Biochem Exam 2 Stuff

Primary Structure (1°)

• Amino acid sequence

• Linear order of AA’s

Secondary Structure (2°)

Local spatial alignment of amino acid backbone without regard to side chains, α-helix, β-strands/sheets, random coil, and β-turns

Tertiary Structure (3°)

The 3-dimensional structure of an entire polypeptide, fold, biological function and catalytic mechanism

Quaternary Structure (4°)

• The manner in which the tertiary structures of two or more polypeptide chains of a protein interact

• Spatial arrangements of subunits (folded chains)

Conformation

Spatial arrangement of atoms that depend on bonds and bond rotations/torsions.

In 1930s-1940s Linus Pauling and Robert Corey determined the structure of the peptide bond by X-ray.

• 40% double bond character.

• The amide or peptide bond [(O=)C-N] is 0.13 Å shorter than Cα-N bond

C=O is 0.02 Å longer then those for ketones and aldehydes

• Planar conformation maximizes pi-bonding overlap

• Resonance gives 85 kJ/mol stability when bond is planar!

Peptide bonds are planar

Most peptide bonds are trans, 10% that follow proline may be cis

This is Cα-N bond or phi angle, Φ

• Start with fully extended protein structure

• Rotate counter clockwise start at +180° and decrease angle

• Rotate clockwise start at -180° and increase angle

Phi and Psi Torsion Angles

• For a trans peptide bond, the dihedral angle is 180° by definition (or -180°, these are the same).

• In a cis peptide bond, the dihedral angle is 0° by definition

Ramachandran Diagram

If you plot Ψ on the Y-axis and Φ on the X-axis, you will plot all possible combinations of Φ, Ψ.

Φ and Ψ Angles Define…

Secondary Structure

The α-Helix

The most favorable Φ and Ψ angles with little steric hindrance

The α-Helix forms repeated hydrogen-bonds along backbone

• n = 3.6 residues per turn

• p = 5.4 Å

• d = p/n = 5.4Å/3.6 = 1.5Å

The C=O of the n^th residue points towards the N-H of the (N+4)^th residue

• The N^……..H^……..O hydrogen-bond is 2.8 Å and the atoms are 180° in plane.

• This is almost optimal with favorable Van der Waals interactions within the helix

The N_m Helix Nomenclature

• N = the number of repeating units per turn

• m = the number of atoms that complete the cyclic system that is enclosed by the hydrogen bond.

What is the designation for an α-helix?

N_m = 3.6₁₃

Helix types: The 2.27 Ribbon

Atom (1) -O- hydrogen-bonds to the 7th atom in the chain with an N = 2.2 (2.2 residues per turn)

Helix types: 3₁₀-helix

• Atom (1) -O- hydrogen-bonds to the 10th residue in the chain with an N= 3.

• Pitch = 6.0 Å occasionally observed but torsion angles are slightly forbidden.

  • Seen as a single turn at the end of an α-helix.

Helix types: π-helix 4.1₁₆

• 4.4 residues per turn.

• Very rare!!

Beta Structures (β-Sheet)

• Hydrogen-bonding between adjacent peptide chains.

• Almost fully extended but have a buckle or a pleat.

  • Much like a Ruffles potato chip

2 Types of Beta Structures

Parallel & Antiparallel

• 7.0 Å between pleats on the sheet

• Widely found pleated sheets exhibit a right-handed twist, seen in many globular proteins

What is the repeat distance of a β-Sheet?

7.0 Å

Where is the R group at for a β-Sheet?

R group on the amino acids alternate up-down-up above and below the plane of the sheet

How long is a β-Sheet?

2 - 15 amino acids residues long

How many strands are in a β-Sheet?

• 2 - 22 strands per sheet

• Avg. of 6 strands with a width of 25 Å

Parallel β-Sheet is less stable than antiparallel

True

Antiparallel β-Sheet needs what?

A hairpin turn

What does a tandem parallel β-Sheet need?

It needs crossover connection which is right handed sense

Non-Repetitive Structures

• Turns - coils or loops: 50% of structure of globular proteins are not repeating structures

• β-bends (b-turns) - type I and type II: hairpin turn between anti-parallel sheets

What type is when the carbonyl is on the same side as sidechains?

Type II

Fibrous proteins

• Highly elongated molecules whose shapes are dominated by a single type of secondary structure.

• No significant tertiary structure arrangements.

• Usually insoluble in water.

