Biochem 320 M2

What are the phi and psi angles for type 1 beta turns?

phi: -60 (pro)

psi: -90

What are the phi and psi angles for type 2 beta turns?

phi: -60 (pro)

psi: 80 (gly)

What amino acids favour an alpha-helical conformation?

met, ala, leu, asp, lys

What amino acids have low propensity and tend to be in irregular structures?

pro and gly

What 4 non-covalent forces are critical in determining tertiary protein structure?

1\.) hydrophobic effect

2\.) van der waals interactions

3\.) H-bonds

4\.) ionic interactions

How do hydrophobic interactions aid in determining tertiary protein structure?

aliphatic molecules like to interact with eachother

\-hydrophobic molecules will clump together and face their hydrophilic regions on the outside

How do van der waals interactions aid in determining tertiary protein structure?

The molecules don’t feel much of a strong attraction to one another, but their repulsion may be strong

How do H-bonds aid in determining tertiary protein structure?

The free electron pairs and empty orbitals can neutralize eachother

How do ionic interactions aid in determining tertiary protein structure?

opposite charged amino acids can neutralize eachother

What is a protein’s “native conformation”?

The low energy functional state

\-created by binding of ligands and creation of allostery

\-flexible portions of proteins

\-kinetic motions of atoms in proteins (breathing)

What is circular dichroism?

spectroscopic technique that uses differential absorption of left and right circularly polarized light

\-sensitive to secondary protein structure

\-characteristic spectra for alpha helicies, beta sheets and random coil/irregular structure

\-useful in denaturation/renaturation experiments

What is x-ray crystallography?

protein preparations used to grow crystals

\-requires very pure samples

\-crystals exposed to X-rays

\-provides information about electron density by generating a diffraction pattern

\-the diffraction pattern is used to generate a 3D electron density map

How are crystals made for X-ray crystallography?

crystal is on a loop and is mounted on a machine to align the crystal in an x-ray beam

\-the crystal is kept cold with liquid nitrogen

what are 3 pros and 4 cons to X-ray crystallography?

pros

1\.) highly detailed structures

2\.) rapid solutions

3\.) useful for large proteins

cons

1\.) requires crystal growth

2\.) crystals must diffract

3\.) structures are static

4\.) cannot see hydrogens

What is 2 dimensional NMR?

looks at the distances of protons

\-off diagonal peaks correlate to close range interactions of protons

\-combined with ideal geometry information

\-provides a range of solutions reflecting motion and error

\-a family of structures is provided in regions where there are many lines and these regions may be in motion or have few determined distances

\-regions where the lines are overlapping well are well defined regions

\-the core tends to be well defined compared to loops and termini

What are 2 pros and 2 cons of protein NMR?

pros

1\.) dynamic information

2\.) proteins are in solution

cons

1\.) difficult for large macromolecules due to overlapping

2\.) synthesis of peptides containing isotopes can be extensive and time consuming (norms of these atoms give poor NMR, so isotopes are preferred)

what is the structure of alpha keratin filaments?

alanine helices and cysteine cross links (for disulphide bonds)

\-harder structures have more disulphide bonds and softer structures have less

\-composed of dimers assembled into a protofilament which make up a microfibril

\-the two right handed chains in a dimer are twisted in a left handed super helix

what are leucine zippers?

use alpha keratin filaments

\-packed together by hydrophobic leucine zippers

\-leucine residues from 1 helix pack into holes between side chains in the other helix

\-interface between superhelixes is stabilized by ionic and polar interactions

what is collagen?

25-30% protein mass of mammals

\-provides structure and rigidity

\-extracellular matrix proteins

\-skin, bones, teeth

\-composed of a helical structure

\-left handed, interchain H-bonds (between not within)

\-three chains assemble into a right handed superhelix

\-only made of 4 amino acids (gly, pro, hydroxypro, hydroxylys)

What are 4 disorders related to collagen deficiencies?

