiently basic to be ~100% protonated at pH

Acidic Side Chains

aspartic acid, glutamic acid

Negatively-Charged Side chains

aspartic acid, glutamic acid -Both amino acids in this category contain a carboxyl group in their side chain that is ~100% deprotonated at pH 7

Dipole-Dipole

attractions between oppositely charged regions of polar molecules

Tryptophan, asparagine and glutamine also contain nitrogen atoms in their side-chains

but are not considered as basic amino acids because their nitrogen atoms are very weakly basic.

Instantaneous Dipole-Induced Dipole

cause all atoms and molecules to be attracted to each other

The double bond character of peptide bond results in...

cis-trans isomerism; the trans conformation is usually much more stable and favored (less steric strain)

Glycoproteins

contain carbohydrate groups

Histidine

contains an imidazole ring (heterocyclic amine) group in the side chain; this group has pKa 6.0 so it will be partially protonated at pH 7

Collagen Fibrils

crosslinked by covalent bonds derived from lysine residues (~5% of residues at positions X or Y).

Water is a hydrogen bond ______ and a hydrogen bond ________ (Hydrogen Bond Distance: ~1.8 Å)

donor; acceptor

Aliphatic Side Chains - nonpolar

glycine, alanine, valine, leucine, isoleucine, proline, methionine

C-terminus of peptide

has an unbound α-carboxylate group

Weak acids (poor H+ donors, small Ka)

have strong conjugate bases (good H+ acceptors)

Strong acids (good H+donors, large Ka)

have weak conjugate bases (poor H+ acceptors)

As with amino acids, peptides are usually least soluble at their...

isoelectric point

D-amino Acids

often found in other classes of biomolecules (e.g., bacterial cell walls, peptide antibiotics)

In non-protein peptides (e.g., glutathione)

other carboxyl or amino groups are sometimes involved in amide bonds

Weak acids dissolved in Buffers

pH- pKa =log([A-]/[HA]) -Final concentrations of the weak acid (AH) and its conjugate base (A-) can be calculated using the Henderson-Hasselbach equation as follows: -pKa = pKa of weak acid added (not the buffer acid) -pH = pH of the buffer solution

pI of amino acids with non-ionizable side chains

pI equals the average of the pKa's of the carboxyl and ammonium groups. For example, the pI of glycine is (2.34 + 9.6)/2 = 5.97). -Amino acids (and peptides) are typically least soluble in water at their pI.

Short Peptides

pK's of ionizable side-chain groups are usually similar to those for the amino acids (in large proteins folded in their native (active) conformations, the pKa's of side chain groups sometimes differ dramatically from the above values)

Histidine basic side chains

pKBH of 6.0 for the heterocyclic aromatic of side chain (imidazole) is comparable to pyridine (nitrogen in sp2 hybridization state) - about 9% protonated at pH 7. -While the imidazole group bears two nitrogen atoms, only one of these sites can be ionized as protonation at the other nitrogen would result in loss of aromaticity.

Buffers are most effective at pH=

pKa -In choosing a buffer, best to use an acid with pKa near the desired pH (pKa± 1)

pKbh for -NH2 =

pKa of -NH3+

pKbh Values

pKa of the conjugate acid of a base

For a conjugate acid/base pair

pKa+pKb=pKw

Some proteins contain only ________, others also contain _________ components.

peptide chains; non-peptide

Cysteine and Tyrosine are considered as

polar neutral amino acids (side chains are much less acidic than those of aspartic and glutamic acid), but can undergo substantial deprotonation at high pH.

Pyridine and Pyridine-like aromatic amines

relatively weak amine bases

Polar Side Chains

serine, threonine, asparagine, glutamine, cysteine

ΔG < 0

spontaneous in forward direction

Stability of proteins depends on a variety of conditions:

temperature, pH, presence various ions, organic solvents, etc

Due to resonance stabilization of the amide (peptide) bond...

the C-N bond has considerable double bond character -impacts shape of peptides and proteins

Ion-Induced Dipole

the charge of an ion induces a temporary partial charge on a neighboring non polar molecule or atom

Ion-Dipole

the charge of an ion is attracted to the partial charge on a polar molecule

D/L System

the configuration a compound is designated by comparison with the two enantiomeric forms of glyceraldehyde (relative configuration) rather than by the R/S system (absolute configuration)

