CHE 4341 Exam 2

Primary Structure of Proteins

amino acid sequence & location of S-S bonds

Main bonding interaction for Primary Structure

Covalent bonds

Secondary Structure of Proteins

Interaction of amino acids close to each other to form specific patterns (eg alpha-helix, beta-sheet)

Tertiary Structure of Proteins

3D structure of polypeptide

Main bonding interaction for Tertiary Structure

Hydrophobic effect → other bonding interactions still help

Quartenary Structure of Proteins

arrangement of multiple folded protein molecules (subunits) in a multi-subunit protein

Main bonding interaction for Quatenary Structure

Hydrophobic effect → other bonding interactions still help BUT no covalent bonds or disulfide bonds between subunits

Peptide bond has _______

40% DB character (so behaves like double bond)

Nearly all peptide bonds are

TRANS; 10% that follow proline may be cis

Peptide bonds are

PLANAR → no rotation → cis/trans isomerism

Why are peptide bonds mostly trans?

Trans bonds are more stable than cis

Since no rotation can occur around a peptide bond,

rotation can only occur around ALPHA carbon

Pauling and Corey figured out the

structure of the peptide bond

Phi (Φ) Angle

For C_a-N = +180º

Psi (ψ) bonds

For C_a-C = -180º

Certain conformations (rotations around ψ and Φ are forbidden

b/c of steric hindrance

Ramachandran Plot

Calculated Plot of all sterically allowed ψ & Φ angles for a polypeptide chain

Beta sheets are on located on

top left side of the Ramachandran Plot

Green terrain

for GLYCINE; has more conformations than rest of AAs

Blue terrain

for rest of AAs (excluding glycine)

What is Protein 2º Structure?

general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids (DNA/RNA) to give regular, recurring local conformations

Types of bonding in Protein 2º Structure?

H-bonding between backbone amide NH of one AA & backbone carbonyl C=O of another

Linus Pauling with Robert Corey proposed

Alpha helix (1951)

In alpha helixes, _____ form between adjacent AA’s to give __________

H-bonds; curled polypeptide helix

Hydrogen bonds in Alpha helices occur between

n and n+4 residues

In Alpha helices, R groups point

outward ~ perpendicular to axis!

Pitch of Alpha Helices

5.4Å (0.54nm)

How many resides does it take to make one turn in a alpha helix?

3.6 residues/turn1.

What is rise?

how far the backbone moves forward along the helix's axis with each new amino acid

Rise per AA in alpha helix

1.5Å per AA

H-bond in alpha helices are

parallel to helix axis

Most alpha helices are/have

RIGHT-handed; have a dipole (polarity)

Alpha Helix Dipole

All C=O groups point toward the C-terminus give entire helix is a dipole with (+) N, (-) C-termini

Stable α-helices typically end (C-term) with a

+ charged AA to neutralize the dipole moment (C=O^d-)

Some proteins can be PREDOMINANTLY α-helices

eg: Myoglobin

Destabilizers of α-helix

1. Proline (Pro) not likely found in α-helix

2. Steric/charge repulsion is caused by adjacent bulky amino acids; also by like charged amino acids

3. R-groups too large (tryptophan, tyrosine)

4. or too small (glycine)

Amphipathic α-helices

Hydrophobic residues on one side and hydrophilic residues on opposite side

α-Keratin

coiled coil* motif of 2 amphipathic alpha helices

Where is α-Keratin found?

key structural material in hair, skin, & nails; horns that cover core of live bone

α-Keratin makes up hair via

Keratin Dimer

Keratin

disulfide crosslinks → α-Keratin in Cys → form -S-S- bonds

How do perms work (same method for relaxing curly hair)?

Reduce -S-S- bonds → 2RSH; Next curl; then re-oxidize to –S-S-

α-Keratin can be used to

detect Heavy Metal Poisoning

β-pleated strand

polypeptide chains almost fully extended

β-pleated strand are found in

β sheets

β sheets are

multiple β strands arranged side-by-side; can come from same or different polypeptide

β strands are in ______ and H-bonds occur between AAs on ________ perpendicular to strand direction

X-plane; adjacent strands

Side chains in β sheets

alternate in pointing above & below plane perpendicular to H-bonds AND strands → Z axis

β sheets can easily

accommodate adjacent bulky / like-charged residues

Antiparallel β sheets

strands run in opposite N to C- terminal direction

Parallel β sheets

strands run in the same N- to C- direction

β sheet structure is ______ conformation with ______ residues involved per strand

Extended zig-zag (ruffled); 6 to 12 residues

In β sheets, there is a repeating unit of

2 AA residues w/ distance = 7 Å (3.5 Å per AA)

β sheets may be amphipathic because

one surface of β sheet may consist of hydrophobic side chains (R gps) while other can have hydrophilic ones

Some proteins are predominantly β sheets

E.g. Concanavalin A

Silk Fibroin has

Extended layers of antiparallel b sheets of (-Gly-Ser-Gly-Ala-Gly-Ala-) → roughly alternate with Ala and Gly

The Ala in slik fibroin

Ala from 1 sheet interdigitates w/ Ala from another sheet:

Spider silk is

biocompatible → because it is protein

What is collagen?

main structural protein in the extracellular matrix (ECM) in connective tissues in animals

What is the most abundant protein in mammals which makes up 1/3 of total protein mass in large animals?