1\.) osteogenesis imperfecta

2\.) atopic dermatitis

3\.) ehlers-danlos syndrome

4\.) scruvy

what is 4-hydroxyproline?

hydroxyl group on carbon 4

ensures appropriate collagen helix conformation

\-hydroxylation occurs after protein synthesis

\-hydroxylation requires ascorbic acid (vitamin C) to occur properly

\-enzyme to catalyze reaction is propylhyroxylase

What is 5-hydroxylysine?

appears at intervals in collagen polypeptides

\-mostly found at the ends of chains

\-requires vitamin c

cross-linking of peptides occurs through lysine and hydroxylysine residues

What are 4 similarities amongst soluble globular proteins?

1\.) mixtures of secondary structures including regular and irregular structures

2\.) have hydrophobic cores and hydrophilic interiors

3\.) have closely packed interiors by van der waals interactions

4\.) maximized H-bonds in the interior

What are 3 differences between soluble globular proteins?

1\.) secondary structure composition

2\.)prosthetic groups

3\.) presence of disulphides (only in extracellular proteins)

What are the 5 tertiary structure rules for soluble globular proteins?

1\.) hydrophobic interactions are critical (at least 2 layers present to bury hydrophobic groups)

2\.) extensive H-bonding within secondary structure (not between)

3\.) knots do not form

4\.) elements close in primary structure are usually close in tertiary structure

5\.) connections between sequential beta strands in a parallel beta sheet are usually right handed

What are the 3 different classification systems for tertiary protein structure?

1\.) SCOP: structural classification of proteins

2\.) CATH: class, architecture, topology, homology

3\.)Pfam: protein family

What are motifs?

supersecondary structure

\-recognizable combinations of secondary structure that appear in a number of different proteins

What are domains?

supersecondary structure

\-discrete, independently-folded compact units within a polypeptide

\-may be composed of or include motifs

What are 2 motifs that generate two layers of secondary structure?

1\.) beta-alpha-beta motif

2\.) alpha-alpha motif

the burial of hydrophobic R groups to exclude water requires at least 2 layers of secondary structure

What is an alpha/beta barrel?

a complex motif that’s built from repetitions of the beta-alpha-beta loop motif

\-composed of 8 simple motifs

What is a domain?

A distinct stable globular folded unit within a given polypeptide

\-more intradomain interactions than interdomain ones

What is the structure of hemoglobin?

a dimer of alpha beta protomers (alpha 2 beta 2)

What is quaternary protein structure?

multiple subunits form a protein

\-subunits form multimers

\-protomoers form oligomers

\-less DNA required, less synthesis

\-errors in translation less critical

\-

What is a multimer?

Subunits of individual polypeptides assembled to form the quaternary structure

what are oligomers?

have repeating structures

\-each repeating unit is a protomer

\-protomers are composed of more than one polypeptide

\-associated with a symmetry form

what are protein symmetries?

individual protomers may be related by rotational and translational symmetry

\-global (whole protein) or local (only some portions of the protein)

\-pseudosymmetry arises when non-identical, homologous subunits are related

what is cyclic symmetry?

Cn is N protomers arranged around 1 rotation axis

what is dihedral symmetry?

Dn is 2N protomoers arranged around 2 axes

\-1 axis is N fold, the second is 2 fold, they intersect at right angles

What is tetrahedral, octahedral and icosahedral symmetry?

12, 24 and 60 protomers arranged around multiple axes

what is helical symmetry?

protomers are related to eachother by both a rotation and a translation

\-ex.) tobacco mosaic virus

What is the subunit composition of E.coli RNAP holoenzyme? What kind of symmetry would it display?

alpha 2, beta, beta’, omega

\-no global symmetry, likely to have local symmetry between similar subunits like alpha, producing a 2 fold axis

What are 4 ways that a proteins native state can be denatured?

1\.) Heat

2\.) PH

3\.) Detergents

4\.) organic solvents/molecules (ex. acetone, guanidine-HCl)

what are 2 examples of denaturants? What are their features?

1\.) Guanidinium ion

2\.) Urea

\-chaotropic agents

\-water soluble

\-disrupt hydrophobic interactions

what are 2 examples of reducing agents? what are their features?