Steric Strain

the interference between two bulky groups that are so close together that their electron clouds experience a repulsion

L-α-amino acids

the structure is a right-handed helix containing 3.6 amino acid residues per turn and a pitch (rise per turn) of 5.4 Å

Cysteine acidic side chains

the thiol is ~4% deprotonated at pH7

The geometry around the nitrogen atom of amides (peptide bonds) is...

trigonal planar

Aromatic Side Chains

tryptophan, phenylalanine, tyrosine

Nonpolar compounds in water

typically have low to very low solubility in water

pH ~7 of phosphate diesters

virtually 100% ionized

Ammonia is a considerably _____ base than hydroxide ion, but a _____ base than carboxylate ions and water

weaker; stronger

Strong Acids and Bases

~100% Dissociated in Water

For a spontaneous process

ΔG < 0

The spontaneity of a process is determined by _______ (change in free energy), not by _______.

ΔG; ΔH

Improper collagen structure is a factor in several disease conditions:

-Genetic Disorders: Mutations in genes encoding collagen peptides or other proteins associated with normal collagen production. -Autoimmune Disorders: In some cases, healthy collagen fibers are degraded. -Bacterial Infections: Certain bacteria secrete collagenase, which catalyzes the hydrolysis of collagen fibers. -Vitamin C Deficiency - Scurvy: Many effects are due to defective collagen, which prevents formation of strong connective tissue; abnormal bleeding/bruising due to rupture of capillaries, loosening of teeth, bones break easily, impairment of wound healing. Hydroyproline is not encoded in the gene for collagen peptides. Peptides are synthesized with only proline, and specific proline residues are later converted to Hyp by the enzyme prolyl hydroxylase. Vitamin C is not required for the conversion of Pro to Hyp, but is for other reactions catalyzed by this enzyme - deactivation of the enzyme via vitamin C deficiency ultimately effects normal collagen production

α-Helix

-Helix containing 3.6 amino acid residues which allows hydrogen bonding of the α-carboxyl of one amino acid with the α-amino group of the amino acid four positions further down the peptide from the N-terminus. -Atoms in interior (core) of the α-helix are tightly packed - important for stabilization of the structure via van der Waals and London forces. -Side-chains of amino acids project outward (and slightly downwards (toward N-terminus)) from the axis of helix - minimizes steric strain between side chains. -The stability of α-helix increases with number of amino acid residues; moderately-sized segments of α-helix (10-20 amino acids) are very common in globular proteins

Hydrogen Bonding

-Hydrogen bonding interactions can exist between a variety of functional groups in proteins - both backbone atoms and side chains. -Hydrogen bonding interactions are fairly weak individually, but large number of hydrogen bonds can yield considerable stabilization. Most hydrogen bonding interactions in proteins occur in the following situations: -Hydrogen Bonding between Backbone C=O and N-H Groups: Accounts for about two-thirds of the hydrogen bonds in many proteins (a-helix, b-sheet, various bends). -Hydrogen Bonding between Side-Chain Groups: Most common between side-chains of residues located within five positions of each other in the primary structure. -Hydrogen Bonding between Side-Chain and Backbone Groups: A large percentage occur at the ends of α-helixes. -At the N-terminal end of a α-helix, the first four α-N-H groups cannot hydrogen bond to C=O groups within the helix -At the C-terminal of a α-helix, the last four a-C=O groups cannot hydrogen bond to N-H groups within the helix

Salt Bridges on Exterior of Proteins (exposed to water)

-Interaction energy is strongly diminished by the dielectric constant of water. -Water: Very large dielectric constant- strongly diminishes electrostatic forces between ions.

Three types of column chromatography commonly used in protein purification

-Ion exchange chromatography -Size-Exclusion Chromatography (Gel Chromatography) -Affinity Chromatography

Functions of Monosaccharides

-Monosaccharides are a key source of energy for many organisms. Energy released upon oxidation of glycose is used for the synthesis of ATP, which is subsequently utilized to provide energy for other cellular functions. -Monosaccharides are components of complex carbohydrates (disaccharides, trisaccharides, ..., polysaccharides) and nucleic acids (DNA and RNA), and provide a source of carbon for the synthesis of other classes of biomolecules.

