Protein Structure and Function

Molecular Biology and Mutations

• Line length in ancestry diagrams of hemoglobin genes is proportional to DNA sequence divergence, illustrating evolution.
• Mutations in DNA can lead to:
  • Defective proteins causing diseases like cancer (kinases), cystic fibrosis (ion channels), and sickle cell anemia (hemoglobin).
  • Changes in gene regulatory elements, altering gene expression levels (overexpression, lower expression, or no expression).
  • Changes in RNA expression, including truncations, missense errors, and splicing errors.

Weak Bonds in Cells

• Determine the shape of macromolecules:
  • The double-stranded helical shape of DNA is maintained by numerous weak hydrogen bonds between complementary base pairs (A-T and G-C).
• Enable reversible self-assembly of subunits:
  • Examples include membrane lipid bilayers and protein polymers like microtubules and actin filaments.
• Determine the specificity of molecular interactions:
  • Crucial for enzyme-substrate specificity and catalysis.
• Environmental changes (pH, temperature, ionic strength) affect the strength of weak bonds, leading to denaturation (unfolding or disassembly) of molecules or aggregates.
• Multiple weak interactions result in highly specific and tight binding, requiring complementary surfaces.

Types of Noncovalent Interactions

1. Ionic bonds
   • Strong attractive forces between positively and negatively charged atoms.
   • Involve the donation/acceptance of electrons rather than sharing.
   • Strong in the absence of water but weak in its presence.
2. Hydrogen bonds
3. Van der Waals interactions
   • Weak forces resulting from fluctuations in electron clouds of closely positioned atoms.
   • Individually weak but significant when two macromolecular surfaces are in close proximity.
4. Hydrophobic interactions
   • Water forces non-polar (uncharged) surfaces out of solution to maximize hydrogen bonding among water molecules.

Protein Learning Objectives

• Familiarity with amino acids, including their codes and properties.
• Understanding the types of interactions that stabilize protein structure and their origins.
• Recognizing and understanding the formation of primary ($1^\circ$), secondary ($2^\circ$), tertiary ($3^\circ$), and quaternary ($4^\circ$) structures.
• Explaining the modular nature of proteins and its evolutionary implications.
• Understanding how proteins are classified and compared by sequence and/or structure.
• Recognizing that proteins have built-in assembly instructions.
• Understanding how protein function is defined by its 3-dimensional landscape.
• Understanding how and why proteins can be regulated (turned on and off).
• Recognizing that proteins often function in networks or multi-molecular complexes.
• Appreciating how allosteric effects lead to conformational changes and altered activity.

Shape and Structure of Proteins

• The shape of a protein is dictated by its amino acid sequence.
• Proteins fold into conformations that minimize energy.
• Proteins exhibit a wide variety of complex shapes.
• Alpha helices and beta sheets are common folding patterns.
• Helices form readily in biological structures.
• Beta sheets form rigid core structures in many proteins.
• Proteins have multiple levels of organization.
• Only a fraction of possible polypeptide chains are useful.
• Proteins can be classified into families.
• Large protein molecules may consist of multiple polypeptide chains.
• Proteins can assemble into filaments, sheets, or spheres.
• Some proteins have elongated fibrous shapes.
• Extracellular proteins are often stabilized by covalent cross-linkages.

Amino Acids

• The 20 amino acids are categorized into four groups based on their side chains.
• L isomers are found in proteins.
• The alpha carbon is a chiral center in an amino acid.
• The 20 amino acids have overlapping properties; small changes can result in big effects.
• Degeneracy of the genetic code means that each of the 61 sense codons can mutate in 9 different ways.
  • 134 of the 549 possible changes are synonymous (do not change the amino acid).
  • The rest are nonsynonymous (change the amino acid).

Primary Structure of Protein

• Formed through a condensation reaction joining amino acids.
• Linear arrangement of amino acids, written from the N-terminus to the C-terminus.
• Amino acid sequence dictates the 3D shape/structure of a protein.
• MDLY represents an example sequence.
• Peptide bonds have partial double bond character, restricting rotation.
• Rotation occurs along the polypeptide backbone.
• Ramachandran plots visualize the sterically allowed phi and psi angles.

Ramachandran Plots

• Alpha-helix: Phi ~ -57 degrees, Psi ~ -47 degrees
• Beta-sheet: Phi ~ -110 to -140 degrees, Psi ~ 110 to 135 degrees

Other Considerations

• L isomer is found in proteins.
• The alpha carbon is a chiral center in an amino acid.
• Alpha helices are almost always right-handed in proteins.

Protein Structure

• Secondary structures like alpha helices and beta sheets form the core elements of protein architecture.
• Beta sheets form rigid structures often found in the core of proteins.
• Protein conformation (shape) is determined by its amino acid sequence.
• All types of noncovalent bonds help a protein fold properly.
• Multiple weak bonds cooperate to produce a strong bonding arrangement.
• Polypeptide chains fold in 3D to maximize weak interactions.
• Hydrogen bonds play a major role in holding different regions together.
• Proteins can be denatured by chaotropic agents like urea.

Levels of Protein Structure

• Primary ($1^\circ$): Amino acid sequence
• Secondary ($2^\circ$): Alpha helices and beta sheets
• Tertiary ($3^\circ$): Overall folding of a polypeptide chain
• Quaternary ($4^\circ$): Assembled subunits
• Similar codons often generate similar amino acids, and mutations in amino acids are usually conservative.
• Some amino acids are found more frequently in helices or sheets.
• Proteins have various functions: binding, catalysis, switching, and structural roles.
• Protein structure is determined by its sequence.