1\.) beta-mercaptoethanol (BME)

2\.) dithiothreitol (DTT)

\-reduce disulphide bonds

\-become oxidized in the reaction

How is denaturation a cooperative process?

transition from folded to unfolded proteins occurs over a small range

\-transition midpoint is characteristic of both the denaturant and the protein

\-unfolding decreases energy required to unfold the rest of the structure

Explain the thermal denaturation of proteins?

temperature can break weak non-covalent forces

\-transition tracked using spectroscopic markers

\-backwards S shape curve

\-cooperative process

How does renaturation of denatured ribonuclease occur?

\-removal of denaturants allows some protein to refold

\-a protein can fold into its native structure as long as no covalent bonds have been affected

\-it disulphide bonds reform before the denaturant is removed the protein may be locked into the wrong conformation

What is the folding pathway for renatured proteins?

stepwise process

\-1.) formation of secondary structures

\-2.) formation of motifs

\-3.) formation of domains

\-4.) final tertiary structures

\-after formation of secondary but prior to completion of tertiary structure, protein adopts molten globule state (contains hydrophobic core, secondary structures and a collection of dynamic structures)

What is the free energy of folding?

\-unfolded states at the top of the funnel have large conformational entropy and high free energy

\-as folding progresses, the number of states decreases and conformational entropy decreases

\-the native, folded state has the lowest conformational entropy

With relation to free energy of folding, what kind of structures form during misfolding?

amorphous aggregates and amyloid fibrils form

\-have even lower conformational entropy than the native state

\-high degree of intermolecular contacts compared to intramolecular contacts in correct folding

what 2 major protein types help to fold proteins?

1\.) molecular chaperones

2\.) isomerases

what are 2 examples of molecular chaperones?

1\.) heat shock proteins

2\.) chaperonins

what are 2 examples of isomerases?

1\.) protein disulphide isomerase (PSI)

2\.) peptide prolyl isomerase (PPI)

what are 5 similarities between chaperones?

1\.) misfolded proteins have exposed hydrophobic regions which may aggregate

2\.) molecular chaperones isolate misfolded proteins so they can’t interact

3\.) energy is typically required via ATP hydrolysis- used to unfold the protein substrate

4\.) process repeated for multiple cycles

5\.) kinetic effect

What are the 4 steps of the GroEL/GroES chaperonin complex?

1\.) GroES cap binds GroEL ring inducing a conformational change in the new cis ring, releasing the improperly folded protein into the large cavity where it can refold

2\.) cis ring catalyzes hydrolysis of its 7 bound ATPs and releases inorganic phosphates which weakens the interactions binding GroES/EL

3\.) Second molecules of improperly folded protein bonds trans ring followed by 7 ATP

4\.) cis ring released GroES cap, ADP, and a better folded protein

What is the activity of protein disulphide isomerase (PDI)?

catalyzes the shuffling of disulphide bonds to form correct bonds of the native conformation

\-has isomerase and oxidoreductase properties

\-active site contains cys

What is the activity of peptide prolyl cis-trans isomerase (PPI)?

proline residues can adopt a cis or trans peptide bond (mostly trans)

\-PPI aids in adopting a cis bond

what are protein-ligand interactions?

ligands are molecules bound to a protein in a reversible manner

\-non-covalent binding

\-binding site is complementary to ligand

\-use of h-bonds, van der waals, hydrophobic interactions, and ionic interactions

\-cooperative: sigmoidal binding curve, multiple binding sites

\-non-cooperative: hyperbolic binding curve

what 4 ways might a binding site have complementary interactions for a ligand?

1\.) charge

2\.) hydrogen bonding

3\.) non-polar/hydrophobic

4\.) shape/size

what is the formula for the dissociation constant for a protein ligand interaction?

Kd= \[P\]\[L\]/\[PL\]

what is the formula for the fraction of protein bound to ligand?

