Protein Structure

Protein Structure Types

1. Fibrous

2. Membrane

3. Globular

4. Intrinsically Disordered

• Need to know the general principals of each type of structure to understand health and disease related structure-function relationships

Fibrous Proteins

• Function: Provide support, shape and structure

• Structure:

  • Polypeptide chains arranged in long segments or sheets

  • Usually consists of a single type of secondary structure (alpha-helix/beta sheet)

  • Usually have a simple, repeating primary sequence pattern

Proline

• Ring structure provides hindrance to the alpha-helical conformation

• Disfavoured in both α-helices and β-sheets because it has no backbone HN to participate in hydrogen bonds

• So, proline fits well in the first turn of an α-helix but anywhere else it usually produces a significant bend in the helix

• Called a “helix-breaker”

Glycine

• Also a “helix breaker”

• Its high conformational flexibility makes it entropically expensive to adopt the relatively constrained α-helical or β-sheet structure

α-Keratin

• Found in hair, wool, skin, hooves

• The two-chain coiled coils combine into higher order structures called protofilaments and protofibrils

• The two helices in the coiled coil are held together by hydrophobic interactions between hydrophobic residues

• Same pattern of 7 residues repeats: HPPHPPP (H=hydrophobic, P=polar)

• Coiled coils found in many kinds of proteins

Epidermolysis bullosa simplex (EBS)

• One α-keratin mutation that leads to a blistering skin condition

• R211P

  • The notation means that an R (arginine), at amino acid 211 in the sequence, is mutated to a P (proline)

    • Proline causes kinks in α-helices - so disrupts the helical secondary structure of α-keratin found in skin

Disease-Related Mutations in Coiled-Coils of other Proteins

• Common

• e.g. familial hypertrophic cardiomyopathy

• Coiled-coil of myosin

Fibroin

• Consists of layers of antiparallel beta sheets

  • Parallel and anti-parallel

  • Hydrogen bonding occurs between beta-strands

  • Side chains alternate up and down

Silk Fibroin

• Protein made by silk moth (Bombyx mori)

• Primary structure → ~3000-4000 amino acids

• 45% glycine

• 30% alanine

• 12% serine

• No cysteine

  • 87% of the protein is composed of only 3 AA

• Repeating unit → -(gly-ser-gly-ala-gly-ala)_n-

• Glycine and alanine have small side chains

• Sheets stack with glycines facing each other, and ala/ser facing each other - stabilized by noncovalent interactions

• How does having small glycine sidechains make silk fibroin strong?

  • Close packing of the β-sheets and an interlocking arrangement of R groups

β –Sheets, Amyloid β Peptide and AD Pathogenesis

• Amyloid beta (Aβ) peptides are aggregation-prone peptides

• These peptides self-assemble into β-sheet–rich fibrillar structures

Collagen

• Main constituent of bones, tendons, ligaments, and blood vessels of higher organisms

• Consists of a repeating tripeptide in which every third residue is a glycine (GXY)

• X & Y are usually proline (although lysine occurs sometimes)

• Lots of the prolines are post-translationally modified to be hydroxyproline

• Secondary structure → polyproline helix:

  • Stabilized by repulsion between prolines/hydroxyprolines

Collagen building blocks:

Collagen requires the amino acids proline and lysine to be chemically modified through hydroxylation to become hydroxyproline and hydroxylysine

Collagen enzyme activation:

• The enzymes prolyl hydroxylase and lysyl hydroxylase carry out these reactions

• Vitamin C acts as a necessary cofactor and electron donor to keep iron (Fe²⁺) in its active ferrous state

Collagen and structural stability:

• Without vitamin C, proline cannot be efficiently converted into hydroxyproline

• Hydroxyproline is vital for forming and stabilizing the collagen triple helix

Membrane Proteins

• Found in the plasma membrane that surrounds a cell

• Found in the membranes of organelles

• ~30% of all genes in eukaryotes code for membrane proteins

• ~>> 30% of drug targets

• Because membrane proteins exist (at least in part) in a hydrophobic lipid environment, the organization of membrane proteins follows different rules from those of soluble proteins which exist in a polar environment

• The surface of the transmembrane regions of membrane proteins are composed primarily of amino acids with hydrophobic side-chains

• Hydrophilic residues are primarily located in the segments that protrude from the hydrophobic bilayer interior

Functions of Membrane Proteins

• Junctions → serve to connect and join two cells together

• Enzymes → fixing to membranes localizes metabolic pathways

• Transport → responsible for facilitated diffusion and active transport

• Recognition → may function as markers for cellular identification

• Anchorage → attachment points for cytoskeleton and extracellular matrix

• Transduction → function as receptors for peptide hormones

Two Types of Membrane Proteins

1. Peripheral Membrane Protein

2. Integral Membrane Protein

Peripheral Membrane Proteins

• Temporarily associated with the membrane

• They can be easily removed, which allows them to be involved in cell signaling

• Can be attached to integral membrane proteins, or they can stick into a small portion of the lipid bilayer by themselves

• Often associated with ion channels and transmembrane receptors Most peripheral membrane proteins are hydrophilic.