  • Examples: Keratin and Collagen

Globular proteins

• More “compacted” molecules, containing several types of regular secondary structures.

• Usually arranged in domains with unique tertiary structure. May also contain irregular, more flexible, segments (coils).

• Usually water soluble.

  • Examples: Globin, Hemoglobin, Cytochrome c…

About 98% of proteins are globular. But some Fibrous proteins are extremely abundant.

Collagen is the most abundant protein in vertebrates!

Keratin: A Quasi α-Helical Protein

• Nails, hair, horns and feathers α or β-forms

• Over 50 variants, tissue specific

2 types of keratin

• Type I: acidic, negative charge 

• Type II: basic, positive charge 

Collagen

A triple coiled helix

Sidechain Locations in Proteins; Non-polar: (Val, Leu, Ile, Met, and Phe)

Occur mostly in the interior of a protein keeping them out of the water (hydrophobic effect)

Sidechain Locations in Proteins; Charged polar: (Arg, His, Lys, Asp, and Glu)

Normally located on the surface of the protein in contact with water

Sidechain Locations in Proteins; Uncharged polar: (Ser, Thr, Asn, Gln, and Tyr)

• Usually on the protein surface but also occur in the interior of the protein.

• Usually involved in hydrogen bonds with neighbors

Hydrophaty scale

Allows an assessment of which amino acid residues would point towards the interior (high numbers) or towards the surface (low numbers)

Protein Domains

• Many single polypeptide chains fold into multiple structural domains, each with their own function

• glyceraldehyde-3-phosphate (GAP) dehydrogenase

• The blue domain binds NAD+

• The orange domain binds GAP

• A glycolysis pathway enzyme

• Note: both domains are part of the tertiary structure of this polypeptide chain!

Supersecondary structural motifs

• Small, recognizable combinations of a few secondary structure elements that occur repeatedly in different proteins.

• They are usually smaller then protein domains and usually don’t have an independent function by themselves, but serve as building blocks for more complex structures

Protein domains

• Larger, compact and independently folding units of proteins’ tertiary structure.

• Often corresponds to a protein unit that is functional (e.g., specific biochemical function) and “evolutionary” (i.e., conserved over the evolution)

Quaternary Protein Structure

4° structure is the relative placement of different polypeptide segments

Additional levels of structural organization

• Primary structure

• Secondary structure

• Supersecondary structural motifs

• Protein domains

• Tertiary structure

• Quaternary structure

Protein structures are stabilized by…

Several different forces

Electrostatics, hydrogen bonds and van der Waals forces hold…

A protein together (tertiary and quaternary structs.)

Hydrophobic effects force global protein conformation and…

Has the greatest effect on structure and stability.

In transmembrane proteins the most hydrophobic residues are found…

In membrane spanning regions.

Peptide chains can be cross-linked by…

Disulfides, salt-bridge networks, metal ions, prosthetic groups, or other ligand compounds

Proteins refold very rapidly and generally…

In only one stable conformation.

Heating disrupts protein structure thermal vibrations and disrupt weak bonding forces.

Note that there are heat stable proteins, which have a few more hydrogen bonds and salt bridges — networks of “weak” interactions

pH changes lead to denaturation.

Protonation of amino acids leads to loss of charge and H-bonding

Detergents associate with the…

Nonpolar amino acids blocking water interactions

Chaotropic agents (e.g., Urea and Guanidinium ions)

Disrupt hydrophobic interactions by increasing the solubility of nonpolar groups (most commonly used protein denaturants, but mechanism of action not well understood).

What is the first step in protein folding?

Secondary structure formation

What is the driving factor in protein folding?

Hydrophobic affect

Proteins fold in a hierarchical way

1. Secondary structure formation (<5 ms)

2. Hydrophobic collapse (release of water molecules from protein core)

3. Formation of tertiary structure (Up to 1 s)

Folded state

Lower protein entropy, but much higher water entropy due to the release of waters from “cages” around exposed hydrophobic surfaces.

Proteins can (usually) fold spontaneously into their native states via directed pathways rather than random conformational searches.

Proteins appear to fold in a hierarchical manner, with small local elements of structure forming and then coalescing to yield larger elements, which coalesce with other such elements to form yet larger elements.

There are several local minimums that could trap a protein however the dynamic nature of a protein allows folding to a global energy minimum.