= \[L\]/ Kd+ \[L\]

what are enzymes?

mostly proteins

\-reduce times for reactions by increasing kinetic rate

\-highly specific

\-operate under mild conditions

\-may require cofactors or be regulated

what are features of an enzymes active site?

provides a favorable sequestered environment for the reaction

\-analogous to ligand binding site except it also catalyses the reaction

what are cofactors?

additional chemical components that assist in enzyme function

\-covalently or tightly associated

\-can be metal ions, coenzymes or prosthetic groups

what is a holoenzyme?

enzyme with its prosthetic group

what is a apoenzyme?

enzyme without its prosthetic group

what determines the rate limiting step in multistep reactions?

the step with the highest activation energy barrier

what is the formula for rate of reaction?

V=k(rate constant) x \[S\]

\-lowering change in activation energy increases the rate constant

what are the 7 classes of enzymes?

1\.) oxidoreductases

2\.) transferases

3\.) hydrolases

4\.) lyases

5\.) isomerases

6\.) ligases

7\.) translocases

what are oxidoreductases?

class 1 enzymes

\-includes dehydrogenases, reductases and oxidases

\-substrate oxidized is an electron/hydrogen donor

\-may see formation of a double bond on the donor

\-may use NAD

what are transferases?

class 2 enzymes

\-transfer a group from 1 compound to another

\-includes kinases

what are hydrolases?

class 3 enzymes

\-catalyze hydrolysis reactions

\-names after the substrate with the suffix -ase

\-protein + water

\-usually goes only in 1 direction

what are lyases?

class 4 enzyme

\-enzymes cleaving C-C, C-O and C-N bonds using methods other than hydrolysis or oxidation

\-will involve 2 or more compounds in 1 direction and 1 fewer in the other direction

\-when acting on a single substrate, a molecule is eliminated generating a new ring or new double bond

\-often called synthases

what are isomerases?

class 5 enzymes

\-catalyze structural rearrangements within a single molecule

\-Ex. D to L forms of amino acids

what are ligases?

class 6 enzymes

\-catalyze joining of 2 molecules with concomitant hydrolysis of the diphosphate bond in ATP or a similar triphosphate

\-often called synthetases

what are translocases?

class 7 enzymes

\-catalyze movement of ions or molecules across membranes or their separation within membranes

how is enzyme classification denoted?

EC.#.#.#.#

\#1: class

\#2: substrate

\#3: mechanism

\#4: specificity

in the 10 steps of glycolysis, what classes of enzymes are used?

1\.) transferase (2)

2\.) isomerase

3\.) transferase (2)

4\.) lyase (4)

5\.) isomerase (5)

6\.) oxidoreductase (1)

7\.) transferase (2)

8\.) isomerase (5)

9\.) lyase (4)

10\.) transferase (2)

what 4 ways do enzymes increase the reaction rate?

1\.) participating in the reaction directly or through cofactors

2\.) desolvation

3\.) proximity and orientation to reduce entropy

4\.) stabilize the transition state for preferential binding

what is general acid-base catalysis?

proton transfer to/from specific side chains in the enzyme active site is faster than transfer to/from a solvent

\-differs from specific acid/base catalysis (H+/OH- as catalyst)

\-base is just catalyst with unpaired electrons

\-acid contains a hydrogen

how does RNase A act as an acid/base catalyst?

1\.) His12 of the enzyme acts as a general base, removing the hydrogen of the OH of C2’

2\.)His119 is a general acid which promotes nucelophilic attack of the phosphate to cleave the bond

3\.) add water

4\.) His12 now acts as a general acid and breaks the bond between the phosphate and oxuygen

5\.) His119 is a general base and removes a hydrogen from water, while the new OH group can attack the phosphate

what is covalent catalysis?

a transient covalent bond is formed between enzyme and substrate

\-alters pathway of the reaction (different intermediates than in the uncatalyzed reaction)

what are nucleophiles vs electrophiles?

nucleophiles: have free electron pairs and donate electrons to attack electrophiles

electrophiles: want electrons, may be positively charged or contain a double bond

what 3 ways can metal ions participate in catalysis?

