Biochem Exam 1

van der waals

• noncovalent IMFs in between dipoles of 2 or more neutral molecules (energy: 0.4-4kJ/mol)

• ex:

  • dipole-dipole

  • dipole-induced dipole

  • london dispersion forces

• strength of x interactions depends on distance btwn 2 molecules

  • repel at <0.4 nm

  • attract at 0.4 nm

  • weaker as distance >0.4 nm

• when many are involved, weak adds up to being strong

• contribute to close packing of hydrophobic core, work together w effect

dipole-dipole

• electrostatic attraction between permanent dipoles of 2 neighboring polar molecules

dipole-induced dipole

• occurs when permanent dipole of one polar molecules induces a weak, temporary dipole in a neighboring nonpolar molecule

london dispersion forces

• induced dipole-induced dipole

• electron clouds can show asymmetrical electron density within an atom or molecule

• Such distortions cause weak, temporary dipoles to form in neighboring molecules, attracting them to one another

aqueous chemistry

• H-bond results in:

  • high bp

  • high mp

  • high heat capacity

  • high heat of vaporization

  • high surface tension

• hydration shells stabilize ions

• ionizes itself

hydrophobic effect

• when hydrophobic molecules cluster together, total nonpolar surface exposed to water decreases

• ordered water is released, increasing S of water

  • S inc makes deltaG more negative, making process spontaneous

  • drives micelle formation, membrane formation, burial of hydrophobic residues in proteins

buffers

• resist change in pH

• consist of weak acid (HA) and conjugate base (A-)

• most effective when pH is within 1 unit of pKa of weak acid

titration curve

• at pKa values, you have half species in protonated and half in deprotonated

• curve relatively flat around pKa (pH doesn’t change much)

• steep at equivalence pts (pH changes drastically)

spon at all temps

• negative deltaH (exo)

• increase in S/ >0

spon at low temps

• negative deltaH (exo)

• decrease in S/ <0

spon at high temps

• positive deltaH (endo)

• S increases/ >0

non spon

• positive deltaH

• decrease in S/ <0

standard

• pH = 0

• [Mg2+] n/a

biochemical standard

• pH = 7

• [Mg2+] = 1-5 mM

Polar aa

• S anta’s

• T eam

• C rafts

• N ew

• Q uilts

• Y early

• plus His, and Gly

nonpolar aa

• A lan

• V isits

• L ondon

• I n

• M ay

• F or

• W inston’s

• P arty

Acidic aa

• net negative at pH 7 bc pka is 4 so protons lost to solvent (carboxyl deprotonated, amino protonated)

• Asp (D)

• Glu (E)

basic aa

• net positive at pH 7 bc pKa is higher so they hold onto protons (but carboxyl deprotonated and amino group protonated)

• Lys (K)

• Arg (R)

isoelectric point

• pH where amino acid has no net charge

• non-charged: average of the 2 pKas

• basic: average of 2 highest pKas

• acidic: average of 2 lowest pKas

protonation states of aa

• pH > pKa = deprotonated

• pH < pKa = protonated (pH low = lots of H+ in solution, lots of things get protonated)

uv absorption

• aa with aromatic side chains will absorb uv light at 200-400 nm due to pi electron systems

  • Phe, Tyr, and Trp

anion exchange

• uses positively charged resin to bind negatively charged aa

• target acidic aa

cation exchange

• used negatively charged resin to bind positively charged aa

• targets basic aa

peptide bond formation

• thermodynamically unfavorable

• ATP/GTP investment needed to drive x during protein synthesis

• amide linkage btwn alpha-carboxyl of 1 and alpha-amino of other w/ release of water (condensation rxn)

• N-terminus to C-terminus

disulfide bond

• 2 cysteine residues can be oxidized to form covalent x (cystine)

• stabilize protein structure, especially in secreted proteins

• formation requires oxidizing conditions (typically outside the cell or in the ER)

• can be intrachain (w/in one polypep) or interchain (btwn polypeps)

ion-exchange chromatography

• aa separated based on net charge

• low pH, aa positively charged and bind to cation exchanger

• gradually inc pH causes aa to elute in order of pI values

  • acidic aa elute first (low pI), basic elute last (high pI)