Muramic Acid and Neuraminic Acid:

-N-Acetylmuramic Acid -N-Acetylneuraminic Acid

Molecular Chaperones

-Newly synthesized proteins are exposed to very high concentrations of other macromolecules (~300 g/L) - large potential to form aggregates. -Molecular chaperones function by inhibiting and/or disrupting incorrect interactions between complementary surfaces - allows peptide to achieve native folding pattern without aggregating with other proteins. -GroEL/ES System (E. coli): E. coli: ~ 2400 Cytosolic Proteins. ~250 Associate with GroEL/ES ~85 do not fold properly without GroEL/ES - 13 are essential for viability of organism.

Glycosides

-O-Glycoside -N-Glycoside

Least favorable amino acids for α-helix formation

-Proline: 1. No N-H group for hydrogen bonding with a backbone C=O group of another amino acid residue in helix 2. Limited rotation around φ bond forces ~30° bend in axis of helix -Glycine: Too much conformational flexibility

Orientation of peptide chains in β-Sheets

-Same direction (parallel) -Opposite directions (antiparallel)

Determination of Amino Acid Composition: Separate and Quantitatively Analyze Amino Acids in Hydrolyzed Sample

-Small Peptides: Paper chromatography or thin layer chromatography (TLC) can be used. -Larger Peptides/Proteins: Ion-Exchange Chromatography

Collagen

-The most abundant protein in vertebrates (~30% of protein by mass) - structural component of skin, bones and teeth, tendons and ligaments, blood vessels, cartilage, cornea, and intervertebral disks. -Individual peptide chains of collagen contain approximately 35% Gly, 11% Ala, and 15-30% of a combination of proline and 4-hydroxyproline (Hyp). -The majority of the peptide chain is composed of the repeating sequence: (Gly - X - Y)n X = Pro in about 28% of cases Y = Hyp in about 38% of cases -The combination of large amounts of Pro/Hyp and Gly allows the peptide chain to form a polyproline II helix. Three peptide chains then coil to form a triple-helical structure (structure similar to rope).

Acid-Base properties of peptides

-The nitrogen atoms of peptide bonds (amides) are non-basic -The C-terminal α-carboxyl group is a little less acidic than in amino acids (pKa ~ 3) -The N-terminal α-amino group is a little less basic than in amino acids (pKBH ~8)

Melting of Water

-Unfavorable Enthalpy Change (ΔHo = 6 kJ/mol): Less hydrogen bonding between water molecules -Favorable Change in Entropy (ΔSo= 22 J/mol∙K): Less Restriction of motion of water molecules

Formation of Solvation Shell

-ΔH< 0 (favorable) -ΔS< 0 (unfavorable)

O-H Bond Distance in water

0.965 Å

O-Glycoside Formation

In the presence of strong acid (or a specific enzyme in the case of carbohydrates) as catalyst, aldehydes and ketones react with two equivalents of alcohol to form acetals. Step 1: Addition of one alcohol to form the hemiacetal. Step 2: Acid-catalyzed condensation of the hemiacetal with a second alcohol to form the acetal.

Alkylation of Sugars (Ether Formation): Other Methylating Reagents

A number of reagents are much better methylating reagents than methyl iodide (more reactive in SN2 reactions - better leaving groups), but their high reactivity renders them particularly hazardous - highly toxic, carcinogenic, mutagenic, etc. -As shown above, methylation of the methyl glycoside of a monosaccharide gives a product in which all of the OH groups of the monosaccharide have been converted to methoxy groups (OCH3). However, it is important to recognize that not all of these methoxy groups are chemically identical. The methoxy group at the anomeric carbon is part of the acetal group, whereas the others are ethers. The chemical reactivity of these groups differs substantially, in particular, with regards to cleavage via acid-catalyzed hydrolysis. -Acetals - Cleaved by dilute aqueous acid (e.g., 1 M HCl (1 M H3O+)). -Ethers - Cleaved only by heating under strongly acidic conditions (e.g., concentrated HBr or HI). Hydrolysis with dilute acid removes only the alkyl group at the anomeric carbon - the ether groups are unaffected under these conditions. -Because only the glycosidic alkoxy group is removed during hydrolysis in dilute aqueous acid, methylation followed by hydrolysis can be used to determine the ring size (pyranose vs. furanose) of a glycoside - the oxygen atom that is part of the furanose or pyranose ring system is the only hydroxyl group that will not be methylated.