1\.) binding/orientation of substrate

2\.) stabilization of transition state

3\.) serving as reduction/oxidation centers

what is PEP carboxykinase?

a lyase

\-oxygen in pyruvate gets phosphorylated by ATP/GTP in the second step

\-assisted by magnesium (stabilizes negative charge on phosphate groups) and manganese (stabilizes negative charge on pyruvate O- group) cofactors

\-oxaloacetate has a carbon dioxide removed to form pyruvate which, with ATP/GTP, becomes PEP

how is entropy reduced by proximity and orientation?

reaction rate increases when two reactive groups are spatially constrained in the correct space and orientation

how is entropy reduced by desolvation?

water forms a shell around small molecules

\-substrate binding removes many waters, and they’re replaced by interactions with polar side chains

how is entropy reduced by induced fit?

enzymes change shape upon substrate binding

\-brings catalytic groups into orientation

\-can be used to exclude water

what is hexokinase?

a phosphotransferase

\-uses non-covalent catalysis

\-enzyme provides general base

\-works in the presence of magnesium

\-uses induced fit, changes shape dramatically when glucose and ATP bind, prevents water from hydrolyzing the phosphoanhydride

what is transition state binding?

enzymes preferentially bind the transition state

what are serine proteases?

hydrolase (protease)

\-uses covalent catalysis, general acid/base catalysis, and intermediate/transition state stabilization

\-240 aa long

\-synthesized as zygomens (inactive precursors) activated by clevage of peptide chain

\-2 domains (beta barrels) with the active site at the junction of the domains

\-extracellular proteins with disulphide bonds

\-three resides (catalytic triad) are critical in mechanism

\-specificity determined by a substrate binding specificity pocket

what is the catalytic triad of a serine protease? Additonal important residues?

1\.) His57: general acid/base

2\.) Ser195: nucleophile, sidechain OH

3\.) Asp102: modulates His57

* Gly193 and Ser195 act as oxyanion hole
* Asp194 and Ile16 cause zymogen activation, main chain cleaved between Lys15 and Ile16

what is the substrate specificity of serine proteases?

determined of the chemical nature of the specificity pocket

\-chymotrypsin cleaves peptides after large hydrophobic residues (Phe, Trp, Tyr)

\-trypsin cleaves peptides after positively charged resides (Arg, Lys)

\-elastase cleaves peptides after small, non-polar resides (Ala)

what is the oxyanion hole’s role in serine proteases?

involved in transition-state stabilization

\-formed by backbone NH groups of residues 193 and 195

\-oxyanion hole is partially positive and able to form hydrogen bonding interactions with a negatively charged oxygen that develops during the reaction

\-ideal interaction is when carbonyl carbon of peptide bond being cleaved is tetrahedral (not planar)

what is the mechanism of chymotrypsin?

1\.) general base catalysis and nucleophilic attack to form tetrahedral intermediate

2\.) general acid catalysis aids breakdown of intermediate to the acyl-enzyme intermediate

3\.) amine product is released and replaced by water

4\.) general base catalysis and nucleophilic attack to form tetrahedral intermediate

5\.) general acid catalysis aids breakdown of tetrahedral intermediate to the carboxyl product and the active enzyme

what is k1 vs k-1?

k1: constant for rate of formation of protein and ligand

k-1: constant rate for deformation of protein and ligand

how can dissociation constant be calculated using rate constants of formation and deformation?

Kd= k-1/k1

what is k2 and k-2

rate constants for formation and deformation of enzyme substrate complex and enzyme and product

\-k2 also called kcat

what are the 4 assumptions of enzyme kinetics?

1\.) steady state conditions, no net change

2\.) initially no product

3\.) excessive substrate compared to enzyme

4\.) rate of product formation =k2\[ES\]

what is the michaelis-menten equation?

V0= Vmax \[S\]/ Km + \[S\]

where Vmax= kcat x \[Etotal\] and

Km= k-1 + kcat/ k1 (represents formation and breakdown of ES complex)

\-forms a hyperbolic curve