Protein Function

What are the five major functional classes of proteins?

• Metabolic enzymes are chemical catalysts that lower the activation energy of a biochemical reaction to increase the rate of product formation without altering the equilibrium constant.

• Structural proteins are often assembled into long filaments that form cytoskeletal structures involved in cell migration, chromosomal segregation, and muscle cell contraction.

• Transport proteins span membranes and function as selective pores. Passive transporters allow molecules to diffuse down a concentration gradient; active transporters act as energy-dependent pumps that transport molecules against a concentration gradient.

• Cell signaling proteins respond to changes in the extracellular environment by undergoing conformational or chemical changes that regulate cellular processes. A common mechanism to control signal transduction in cells is the phosphorylation and dephosphorylation of signaling proteins.

• Genomic caretaker proteins maintain the integrity of genetic information encoded in DNA and control gene expression. DNA metabolizing enzymes are required for DNA replication, repair, and recombination. RNA metabolizing enzymes are required for RNA transcription, processing, and stability.

How do myoglobin and hemoglobin bind and transport oxygen?

• Myoglobin has a single polypeptide chain and functions as an O₂ storage protein in muscle tissues. Hemoglobin is an O₂ transport protein that contains two α-globin subunits and two β-globin subunits; together they form a heterotetramer (α₂β₂) capable of binding and transporting four O₂ molecules at a time from the lungs to the tissues.

• Each globin polypeptide contains a single heme group that reversibly binds O₂ as a ligand. Two histidine residues in the protein—His F8 (proximal histidine) and His E7 (distal histidine)—play a critical role in O₂ binding to the heme.

• A plot of the fraction of occupied O₂ binding sites in myoglobin as a function of the O₂ concentration (partial pressure of O₂) generates a hyperbolic binding curve. An O₂ binding curve for hemoglobin generates a sigmoidal curve because of cooperative O₂ binding to the four globin subunits.

• Carbon monoxide (CO) forms a bond with the Fe²⁺ in hemoglobin to generate carboxyhemoglobin with an affinity 200 times that of O₂ binding to heme, which limits the amount of O₂ transported from the lungs to the tissues.

• O₂ delivery to tissues is also limited in the presence of CO because O₂ has a higher affinity for the heme group in carboxyhemoglobin molecules (mixture of CO + O₂) than in oxyhemoglobin (O₂ only), thereby inhibiting the conformational shift from oxyhemoglobin to deoxyhemoglobin in the tissues.

• Hemoglobin without bound O₂ molecules (deoxyhemoglobin) is in the T-state conformation (tense), whereas hemoglobin with bound O₂ (oxyhemoglobin) is in the R-state conformation (relaxed). The T-state conformation has a low affinity for O₂, whereas the R-state conformation has a high affinity for O₂.

• The T → R conformational shift is triggered by small structural changes in the F helix. These changes occur when O₂ binds to the Fe²⁺ atom and reduces its effective ionic radius so that it can move into the plane of the heme. This translocation of the Fe²⁺ ion displaces the proximal histidine (His F8) and tilts the entire F helix, resulting in numerous conformational changes throughout the tetrameric complex.

• Allosteric mechanisms control the relative amounts of hemoglobin in the T and R conformational states by shifting the equilibrium. The O₂ molecule is a positive homotropic allosteric regulator that facilitates the binding of additional O₂ molecules to other globin subunits by shifting the equilibrium toward the R state (oxyhemoglobin). Conversely, CO₂, H⁺, and 2,3-bisphosphoglycerate (2,3-BPG) are all negative heterotropic allosteric regulators that shift the equilibrium toward the T state (deoxyhemoglobin).

• The Bohr effect describes the pH and CO₂ dependence of O₂ binding to hemoglobin, in which decreased pH and increased CO₂ lead to decreased O₂ binding.

• The molecular basis of the Bohr effect is the protonation of key residues at the subunit interfaces, resulting in stabilization of the T-state conformation at low pH and CO₂ binding to the N termini of the hemoglobin subunits. Elevated pH causes deprotonation of these same residues and favors the R-state conformation.

