Biochemistry: Protein Folding, Analytical Techniques, Hemoglobin/Myoglobin, and Antibodies

Comprehensive vocabulary flashcards covering protein folding thermodynamics, structural methods, analytical separation techniques, hemoglobin and myoglobin allostery, and antibody structure and immunological assays.

Tertiary Structure ($3^\circ$)

The overall three-dimensional arrangement describing the positions of all atoms in a protein, including the polypeptide backbone and amino acid side chains, built upon secondary structure elements.

Alpha helices, beta sheets, and a mixture of both

Major forms of tertiary structure

Globular and fibrous

Globular Proteins

round, compact, and water-soluble conformations that perform diverse functional roles and comprise the majority of known proteins.

Fibrous Proteins

Proteins characterized by extended secondary structures that are water-insoluble and serve primarily structural roles in biological tissues.

Collagen

A fibrous structural protein

Has an extended secondary structure

Primary component of connective tissue

composed of three left-handed helical polypeptide strands ($3\,\text{amino acids/turn}$) wound together into a right-handed triple helix.

Collagen Primary Sequence Repeat

The repeating tripeptide motif $\text{Gly-X-Y}$, in which $\text{X}$ is frequently proline and $\text{Y}$ is frequently hydroxyproline.

Glycine in Collagen

The smallest amino acid residue is positioned at every third position to fit inside the tight inner spaces of the triple helix

mutation or substitution with alanine causes structural distortion.

Proline and hydroxyproline

Force twists

stabilized core of helix via hydrophobic interactions

Create H-bonds between backbone atoms

Collagen Interstrand Hydrogen Bonding

Backbone hydrogen bonds formed specifically between the carbonyl oxygen ($C=O$) of a proline residue on one strand and the amide hydrogen ($NH$) of a glycine residue on an adjacent strand.

Occur between polypeptide strands

Globular proteins

Majority of proteins

Location of AAs varies based on polarity

4 structures = motifs and domains

Motif (Supersecondary Structure)

A recognizable folding arrangement involving multiple secondary structural elements and their connecting loops, such as $\beta\text{-}\alpha\text{-}\beta$, $\beta$-hairpins, $\alpha\text{-loop-}\alpha$, or $4\,\beta\text{-strands}$ folding onto one another.

Protein Domain

Portion of a protein with a defined fold

Fold = 3 prime structures

Provides clues about function

Rossmann Fold

A specific protein domain motif/fold that specializes in binding dinucleotide cofactors.

Primary sequence dictates what

Dictates 3 prime

Native protein

correctly folded state

RfaH

A transcription factor known for its ability to dramatically switch its tertiary fold between alternative conformations.

Arc

A repressor protein whose overall fold changes drastically upon a change in its primary amino acid sequence.

Protein stability

Can undergo reversible denaturation and renaturation

Equilibrium is reached between folded and unfolded forms

Stability equation

($U \rightleftharpoons N$), calculated as $\Delta G = -RT \ln(K_{eq})$ ;

where unfavorable G = positive and less stable protein

Favorable G = negative and more stable protein

Steps of polypeptide folding into a protein

1. 2 prime structures form in unfolded chain

2. 2 prime structures interact forming 3 prime (molten globule)

3. Native 3 prime forms = protein folded

Molten Globule

well-defined 2 prime and loosely defined 3 prime

Free energy funnel

Higher delta G = unfolded + less stable

Negative delta G = native and stable

Width free energy funnel

entropy of polypeptide chain

Height of energy funnel

Free energy difference between folded and unfolded

Local minima free energy funnel

Intermediate folded states

Top and descend free energy funnel

Top = sequences are unfolded so there are fewer conformational restrictions

Descend= polypeptide becomes more restricted

Thermodynamic contributions to folding (entropic)

Stabilized by non-covalent interactions, hydrophobic effect

Hydrophobic effect: burial of nonpolar AA side chains (biggest contributor)

Chain entropy: loss of conformational freedom in polypeptide (negative contributor)

Thermodynamic contributions to folding (Enthalpic)

formation of H bonds, ionic, van der walls (small contribution)