However certain proteins, and larger complexes, require help in folding

Protein disulfide isomerase (PDI)

Either reforms disulfide bonds as a shuffle mechanism or oxidizes new disulfide bonds

Molecular Chaperones

• Bind unfolded proteins and prevent improper folding.

• Especially important in multi-subunit complexes

Hsp70

Present in both eukaryotic and procaryotic cells

Trigger Factor

Prokaryotic ribosome-associated chaperon

Chaperonins

Form large multisubunit assemblies, both in eukaryotic and prokaryotic cells.

Hsp90

Among the most abundant proteins in eukaryotic cells

Some proteins have unfolded regions

An entire protein, or a protein domain, may lack a defined structure in its native state (despite of standard physiological conditions).

Such intrinsically disordered proteins (IDPs) are rich in certain hydrophilic amino acids, and lack a strong hydrophobic core.

• Sequence analysis suggests that nearly half of human proteins contain disordered segments.

• Not common in prokaryotic proteins.

IDPs tend to participate in signaling and regulation.

IDPs often adopt specific secondary or tertiary structure when they bind to other molecules (e.g., ions, proteins, nucleic acids, etc)

Misfolded proteins can…

Cause diseases

Transmissible spongiform encephalopathies (TSE) are not caused by virus or bacteria but…

Are associated with an “infectious” protein (prion)

The protein PrP has two different folds

• The “normal” fold (PrP^C) is essential to neuritogenesis, neuronal homeostasis, cell signaling, cell adhesion, and a protective role against stress.

• However, the misfolded protein (PrP^SC) causes the disease.

Contact between the (PrP^C) and (PrP^SC) leads to…

(PrP^SC)

Eating nerve tissue of infected humans or cows but not sheep leads to the disease.

The incorrect fold leads to amyloid fibrils that causes mental regression

Sheep TSE

Scrapie

Cow TSE

Bovine spongiform encephalopathy (mad cow disease)

Human TSE

Creutzfeldt-Jakob Disease

PrP^C and PrP^SC have the same primary structure, but…

Alternative tertiary structures

1958

John Kendrew, 3D model of sperm whale myoglobin from X-ray crystallography

X-ray Crystallography

X-rays are bounced off of the protein and deflected by electrons in the various atoms/bonds.

The diffraction pattern of the X-rays is measured and an electron density map is created

Amino acids structures are fit into the electron density.

Given the crystal consistency, the density maps are not as precise as they could, and…

The crystal is said to have a “resolution limit”.

X-ray Crystallography advantages:

• Crystalline proteins assume conformations that are very similar to…

That of the protein in solution (near-native structure)

X-ray Crystallography advantages:

• Most independent X-ray crystallography experiments describe…

The same conformation for the same structure (consistency/reproducibility)

X-ray Crystallography advantages:

• Many enzymes are catalytic active in the crystalline state.

Since activity is highly dependent on structure, this is strong evidence that the crystalline conformations must indeed be near-native.

X-ray Crystallography limitations:

• Need for a protein crystal

Not always possible, very specific conditions are required for each different protein/complex to crystalize

X-ray Crystallography limitations:

• There are types of molecules that are harder to crystalize or to analyze

i.e., transmembrane proteins, carbohydrates, IDPs, etc

X-ray Crystallography limitations:

• Resolution limit of…

Obtained crystals (additional data helps!)

X-ray Crystallography limitations:

• Protein structures are determined in…

A static state (conformation)

X-ray Crystallography limitations:

• Parts of protein structure might be…

Distorted by crystal packing effects

NMR Spectroscopy

Paramagnetic nuclei have interactions with external magnetic field (¹H, ²H, ¹³C, ¹⁵N and ³¹P).

Nuclei can absorb energy at particular frequencies (resonance frequencies).

Resonance frequencies are sensitive to chemical environment and nearby nuclei.

Correlation spectroscopy (COSY)

Provides interatomic distances between protons that are covalently connected through one or two atoms.

Nuclear Overhauser spectroscopy (NOESY)

Provides interatomic distances for protons that are close in space, but not necessarily connected

NMR spectroscopy advantages:

• No need for crystallization and…

Can be used to determine the protein structure in solution

NMR spectroscopy advantages:

• Provides not a single conformation, but…

An ensemble of conformations

NMR spectroscopy advantages:

• Can probe motions over time scales spanning…

10 orders of magnitude

NMR spectroscopy advantages:

• Resolution can be comparable with X-ray crystallography

NMR spectroscopy results are usually consistent with crystallographic data