Collagen

Collagen is present in

Bone matrix, tendons, cartilage, blood vessels, skin

The Collagen Triple Helix (“Proline Helix”)

3 strands in a triple helical coil (Strong); made up of general repeating unit in each strand = G-P-P/Hyp

Why GLY in Collagen Triple Helix?

Gly residues invariant; located along central axis of triple helix and very small

Main bonding interaction in Collagen Triple Helix

H-bonding only; INTERCHAIN bonding involves HydroxyPro (Hyp) also helps stabilize helix 

Collagen Primary Structure

General Repeating unit in each strand =  …G-P-P/Hyp…

Osteogenesis imperfecta (Brittle bone disease)

a mutation in a gene for collagen leads to the substitution of Gly for another amino acid → prevents the normal production of mature collage, causing brittle bones

Formation of hydroxyPro (by prolylhydroxylase) and hydroxyLys (by lyslylhydroxylase)

requires O₂ and ascorbic acid (vitamin C)

What does Vitamin C deficiency (scurvy) impact?

• lack of hydroxyPro 

• not enough interchain H-bonding

• weak collagen (skin lesions, fragile blood vessels, bleeding gums)

Collagen Fibrils are also strengthened by

intrachain lysine-lysine and interchain hydroxypyridinium covalent crosslinks

Collage cross links accumulate over time, which causes

↑ cross-links = less elastic collagen = more brittle bones /tendons

Non-regular secondary structure turns and loops allow _____ and are found on _______

chain reversal; surface of globular protein  (hydrophillic)

The β turn allows

polypeptide in antiparallel b-sheet to reverse direction

β turns are made up of

carbonyl O’s are H-bonded to amide H’s between residues 1 and 4

What is the similarity between Type I and Type II beta turns?

Pro often 2nd residue in both types

Tertiary Structure is a result of

folding of a polypeptide chain into a closely-packed three-dimensional (3D) structure mainly b/c of  hydrophobic interactions/effect

What MAY occur due to formation of 3º structure?

Amino acids far apart in the primary structure may be brought together

3º Structure Globular (Cytosolic) Proteins are

Hydrophilic outside; hydrophobic inside

What is X-ray crystallography?

Reveals 3D structure in atomic detail → Need crystals of protein

Method of Calculating Protein Structure

diffraction pattern → Fourier transform → Computer fit map to protein sequence → Fixed (frozen) picture of protein

Resolution is measured in

Å (= 10^-10m) → Smaller the better

Negatives of X-ray crystallography

For X-ray crystallography → need protein crystals → difficult to get for many proteins

Limitations of 2D NOESY NMR spectroscopy

need high concentrations of protein, time consuming, only for small (low molecular weight) proteins

NMR Structure of

64 residue polypeptide of Src protein SH3 domain; proteins are dynamic (‘breathe’ → can be displaced up to 2Å)

What is Cryo-Electron Microscopy (Cryo-EM)?

form of transmission electron microscopy (TEM) where the sample is studied at cryogenic temperatures (generally liquid N₂ temp. = -196 ^oC!)

How are proteins categorized?

Globular, Fibrous and Membrane

Globular Proteins are

Aqueous (water soluble)

Fibrous (Rod/fiber-like) Proteins are

Normally insoluble

Protein denaturation occurs via

heating and chaotropic agents which disrupt interactions that stabilize 3º structure → Some (but not all) proteins can be refolded or renatured

Misfolded proteins get refolded or proteolyzed immediately (by proteasome) via

Polyubiquitination (Ubiquitin conjugation) and Protein degradation (proteolysis)

Who made discovered Denaturation and Renaturation of Ribonuclease A (RNase A)?

Christian Anfinsen: Showed that 1º structure determines 3ª structure →

What happened when urea and most β-ME were dialyzed out (leaving only a trace of β-ME)?

The protein spontaneously refolded into its native, catalytically active conformation. The trace β-ME prevented incorrect disulfide pairs from locking in, allowing the correct –S–S– bonds to form.

What reagents were used to denature and reduce RNase A, and what did each do?

• 8 M urea → disrupts non-covalent interactions (H-bonds, hydrophobic effects) → unfolds the protein

• β-mercaptoethanol (β-ME) → reduces disulfide bonds → converts –S–S– to free –SH groups

Its ______ to fold in vitro

EASY → where folding by dilution in buffer

Its ______ to fold in vivo

DIFFICULT → folding occurs via co-translational folding

Why difficult to fold in vivo?

cellular environment is crowded → high potential for misfolding and precipitation (aggregation)

Chaperone

proteins that help other proteins fold in vivo (although most proteins can fold without them) → also called Heat Shock Proteins (HSPs)

Large Chaperones called Chaperonins like Hsp60 (GroEL/GroES complex in E. coli)  actively fold

proteins in prokaryotes, cytosol of eukaryotes & mitochondria.

Precipitation (aggregation) is prevented/folding assisted by

DnaJ&DnaK coating the unfolded protein and preventing denaturation/precipitation

GroEL/GroES Folding Mechanism

requires energy from ATP hydrolysis

Protein disulfide isomerase facilitates

formation of correct disulfide bridges