Net protein stability

Difference between favorable and unfavorable aspects

Denaturing of protein

reversible as long as AA chain stays intact

heat

breaks noncovalent bonds

pH

Alters ionization states, affects ionic and H bonds

chemicals

disrupts hydrophobic effects

Chaotropic agents

Salts that disrupt hydrophobic effects

disorders water around unfolded protein

6M guanidinium chloride

Tm

melting temp 50% of protein has become unfolded

Chaperones

help fold native states

has various roles

Roles of chaperones

bind to folding intermediates to prevent aggregation

Closed chamber for folding individual protein

Catalyze disruption and reformation of covalent bonds or isomerization

Provide disulfide isomerase

prolyl cis/trans isomerase

Disulfide isomerase

Break and reform S-S bonds

Prolyl Cis/Trans isomerase

Catalyze the switch between the two

slow process

Protein structure prediction

based on 3 prime

Homology: template with similar proteins

De nova: AA

AlphaFold: 3D based on sequences

Misfolding

dangerous

Hydrophobic regions exposed = aggregated

chaperones prevent this

Amyloid

Extended oligomer beta-sheet structure = protein aggregate

Occurs in structures with beta strands in native states

Proteins unfold to molten globule form

Fibrils

partially unfolded

Purification

Separate from other proteins

Based on size, charge, and sequences

SDS page

Protein separation

Common with nucleic acids

Positive electrode at the bottom

used to unfold protein and coat with negative charged

• Reductant used to break covalent disulfide bonds

Separates based on mass

Steps of an SDS page

1. proteins unfolded with SDS

2. Coated with negative charge via SDS

3. Run through gel

4. Proteins visualized by Coomassie blue

pI

= pH values where net charge = 0

Relationship between pH and pI

pH > pI = protein more protonated (lower pH)

pH < pI = protein less protonated (higher pH)

Isoelectric focusing gel

pH instead of SDS to induce protein migration in electric field

stable pH gradient is established

Mixture added and field applied

Proteins migrated towards opposite electrode

Stops moving when net charge = 0

pH and pI on charge

pH < pI = positive

pH > pI = negative

2-D gels

combines SDS page and IEF

Separates based on charge and size

isoelectric = separate by pI / charge

SDS = separates by size

Spec detection

Aromatic AA absorb 275-280 nm

W, Y, F

Beers Law

determine concentration of proteins

A = e * c * I

A= abs

c = conc

I = length

E = proportional to W, Y, F

Fractionation by Ammonium sulfate

Salting out based on solubility

more salt = more soluble

Column Chromatography

separate proteins

mobile phase: buffer used to wash

stationary: solid mixture, interact with/bind protein

eluate: buffer/protein coming off column

Ion Exchange Chromatography

separates proteins based on charge;

anion exchangers utilize a positive stationary phase to bind anions (-)

whereas cation exchangers utilize a negative stationary phase to bind cations (+)

elution: high salt = greater net charger

• less bound = first

• more bound = last

Gel Filtration Chromatography

Size-exclusion chromatography

using porous beads in which large proteins cannot enter the pores and elute first

whereas small proteins penetrate the pores and elute last.

Affinity Chromatography

A purification technique

relying on immobilized target-specific ligands (stationary phase)

• such as nickel or cobalt matrices binding polyhistidine tags

• Bound proteins can be eluted with imidazole

which are eluted competitively using free ligand or imidazole.

Protein sequencing

based on level of structures

1 = AA sequences

2 + 3 = 3D fold

4 = number of polypeptides and arrangement

Primary structure determination steps

1. reduction of disulfide bonds

   1. Use DTT or BME to break

   2. Add Iodoacetate to prevent reformation

2. Fragmentation of protein chain

   1. Endopeptidases that recognize certain AAs and cleaves them

3. Determine sequence

   1. Mass spec

   2. ESI = size

   3. Tandem = sequence

Electrospray Ionization (ESI) Mass Spectrometry

A soft ionization mass spectrometry

Mass of protein

that ionizes intact polypeptides without fragmentation, generating a characteristic Gaussian distribution of peaks that each differ by a charge of $+1$.

Tandem Mass Spectrometry (MS/MS)

Determine protein sequence

Each peak represents mass of one AA

1. Fragment with peptidase

2. MS-1 used to isolate peptides

3. Break peptide bond by He (collision cells)

   1. Generates b and y-series ion fragments

   2. N-terminal ($b$-series)

   3. C-terminal ($y$-series)

Peptide sequencing by MS/MS

MS-1: Fragmented protein into peptides

MS-2: Fragments individual peptides from MS-1 to give same charge

y= 1st peak = C term

b = 1 st peak = N term

• Can’t determine Leu or Ile or Gln and Cys

Oxygen binding proteins

Proteins can bind ligands

Binding can induce conformational changes critical for protein function

Mb and Hb both bind O2

Protein ligand binding

P + L —> PL

Ka

$$K_{a}=\frac{[PL]}{[P]+\left\lbrack L]\right\rbrack}=\frac{1}{K_{d}}$$

Protein ligand dissociation

PL —> P + L

Dissociation Constant ($K_d$)

The equilibrium constant defined as $K_d = \frac{[P][L]}{[PL]} = \frac{1}{K_a}$, which numerically equals the free ligand concentration ($[L]$) required to achieve half-maximal fractional saturation ($Y = 0.5$).