• unbound elute

• bound eluted w/ buffer (raise salt conc, competing ions for resin)

polypeptide

• 20 residues

oligopeptide

• 4-20 residues

protein

• one or more polypeptides folded into functional unit

peptide backbone

• rigid; C-N bond cannot rotate freely

  • C-N bond shorter than typical but longer than C=N

• planar; all 6 atoms of group lie in same plane

  • stabilized by 80-90 kJ/mol of resonance energy

• φ (phi) and ψ (psi) rotate

  • conformational flexibility arises

• ω is essentially locked

• EACH a-C ACTS LIKE HINGE, CONNECTING 2 RIGID PLANES, ALLOWING ROTATION ONLY AROUND PHI AND PSI

polypeptide backbone

• each peptide bond has permanent dipole moment

• dipoles align in alpha helices and create net dipole along helix axis

  • can stabilize charged groups at helix termini

• chemically unreactive under phys conditions despite polarity

primary

• determined by gene sequence

• linear aa sequence

  • order dictates folding

secondary

• alpha helices and beta sheets

• stabilized by backbone H-bonds from amino H to carboxyl O

tertiary

• 3D folding of single peptide to max stability

• helices and sheets pack together closely

  • minimize empty space

  • maximize VDW contacts

  • connecting loops are short

• stabilized by:

  • H-bonds

  • ionic interactions

  • van der Waals

  • hydrophobic interactions

  • disulfide bonds

quaternary

• subunit organization

• assembly of multiple polypeptide subunits

• stabilized by:

  • H-bonds

  • ionic interactions

  • van der Waals

  • hydrophobic interactions

  • disulfide bonds

globular protein

• marginally stable, net deltaG -20 to -40 kj/mol

  • facilitates motion, conformational dynamics

• compact, roughly spherical

• water-soluble

• surface includes water molecules, backbone and side chain make H bonds with them

• enzymes, transport, regulation

fibrous protein

• long, rod-like shape

• water-insoluble

• primarily structural roles

hydrophobic protein core

• nonpolar residues buried in x

  • primary driving force for protein folding

• releases ordered water

• entropy gain

ionic interactions

• form between oppositely charged side chains (salt bridges/ion pairs)

• found on protein surfaces (burying charges in core is unfavorable)

• pH-dependent

  • changes in pH can disrupt x and cause conformational changes or denaturation

steric constraints of psi/phi

• only 20% of phi/psi space is sterically allowed

• proteins adopt specific, repeatable a-helix, b-sheets

ramachandran plot

• maps phi vs psi for all residues

phi

• closed loop, φ

• rotation around a-C—N

• where conformational flexibility arises

psi

• trident looking, ψ

• rotation around a-C—C

• where conformational flexibility arises

a-Helix plot

• phi: -60

• psi: -50

B-sheet plot

• phi: -120 to -140

• psi: 120-140

• inter-strand H bonds

left-handed a-Helix

• phi: 60

• psi: 50 (rare)

B turn

• reverses polypeptide chain direction

  • connect adjacent strands in antiparallel

• 4 residues stabilized by i+3 H bond

• enriched in proline and flucine

• found on protein surface, exposed to solvent

a helix

• 3.6 residues per turn

• 1.5 A rise per residue

• 5.4 A pitch per turn (vertical distance to make 360)

• R groups project outward

• H bonds nearly parallel to helix axis

• i+4 H bond pattern

  • C=O forms Hbond w N-H, four residues ahead

• dipole: N-terminus (+), C-terminus (-)

proline

• helix breaker

• causes kink

• no N-H for H bond

glycine

• helix breaker

• too flexible

• destabilizes helix

• only achiral aa

helix formers

• A; small

• L; hydrophobic, good packing

• M; flexible hydrophobic

• E (glutamate); can form salt bridges

• K (lysine); can form salt

b sheet

• composed of B strands in extended conformation

  • connected by inter strand H bonds

• antiparallel

  • strands run in opposite directions

  • linear H bonds

• parallel

  • strands run in same direction

  • angled H bonds (weaker)

• R groups alternate above and below plane

• amphipathic sheets; one face hydrophobic, one hydrophilic

unfolded state

• high entropy, many conformations

• high energy

• exposed hydrophobic residues

folded state

• low entropy, single conformation

• lower energy

• buried hydrophobic residues

folding funnel

• wide at top; many unfolded conformations

• narrow at bottom

  • single native state

  • proteins roll downhill to native state

• CHAIN MOVES FROM HIGHLY DISORDERED TO MORE ORDERED NATIVE CONF

molten globule pathway

1. unfolded

2. molten globule; intermediate w native like 2nd structure, loose 3 packing

3. native

Common for medium proteins

domain by domain

• each domain folds independently

• domains then assemble

• common for large, multi-domain proteins

two state/cooperative

• unfolded → native

• no intermediates

• common for small proteins

motif

• supersecondary structure

• combo of 2nd structure elements that recurs across many proteins

  • B-a-B, helix-turn-helix

• not independently stable

• don’t function alone

• role is structural organization

domain

• 3 structure

• compact region of polypeptide that folds independently

• maintains its own stability thru side chain interactions (H bonds, ionic, disulfides)

• role is functional specialization

  • unique protein specific functions

• single protein can have multiple domains

coiled coil

• superhelical motif

• 2 or more right handed helices twist together form left-handed superhelix

• 7 aa repeat, hydrophobic at a and d, form interlocking stripes

• 3.5 residues/turn (3.6 in regular)