• 2,3-BPG is a negatively charged metabolite that binds to positively charged Lys and His residues at the interface of the two β subunits in deoxyhemoglobin, thereby shifting the equilibrium toward the T-state conformation.

How do globin mutations cause diseases such as sickle cell anemia?

• The human α, β, and myoglobin genes are paralogs; that is, they all arose from a common gene, but their protein products now perform independent functions in the same organism. Hemoglobin and myoglobin genes diverged from a common ancestral gene ~800 million years ago, and the human α- and β-globin genes diverged ~300 million years later.

• About 5% of the human population contains DNA point mutations in the α- or β-globin genes; some of these mutations lead to amino acid changes in hemoglobin proteins that result in reduced O₂ transport from the lungs to the tissues (anemia).

• Sickle cell disease is a genetically inherited blood disorder caused by a single amino acid change (E6V) in the β subunit of hemoglobin. Interactions due to the hydrophobic effect between β^S subunits on different hemoglobin tetramers lead to the formation of large polymeric fibers that result in defective O₂ transport by erythrocytes.

• The β^S mutation provides a measure of resistance to malaria in heterozygous β^S individuals because red blood cells that are infected by the malarial parasite are preferentially sickled as a result of low pH. The infected sickled cells are removed by the spleen, thereby protecting these individuals from severe malaria while they have only a mild case of sickle cell disease.

How does the actin-myosin motor mediate ATP-dependent muscle contraction?

• According to the sliding filament model of muscle contraction, Ca²⁺- and ATP-mediated conformational changes in proteins that make up the thick and thin filaments in muscle cells lead to muscle contraction as a result of filaments physically sliding past one another.

• Thick filaments contain myosin protein molecules arranged tail to tail within the fiber in such a way that their globular head domains are oriented toward the two ends. Titin, the largest protein found in nature, connects the two ends of the thick filaments to anchor proteins located in regions of the sarcomere called the Z disks.

• Thin filaments consist of a polymerized actin protein complex that provides myosin binding sites for muscle contraction. Two other protein complexes are troponin and tropomyosin. Troponin is a heterotrimeric protein containing the TnC, TnT, and TnI proteins, of which TnC binds Ca²⁺, and tropomyosin is a dimeric α-helical protein that mediates myosin binding to actin.

• Muscle contraction is initiated by neuromuscular signals that lead to the release of Ca²⁺ from the sarcoplasmic reticulum, an intracellular membrane-bound compartment that contains high levels of a Ca²⁺ transport protein called SERCA. The constant pumping of Ca²⁺ back into the sarcoplasmic reticulum by SERCA ensures that muscle relaxation occurs once the neuromuscular signals are turned off.

• The muscle contraction cycle is controlled by the binding of ATP, ADP, and Pi to the nucleotide binding site in the myosin head. Following Ca²⁺ release from the sarcoplasmic reticulum, the (ADP + Pi)-bound myosin head binds to actin subunits, leading to the release of Pi, which induces the power stroke conformational change in myosin (contraction).

• The five-step muscle contraction cycle consists of the following. Step 1: The myosin head domains, which contain ADP + Pi in the nucleotide binding site, bind to the actin subunits. Step 2: Release of Pi induces the power stroke conformational change in myosin, which pulls the actin filament toward the center of the sarcomere (contraction). Step 3: Release of ADP empties the nucleotide binding site. Step 4: A new ATP molecule binds to the empty nucleotide binding site, causing myosin to be released from the actin polymer. Step 5: The ATP molecule is hydrolyzed, inducing the recovery conformational change.

• Muscle relaxation occurs when neuromuscular signals are terminated and Ca²⁺ levels in the cytosol decrease, which blocks myosin binding sites on actin. The thick and thin filaments then slip past one another in the opposite direction as the titin spring protein “uncoils” and the sarcomere returns to its elongated state.