Most favorable amino acids for α-helix formation

Glutamic Acid, Alanine, Leucine, Methionine, Glutamine, Lysine and Arginine - Except for alanine, all have two methylenes (-CH2CH2-) between backbone and other groups (below left).

Benedict's and Tollen's non-reducing sugars

Glycosides - since the open chain form of the sugar is required for oxidation to occur, glycosides are not oxidized by these reagents (glycosides are stable in aqueous base)

Typical Strong Acids

H2SO4, H3PO4, HCl

Arginine basic side chains

Has a very large side chain pKBH owing to resonance stabilization of the gaunidinio group-deprotonated only at very high pH.

Detergents in denaturing proteins

Hydrocarbon tails of detergents can associate with nonpolar side chains, which disrupts hydrophobic interactions

Sequencing Proteins

In the mid-1980's, techniques were developed that allowed for the routine study of proteins using mass spectrometry. While the methods used for vaporization and ionization of proteins are much different than with small molecules, determination of structure (sequencing) is achieved by the analysis of fragmentation patterns. Example: Fragmentation occurs primarily between C and N of peptide bond. When the peptide bond is cleaved, one fragment will be positively charged (detected) and the other will be electrically neutral (not detected). While having the positive charge on the carbonyl carbon is more favorable, both types of cleavage can be observed - cleavage at each peptide bond gives two detectable fragments with different masses; computers can quickly analyze mass data to determine peptide sequences. Starting at N-Terminus: -Cleavage of 1st Peptide Bond: Fragments with masses 73 and 606. -Cleavage of 2nd Peptide Bond: Fragments with masses 160 and 519

Christian Anfinsen - Studied the denaturation/renaturation of ribonuclease A

RIbonuclease A (RNase) - Catalyzes hydrolysis of RNA -- 124 Amino Acids- single peptide, 4 disulfide bonds -Mercaptoethanol: Reduces disulfide bond to sulfhydryl (-SH) groups -Oxygen: Oxidizes sulfhydryl to disulfide under basic conditions. -Ribonuclease was treated with mercaptoethanol (RSH) to reduce the disulfide bridges in the presence of high concentrations of urea to denature (unfold) the peptide - the denatured protein has no catalytic activity -Mercaptoethanol and urea were then removed by dialysis, and the denatured protein was allowed to stand exposed to air (O2 oxidizes thiol groups to disulfides) in pH 8 buffer. Within a few hours, the denatured ribonuclease regained its original catalytic activity (i.e., all protein was refolded into native conformation). *Conclusion: The information required for proper folding of a protein (3° structure) is contained in the amino acid sequence (1° structure).

Native Gel Electrophoresis

Run under conditions such that the native conformation of the protein is not disrupted. Basis of Separation: Net Charge: For example, in a mildly basic buffer solution (pH ~9), most proteins will have a net negative charge; proteins with a higher degree of negative charge will migrate towards the anode at faster rates. Size and Shape: Smaller proteins migrate at faster rates, as do those with more spherical shapes. Gives useful information about the structure of the protein, and can be used as a separation method, but not useful for the determination of molecular weights - too many variables; charge, size, and shape

SDS-PAGE: SDS: Sodium Dodecyl Sulfate (CH3(CH2)11-O-SO3-Na+)

SDS is an anionic detergent that denatures proteins via disruption of hydrogen-bonding and hydrophobic interactions. Mercaptoethanol or dithiothreitol is also often added to the sample to reduce disulfide bonds. Sample is heated to promote complete denaturation of the protein. SDS binds tightly to denatured peptides; most proteins bind about 1.4 g SDS per gram of protein (one SDS per two amino acid residues)

Williamson Ether Synthesis

SN2 Reaction of an alkoxide ion with an alkyl halide -Treatment of a glycoside* with methyl iodide or other methylating agent (below) in base converts the hydroxyl groups of carbohydrates to methyl ether groups -*Reaction cannot be used to alkylate the monosaccharide (disaccharide, etc.) due to isomerization/cleavage in basic solution; the sugar must be converted to the glycoside before alkylation to prevent these unwanted reactions

Molecular Weight Determination (SDS-PAGE)

Sample of protein to be analyzed is run side-by-side with a sample containing mixture of proteins with known molecular weight; following separation, proteins are stained. Plot of the logarithms of the molecular weights of known proteins (Mr) against migration distance is linear. -Accuracy of molecular weights determined by SDS-PAGE is on the order of 5-10%