Integral Membrane Proteins

• Permanently embedded within the plasma membrane

• Can be classified according to their relationship with the bilayer:

  • Integral monotopic proteins

    • Span only one leaflet of the bilayer

  • Transmembrane proteins

    • Span the entire plasma membrane

Integral Membrane Proteins: Transmembrane Proteins

• Transmembrane proteins

  • Span the entire plasma membrane : Bitopic or Polytopic

• Bitopic Integral Membrane Protein

  • cross the membrane once (single-pass)

• Polytopic Integral Membrane Protein

  • cross the membrane more than once (multi-pass)

  • either an alpha-helical protein or a beta-sheet protein

Membrane Protein Organization

• The thermodynamic cost of transferring charged or highly polar uncharged compoundsi nto the non-polar hydrocarbon interior of bilayer membranes is very high.

• Two consequences:

1. Most of the amino acid sidechains of transmembrane segments must be non-polar

2. The very polar peptide bonds of transmembrane segments must participate in hydrogen bonds in order to lower the cost of transferring them into the hydrocarbon interior

• This hydrogen bonding is most easily accomplished with α-helices, for which all peptide bonds are hydrogen-bonded internally

• It can also be accomplished with beta-sheets provided that the β-strands form closed structures such as a β-barrel

Computational Studies: Predicting Membrane Protein Structure

• Transmembrane helices in integral membrane proteins are composed of stretches of about 20 predominantly hydrophobic residues

• Separated by polar connecting loops

• Transmembrane helix predictions make use of a hydrophobicity scale

• Hydrophobicity scales are composed of experimentally determined transfer free energies for each amino acid

• There are several published scales that all vary from each other a little bit - some take into account just the sidechain, some backbone plus side chain, some measure transfer to an organic solvent, some to an actual bilayer

If there’s a non-hydrophobic sidechain in a transmembrane helix…it’s there for a reason:

• Enzymatic Activity and Ion Transport

• Gly and Ser residues often form hydrogen bonds that stabilize helix-helix interactions

  • In general, seeing a polar, hydrogen-bonding capable side chain in the middle of a transmembrane helix is a big clue that it’s doing something important

Globular Protein Organization

• Globular proteins (water soluble) are arranged so that most of the hydrophobic residues (e.g. I,L,V, F, etc.) are on the inside, and most of the hydrophilic residues are on the outside

• Mutating a hydrophobic residue from the core of the protein to a polar or charged residue usually completely disrupts the structure and function of the protein

How do we use sequence data (e.g. the sequences of several protein homologs) to identify the likely hydrophobic core amino acids (even if we don’t have a 3D structure)?

Align the sequences of the homologs and look for conserved hydrophobic residues

Hemoglobin and Sickle Cell Anemia

• Glu6 → Val6 Mutation

• The mutation creates a hydrophobic patch on the surface of the beta subunit that causes hemoglobin tetramers to polymerize into long fibrils

• The mutation also confers resistance to malaria (which is why it has persisted) - sickling increases the permeability of the red blood cell to potassium ions, the concentration of potassium inside the cell decreases, this decreases kills the parasite

• The long fibrils distort the shape of the red blood cell (into a “sickle” shape)

• The distorted cells don’t fit through the capillaries and can block them and hence interrupt the delivery of oxygen to the tissues

Homology

Refers to biological features including genes and their products that are descended from a feature present in a common ancestor. Homologous features such as genes are referred to as homologs (or homologues)

Homologous genes become separated in evolution in two different ways:

1. Separation of two populations with the ancestral gene in two species → orthologs

2. Gene duplication of the ancestral gene within a lineage → paralogs

What’s the term for two related proteins from the same organism?

Paralogs

Arg 1 (Arginase 1):

• A cytosolic enzyme predominantly expressed in the liver

• Catalyzes the final step of the urea cycle—hydrolyzing L-arginine into L-ornithine and urea—to detoxify ammonia

• Deficiency in Arg 1 leads to hyperargininemia and urea cycle impairment

Arg 2 (Arginase 2):

• A mitochondrial enzyme found mainly in extrahepatic tissues (such as the kidneys)

• Primarily involved in regulating L-arginine availability, nitric oxide (NO) metabolism, and the synthesis of polyamines rather than the core hepatic urea cycle

Why make a multiple sequence alignment?

Figure out which residues have been conserved over evolutionary time-spans and thus critical for structure and function

• Infer evolutionary relationships [More a biology aspect than a human health and disease aspect]