Protein Biology Lecture: Key Terms and Concepts (Ch 1–7)
Feedback inhibition and allosteric regulation
Enzymatic reactions continue to generate new products (e.g., methionine formation) and a negative feedback loop can block the conversion of earlier intermediates (e.g., homoserine) when methionine or downstream products are in excess.
The solid line in diagrams represents feedback inhibition: enough end product stops earlier steps in the pathway.
In isoleucine biosynthesis there are two internal feedback loops:
One involves threonine (which is a precursor to isoleucine).
The other involves isoleucine itself.
Threonine is converted to isoleucine, so the regulation is quite interconnected and somewhat complex.
There can be feedback from homoserine to the original reaction to prevent overproduction of homoserine if downstream products are abundant.
Feedback can also originate from other molecules (e.g., creatinine) back to the original step, or from lysine back to the conversion of aspartate semialdehyde to downstream products.
Another example: a feedback loop from lysine can influence the conversion from aspartate to aspartate semialdehyde, and then up the pathway.
The lecturer emphasizes that the goal is not to memorize every amino acid or precursor in a diagram, but to understand how to interpret such feedback-inhibition diagrams.
Allosteric enzymes and conformational change
Allosteric enzymes have two or more binding sites that can influence one another.
Allosteric means proteins can adopt two or more conformations (slightly different shapes).
The shift between conformations affects enzyme activity and the binding site's availability.
In one common representation, the enzyme can be in an "open" form (active site accessible) or a "closed" form (active site less accessible).
ADP can bind to the enzyme and trigger a conformational change that closes or opens the active site, depending on the system; ADP binding is a form of regulation.
When the enzyme is in the open form and nothing is bound, there can be some activity; binding of ADP can activate the enzyme ensemble, i.e., positive regulation.
A larger diagram shows an active enzyme (on box) versus an inactive enzyme (off box); CTP can bind in four different places, causing the enzyme to change conformation such that the active sites are no longer accessible to the ligand.
Real-world example of allosteric regulation (ADP vs ATP)
Inactive-to-active transitions can be driven by ligands like ADP/ATP; phosphorylation state and ligand binding influence the shape and activity of enzymes.
Phosphorylation can be used as another control layer that influences allosteric behavior (see below).
Phosphorylation and covalent modification (control of protein activity)
Phosphorylation: the addition or removal of phosphate groups, changing protein conformation and activity.
A kinase transfers a phosphate from ATP to an amino acid side chain (usually serine, threonine, or tyrosine) causing a conformational change.
Example: a serine side chain is phosphorylated by a protein kinase. The ATP donates the third phosphate; ATP has 3 phosphates, while ADP has 2.
ATP: ext{ATP}
ightarrow ext{ADP} + ext{P_i} (the third phosphate is transferred to the amino acid).Resulting conformational change alters activity.
Reversibility: a protein phosphatase can remove a phosphate group, restoring the previous conformation and activity.
Covalent modifications and regulatory examples (p53, GTP-binding proteins)
Covalent modifications can alter protein location and interactions:
p53: DNA-binding domain (green) contains ~50 amino acids and is paired with a transcription activation domain; a grey domain is present but not discussed in depth.
Modifications such as phosphorylation (P), acetylation (AC), and ubiquitination affect p53’s shape and its interactions with DNA and other proteins, thereby regulating function.
Regulatory GTP-binding proteins (GTPases): act as molecular switches controlled by phosphate-state changes on GTP/GDP.
Active form: GTP-bound.
Inactive form: GDP-bound after hydrolysis; a separate process is required to exchange GDP for GTP to reactivate.
Hydrolysis: ext{GTP}
ightarrow ext{GDP} + ext{P_i} (slower intrinsic rate for GDP dissociation; quicker reactivation when GTP is available).
ATP hydrolysis and motor proteins
ATP hydrolysis enables directed movements in cells via motor proteins that walk along cytoskeletal tracks.
Conceptual cycle:
Resting motor protein has a baseline conformation.
Binding ATP induces a conformational change (a “step”).
Hydrolysis of ATP to ADP + P_i leads to a second conformational change, allowing a second step.
Release of phosphate and ADP resets the motor to the starting conformation.
This cycle produces directional, processive movement along filaments (e.g., chromosome segregation during mitosis).
Protein complexes and motor assemblies
Proteins can form large complexes using ATP hydrolysis to drive conformational changes that enable complex functions.
Example: three proteins bind ATP, change shape to grip a substrate (like a key), another ATP binds to induce another shape change, ATP hydrolysis occurs, and the complex completes a movement; ADP and phosphate are released, returning to the original state.
This illustrates how ATP-driven shape changes can produce coordinated mechanical actions within the cell.
Scaffolds and assembly of protein complexes
Scaffolds are proteins that bring other proteins together to form functional complexes.
Structure: a green scaffold with unstructured regions and structured domains (binding sites) for other colored proteins.
When bound, the scaffold helps assemble a complex; after function, the scaffold can release and be reused.
The rapid, random collisions and interactions in the cellular milieu drive assembly and disassembly of these complexes.
Video: Proteins and the protein design revolution (David Baker)
Proteins perform essentially all important cellular functions: digestion, muscle contraction, neuron signaling, immune responses, etc.
Proteins are linear chains of amino acids; there are 20 standard amino acids.
Folding: amino acid sequences determine precise 3D structures in fractions of a second; shapes enable functions.
Hemoglobin example: oxygen binding in lungs and slight conformational change to release oxygen in muscle; function is shape-dependent.
Protein folding problem: the sequence determines structure and function, but mapping between sequence and structure is extremely complex.
Protein design from scratch:
Rosetta software and computational protein design allow design of completely new proteins from computer models.
A synthetic gene is needed because there is no natural gene encoding the designed protein yet.
Once designed, a synthetic gene is inserted into bacteria to produce the protein and test function and safety.
The protein-sequence space is enormous:
A typical protein is about 100 amino acids long, and with 20 possible amino acids per position, the total number of sequences is 20^{100} \,\approx\, 10^{130} (an astronomically large space).
Why design proteins now?
Nature’s amino-acid alphabet and natural evolution sample only a tiny fraction of possible proteins; design expands possibilities to address modern challenges (disease, energy, ecology).
Examples of designed-protein applications:
Vaccines: protein particles carrying viral proteins (e.g., RSV) to enhance immune response; stronger responses than prior vaccines.
Gluten digestion: designed proteins to help in celiac