Protein Domains

• Protein domains consist of independently folding stretches of amino acids.
• A single polypeptide chain can fold into one or more domains.
• Linear stretches of approximately 20 amino acids are required to span a membrane, which can help identify integral membrane proteins.

Coiled Coils

• Stabilized by the hydrophobic effect and can be amphipathic.
• Leucine zippers occur when every 3rd or 4th residue is leucine.

Quaternary Structure

• Results from interactions between multiple polypeptide chains.
• Protein domains are modular units from which larger proteins are built.

Modularity

• Proteins can be made from several domains, like the Src protein with ATP bound.
• Different sequences can lead to different architectures, such as cytochrome C, lactic dehydrogenase, and immunoglobulin fold.

Protein Families

• Serine proteases share similar active sites, even with different sequences.
• Homeodomains are separated by billions of years of evolution.
• Sequence databases can be searched using sequence motifs to find related proteins.

Evolutionary Trace of SH2 Domain

• Amino acids are colored by proximity to ligand.
• Important residues are conserved.
• Protein domains are often swapped or shuffled (e.g., EGF domain, calcium-binding domain, kringle).
• Many proteins are built from different combinations of domains.

Protein Assemblies

• Proteins can assemble into various structures such as phage tobacco mosaic virus and viral capsids (e.g., tomato stunt, polio, SV40, tobacco necrosis).
• Collagen is tough and inelastic, while elastin is stretchy, demonstrating that structure reflects function.

Disulfide Bonds

• Covalent disulfide bonds stabilize extracellular proteins.
• The question of whether insulin can refold properly after reducing disulfides and denaturing the protein is posed.

Antibodies

• Have repeated framework domains and special binding domains.
• Protein interactions typically require many weak bonds.

Enzyme Kinetics

• $Keq = 10^{10} = [AB]/[A][B]$
• A bigger $K_m$ means weaker binding.
• $K<em>m$ is the substrate concentration at half the maximal rate, $V</em>{max}$.
• The energy required to reach the transition state limits the reaction rate.

Significance of $K<em>m$, $k</em>{cat}$, and $k<em>{cat}/K</em>m$

• $K<em>m$ approximates substrate affinity; lower $K</em>m$ means tighter binding.
• $k_{cat}$ is the turnover number for the enzyme.
• At low substrate concentrations ([S] << Km), $V = (k</em>{cat}/K_m)[E][S]$.
• The ratio $k<em>{cat}/K</em>m$ is equivalent to the rate constant for the reaction between free enzyme and free substrate and measures enzyme effectiveness.

Catalytic Strategies

• Includes antibody catalysis and various enzymatic mechanisms involving cofactors like retinal and heme.
• Carbamoyl phosphate synthetase is an example of an enzyme employing specific catalytic strategies.

Multi-Enzyme Complexes

• Pyruvate dehydrogenase is a multi-enzyme complex.

Protein Networks

• Allosteric regulators bind to enzymes and alter their activity by changing the enzyme’s 3D structure.
• Regulatory binding sites are separate from the substrate-binding site (active site).
• Positive and negative regulation mechanisms exist.
• Cooperative binding by multisubunit enzymes enables quicker response to concentration changes.

Allosteric Effects and Regulation

• ATC (aspartate transcarbamoylase) converts carbamoyl phosphate + aspartate into carbamoyl aspartate.
  • Regulated allosterically, similar to hemoglobin.
  • Activated by ATP (a purine) and inhibited by pyrimidines (feedback inhibition).
• CTP (an end product) inhibits ATCase through feedback.
  alters between a relaxed state (R, active) and a tense state (T, inactive).

Protein Control Mechanisms

• Catalytic activities of enzymes are regulated by other molecules.
• Allosteric enzymes have binding sites that influence one another.
• Phosphorylation controls protein activity by triggering conformational changes.
• GTP-binding proteins are regulated by cyclic gain and loss of a phosphate group.
• Nucleotide hydrolysis allows motor proteins to produce large movements in cells.
• Proteins often form large complexes that function as protein machines.
• Covalent modifications control the location and assembly of protein machines.

Protein Phosphorylation

• Kinase insertion/addition sites are crucial for regulation.
• GTPase acts as a switch; GTP/GDP changes activation state.
• Hydrolysis of GTP leads to conformational changes that can pass along a signal.

Protein Kinases

• Src family protein kinases are mapped according to sequence, with activation occurring by sequential events.

Regulation of Protein Activity

• Proteins may be regulated by multiple mechanisms:
  1. Phosphorylation
  2. Binding to GTP or ATP
  3. Allosteric regulation
  4. Feedback inhibition

Motor Proteins

• Utilize leverage for directional movement (e.g., kinesin).
• Examples include ABC transporters.

Protein Assembly

• Assembly by proximity greatly speeds up the assembly line (e.g., PKC moving to the membrane).

Protein Degradation

• Occurs via ubiquitination.

Protein Folding

• Involves chaperones like DnaJ, DnaK, and GroEL/ES systems.
• Energy landscapes illustrate the folding process from unfolded polypeptide to native structure.

X-Ray Crystallography

• Used to determine protein structures.
• Involves diffraction patterns, electron density maps, and model building using Fourier series.