• ex: a-Keratin

collagen

• fibrous protein

• forms unique triple helix bc:

  • Glycine every 3rd residue

    • no side chain allows tight packing for x helix

    • essential bc R group small enough to fit center of tightly packed helix

  • proline, hydroxyproline (not codon encoded, post-translational mod) helix breakers

    • no amino H for bonding

    • suit constraint of phi -60, psi 120

protein surfaces

• x are complementary to ligands

  • shape

  • chemical

    • donors and acceptors

    • (+) and (-)

    • hydrophobic patches and hydrophobic ligands

metamorphic proteins

• proteins exist as ensemble of structures w similar energies and stabilties

• consequence of marginal stability and dynamism

• mutations can change structure dramatically

  • strep, prion

multimeric proteins

• symmetric arrangements of asymmetric objects

• reasons:

  • error reductions

  • allosteric reg

  • cooperativity

  • smaller genes encode smaller subunits, less DNA required to build large complexes

anfinsen’s RNase A experiment

• proved that sequence determines native, bio active structure

• unfolded ribonuclease A by disrupting weak interactions and disulfide bonds

• reoxidize while denatured, random disulfides, inactive

• showed it could spontaneously refold back into active form when normal conditions restored

chaperones

• prevent misfolding and ptn aggregation

• shield hydrophobic regions of nascent/unfolded ptns

• provides protected folding environments

• rescues misfolded proteins

protein isolation

• cells collected from tissue or cell culture

• cells lysed on buffered solution, sonicated, sheared, or incubated in mild detergents to disrupt membranes

• protease inhibitors, reducing agents, and cold temp used to keep protein intact and active

gel filtration chromatography

• molecules separated based on size

• large proteins elute first

affinity chromatography

• separates proteins (purification) based on ptn-ligand interactions

• ligands chemically immobilized on beads and bind to protein (so it doesn’t elute yet)

• non-binding proteins elute first

• bound are eluted by adding competing ligand to column

gel electrophoresis

• native gels separate by size and charge

  • denaturing gels separate primarily by size

• polyacrylamide or agarose gels prepared and samples added into wells on gels

• electric current applied thru gel to pull molecules thru system

• molecules visualized by staining

• smaller molecules move faster

SDS-Page

• SDS-polyacrylamide Gel Electrophoresis

• proteins boiled on a solution of SDS to denature them and give them a net (-)

• reducing agent (DTT) also added to break disulfide

• most accurate for size estimate

• protein shape not a factor bc denatured ptns coated by sds

2-D gel electrophoresis

• ptns first separated according to charge as a function of pH by isoelectric focusing

• they loaded on SDS PAGE gel and separated by mass

x-ray crystallography

• for protein structure determination

• x-ray beam aimed at ptn crystal, e- in it will diffract x-ray

• patterns captured by detector, dataset interpreted by software

• limitations:

  • growing crystals is difficult

  • captures static, non-phys state

  • may not represent solution dynamics, ptn locked in crystal lattice

nmr

• reveals relative locations of atoms w magnetic properties of nuclei

• 1H, 15N, 13C most common nuclei used in biochem

• provides solution state structures and uniquely suited to study dynamics and partially folded states

• limitations:

• large molecules difficult to be easily elucidated

• high protein conc required

• many spectra needed to map a structure

• distances btwn atoms approximated; can be multiple allowed structures

alphafold

• machine learning predicts 3d structure from sequencce

• database w known proteins

• does not capture dynamics, alt states, or ligand binding

• x-ray, cryo-em, nmr remain essential for validation

proteome

• gives more accurate reflection of what cell is doing at any moment in time than genome/transcriptome

  • bc ptns are agents of cellular function

• genome→transcriptome→(post-translation mods) x

  • 20-25k genes→100k transcripts→1M ptn species

diabetic ketoacidosis sxs

• rapid, deep breathing

• sweet/fruity odor on breath

• excessive thirst/urination

• nauseated + confused

diabetic ketoacidosis labs

• low blood pH

• low pCO2

• low [bicarbonate (HCO3-)]

• high blood glucose

• strongly (+) serum ketones

• high anion gap

  • diff btwn measured Na+ cations and Cl- + HCO3- anions = unmeasured anions

t1d

• no insulin

• cells can’t take up glucose

• liver breaks down fatty acids

• ketone bodies produced

  • almost entirely deprotonated at phys pH

  • releasing h+ into blood, making it more acidic, overwhelming buffer

bicarbonate buffer equil

CO₂ + H₂O = H₂CO₃ = H⁺ + HCO₃^-

dka

• ketone bodies release H+

• H+ reacts w bicarbonate buffer, breaks down into CO2

• bicarbonate consumed

• CO2 blown off by lungs, pCO2 drops