β-Turn

Segment of four amino acids that gives 180° turn in peptide chain; glycine and proline are often involved in bends/turns in peptide chain

1111111111111111111111111111111111111111111111111111111111111Primary Structure

Sequence of amino acids in the peptide chain(s) of the protein

Denaturation of Proteins

Several conditions can result in the unfolding of a native protein to some other conformation (random coil). In many cases, the unfolded protein chains aggregate and precipitate from solution as an insoluble solid - in some cases denaturation of proteins is irreversible

Result of SDS-PAGE

The large number of uniformly-bound, negatively-charged SDS ions gives all peptides in the sample approximately the same mass-to-charge ratio. Separation of SDS-proteins by PAGE results primarily because of differences in size (not size, shape and charge).

pKBH

The larger the value of pKBH, the stronger the base. -common to use pKBH to represent the pKa of the conjugate acid of a base

Secondary Structure

The local spatial arrangement (conformation) of the peptide backbone.

Acidic Side chains of Tyrosine

The phenolic group ~100% unionized at pH 7, but will be deprotonated at high pH.

Acid-Base Equilibria

The position of equilibrium in a Bronsted-Lowry acid-base reaction favors formation of the weaker acid-base pair. For the example shown below, since CH3SH is the stronger acid (smaller pKa), the position of equilibrium lies in favor of the products.

Primary Structure of Proteins

The sequence of amino acids in peptide chain. The peptide chains of proteins, which contain hundreds of bonds along the peptide backbone, fold into specific, three-dimensional conformations in the functional protein (secondary and tertiary structure). Multi-subunit proteins require proper orientation of individual peptides (quaternary structure)

Asparagine and Glutamine side chains

The side chain amide groups are very weak bases due to loss of resonance stabilization upon protonation; in strongly acidic solution, amides are protonated at oxygen rather than nitrogen

Lysine basic side chains

The side chain amino group (pKBH 10.5) is comparable to primary aliphatic amines.

Typical Acidities/Basicities of Functional Groups in Amino Acids:

The α-carboxyl group is considerably more acidic than aliphatic carboxylic acids (e.g., acetic acid (pKa 4.76) -Conversely, α-amino groups are usually somewhat weaker bases than aliphatic amines (e.g., ethylamine (pKBH 10.8).

Linus Pauling and Robert Corey

Used x-ray diffraction to study structures of small peptides -Built accurate models of small peptides and pieced them together to predict particularly stable conformations of peptide backbones; also used x-ray diffraction data from α-keratin, a fibrous protein which has a regularly-repeating pattern in its structure.

Liquid Water

To have a higher density in the liquid state, water molecules must be more close-packed than in the solid - must have less hydrogen bonding in liquid water than in ice; each water molecule is hydrogen bonded to ~3.4 water molecules. Hydrogen bonds in liquid water are broken and formed at extremely rapid rates (lifetime of ~10^-12s). -Molecules of liquid water are highly associated, but short lifetime of individual hydrogen bonds gives fluidity

111111111111111111111Secondary and Tertiary Structure

Together, the overall three-dimensional structure of the peptide chain(s) in the protein

Sequencing Larger Peptides: Edman Degradation

Treatment of peptide with phenylisothiocyanate under basic conditions followed by strong anhydrous acid cleaves the N-terminal amino acid from the peptide as its thiazolinone derivative, which is then converted to the more stable phenylthiohydantoin with aqueous acid. Since the rest of the peptide chain is unaltered, the reaction sequence can be repeated on the shorter peptide to determine the next amino acid from the N-terminus and so forth. Automated amino acid sequencers utilize this reaction cycle to routinely sequence peptides of 20-30 amino acids.

Water and Nonpolar Compounds

Typically have low to very low solubility in water.

Several methods have been developed to determine the molecular weights of very large molecules.

Ultracentrifugation -Dynamic Light Scattering -Mass Spectrometry - Can give very accurate determinations of molecular weights of large proteins (e.g., molecular weights of > 100,000 D with accuracy > 0.01%), but require costly instrumentation and a good deal of technical expertise. Reasonable estimates of molecular weights of proteins can be obtained using electrophoresis (SDS- PAGE)

Polyproline and Polyglycine Helices

Under proper conditions, polyproline will precipitate from solution as a left-handed helix with 3.0 residues per turn and a pitch of 9.4 Å (polyproline II helix). Polyglycine can form similar structure.