Bound fraction

$Y=\frac{[PL]}{[PL]+\left\lbrack P\right\rbrack}$ or $Y=\frac{[L]}{[L]+Kd}$

Binding graph

Y vs [L] = hyperbola

[L] = required to occupy 50% of sites

Myoglobin

1 polypeptide (no 4 prime structure)

binds strongly to O2

O2 storage is the function

Hemoglobin

4 polypeptides

Has a 4-prime structure

Has variable binding to O2

O2 transport

Globin

Myoglobin and hemoglobin

similar 3D structure and can bind to heme

Globin Fold

A conserved tertiary architecture consisting of 8 $\alpha$-helices (labeled A through H), where helices E and F sandwich the heme group and helices C and D cap the heme pocket.

A is closest to the N-terminus

Globin protein conservation

Share the same fold

Sequence conservation is low

Conserved residues is His f8 and his E7

Proximal Histidine (His F8)

The conserved 8th amino acid residue of helix F that coordinates directly to the $Fe(\text{II})$ atom of the heme prosthetic group.

Distal Histidine (His E7)

The conserved 7th amino acid residue of helix E that forms a stabilizing hydrogen bond with the bound diatomic oxygen ($O_2$) molecule.

Globin’s bind O2

O2 has low solubility in aqueous solution

O2 reacts with AA to form free radicals

O2 binds to proteins using heme cofactor

O2 binds reversibly to heme

Heme group

binds O2 when in Fe (II) state

makes 6 covalent bonds

• 5 with heme and protein

  • 4 directly to heme

  • 1 by protein

• 1 is available for O2 binding

Then it is covalently anchored by proximal His

Heme - O2 complex

Proximal + heme = covalent

distal = H-bonds to O2 molecule

O2 binding curve myoglobin

hyperbolic

O2 interacts independently with binding sites on Mb

YO2 (y) and PO2 (x)

Hemoglobin

4 subunits

2 alpha and 2 beta

4 primary structure is noncovalent between alpha and beta subunits

can be in oxy or deoxy form

Conformational change happens under O2 binding

Conformational changes in hemoglobin

Change in interaction between alpha and beta

Structural changes within a single globin subunit

Deoxy (T state) and then O2 binds = Oxy (R state)

Steps for hemoglobin change

1. When O2 binds iron = heme goes from domed to planar form

   1. Flattening of heme and pulls proximal His

2. Pulling on prox His residues shifts the entire F helix

3. Shifts helix F, and alters noncovalent interactions across the $\alpha_1\beta_2$ interface.

   1. Ionic interactions in T state are disrupted and weakened = T to R state

4. C term His on beta sheet falls into central cavity = R state

O2 binding curve Hb

sigmoidal

due to cooperativity

Cooperativity (in Hemoglobin)

binding of 1st ligand changed affinity at other site

• multiple binding sites for same ligand

Positive cooperativity

binding of 1st ligand increase affinity for 2nd

Negative cooperativity

Binding of 1st decreases affinity for 2nd

Allostery

Binding of a ligand at one site causes a change that affects others

Homotopic allostery

Allostery between 2 of the same ligand

Binding of X changes affinity for next X

Heterotopic allostery

Allostery between 2 different ligands

Binding of X affects affinity for Y

CO2 + BPG + H+ decreases affinity of O2

Bohr Effect

Impact of pH on Hb affinity for O2

pH affects stabilization of T vs R

T: low O2 affinity

R: high O2 affinity

Higher pH vs Lower pH Bohr effect

higher = stabilizes R state and increase O2 affinity

lower = stabilizes T state and promotes O2 release

2,3-Bisphosphoglycerate (BPG)

A glycolytic metabolite formed during glycolysis

binds to the central cavity of hemoglobin via hydrogen and ionic bonds

stabilizing the low-affinity T (deoxy) state and facilitating oxygen unloading

Antibodies

Proteins made by B-cells

Ig

Bind antigens

Antibody structure

Y-shape

2 heavy and 2 light

H2L2

chains linked via disulfide bonds

Disulfide bonds

link heavy chain to heavy chain

heavy chain to light chain

Ig types

many type

each differ in heavy chain = isotypes

different 1 prime and 4 prime structure

same 2 prime and 3 prime structure

different functions

Immunoglobulin Fold (Ig Fold)

Motif that consists of a pair of antiparallel $\beta$-sheets,

• with one sheet containing $4\,\beta\text{-strands}$

• other containing $3\,\beta\text{-strands}$.

All have Ig fold

Constant region

AA sequence is same in all antibodies from same organism

Variable region

AA differs based on antigen that it binds

Has antigen-binding sites