Molecular Biology of the Cell Exam Review

These flashcards cover key vocabulary terms and concepts including protein structure, function, regulation, and techniques used in molecular biology.

Protein

A molecule made from a long unbranched chain of amino acids.

Polypeptide Backbone

The repeating sequence of atoms along the core of a protein.

Steric Requirement

Spatial arrangement of atoms that influences the folding of proteins.

Noncovalent Bonds

Interactions between molecules that do not involve the sharing of electron pairs.

Amino Acid

Organic compounds that combine to form proteins, containing an amino group and a carboxyl group.

Peptide Bond

A covalent bond formed between amino acids during protein synthesis.

Optical Isomers

Compounds that are mirror images of each other, such as L and D amino acids.

Alpha Helix

A common folding pattern in proteins formed by hydrogen bonds.

Beta Sheet

A secondary structure of proteins, consisting of parallel or antiparallel strands.

Allosteric Regulation

Regulation of an enzyme or protein by binding an effector molecule at a site other than the active site.

Feedback Inhibition

A process in which the end product of a metabolic pathway inhibits an earlier step.

Phosphorylation

The addition of a phosphate group to a protein or molecule that can change its activity.

Ubiquitin

A small protein that tags other proteins for degradation by the proteasome.

Mass Spectrometry

An analytical technique used to measure the mass-to-charge ratio of ions.

Fluorescence Microscopy

A microscopy technique that uses fluorescence to study properties of organic or inorganic substances.

SDS-PAGE

A method for separating proteins based on their molecular weight through gel electrophoresis.

Hybridoma

Cells produced by the fusion of an antibody-producing B-cell and a myeloma cell.

Chaperones

Proteins that assist in the folding of other proteins.

Quantum Dots

Nanometer-sized semiconductor particles that are used in fluorescence imaging.

Coiled-Coil

A structural motif in proteins where two or more alpha helices twist around each other.

Epitopes

The part of an antigen that is recognized by the immune system, specifically by antibodies.

1. What is the primary structure of a protein, and how does the sequence of amino acids affect its final structure?

1. The primary structure is the linear sequence of amino acids in a polypeptide chain. The specific sequence of amino acids determines how the protein will fold into its three-dimensional structure, which dictates its function.

2. Describe the types of non-covalent interactions (hydrogen bonds, electrostatic interactions, and van der Waals forces) that contribute to protein folding.

2. Hydrogen bonds: Occur between the backbone amide hydrogen and carbonyl oxygen of the polypeptide, stabilizing α-helices and β-sheets.

   • Electrostatic interactions: Attractions between oppositely charged side chains (acidic and basic amino acids).

   • Van der Waals forces: Weak attractions between non-polar side chains that help proteins maintain compact structures.

3. What is the difference between an α-helix and a β-sheet in terms of their folding patterns and hydrogen bonding?

3. α-helix: A right-handed spiral structure where each amino acid forms a hydrogen bond with the fourth amino acid in the chain.

   • β-sheet: A structure formed by hydrogen bonds between extended strands of polypeptides, which can be parallel (same direction) or antiparallel (opposite direction).

4. Explain the role of molecular chaperones in protein folding.

4. Molecular chaperones assist in the proper folding of proteins by preventing misfolding and aggregation. They help proteins reach their final, functional structure by stabilizing intermediate states and facilitating correct interactions.

5. What are the key differences between the secondary, tertiary, and quaternary structure of proteins?

5. Secondary structure: Local folding patterns like α-helices and β-sheets.

   • Tertiary structure: The overall 3D shape formed by interactions between secondary structures.

   • Quaternary structure: The arrangement of multiple polypeptide subunits into a functional protein complex.

6. How does domain shuffling contribute to the evolution of new proteins?

6. Domain shuffling involves the recombination of pre-existing protein domains, allowing for the creation of new proteins with different functions. This promotes evolutionary diversity by generating proteins with novel functional properties.

7. What are some examples of proteins that contain multiple domains and how do these domains contribute to the protein’s function?

7. Kinases, which have both a catalytic domain and a regulatory domain.

   • Transcription factors, which contain a DNA-binding domain and an activation domain.

8. How do proteins bind to their specific ligands, and how does the binding site’s structure contribute to this specificity?

8. Proteins bind to specific ligands through complementary shapes and chemical properties of the binding site. The structure of the binding site ensures high specificity by matching the ligand’s size, shape, and charge distribution.

9. What is feedback inhibition, and how does it regulate metabolic pathways in cells?

9. Feedback inhibition occurs when the end product of a metabolic pathway inhibits the activity of the first enzyme in the pathway, preventing overproduction of the product and maintaining balance in the cell.

10. Describe the concept of allosteric regulation. How does the binding of a regulatory molecule to an allosteric site change protein activity?

10. Allosteric regulation involves the binding of a molecule to a site other than the active site (the allosteric site), inducing a conformational change in the protein that either increases or decreases its activity. This regulation is often used for enzymes to control their function in response to environmental changes.

11. What are the differences between negative and positive regulation of enzymes? Provide an example of each.

11. Negative regulation: Inhibits enzyme activity (e.g., feedback inhibition).

    • Positive regulation: Stimulates enzyme activity (e.g., phosphorylation activates certain enzymes like protein kinases).

12. Explain the process of phosphorylation in protein regulation. How does the addition of a phosphate group alter protein function?

12. Phosphorylation involves the addition of a phosphate group (often from ATP) to the hydroxyl group of specific amino acids (e.g., serine, threonine, or tyrosine). This modification can induce a conformational change in the protein, either activating or deactivating its function.

13. What is the “catalytic triad” in serine proteases, and how does it contribute to the enzyme’s function?

13. The catalytic triad in serine proteases consists of three amino acids (usually Asp, His, and Ser) that work together to catalyze the hydrolysis of peptide bonds. The serine residue acts as the nucleophile that attacks the substrate, aided by the other two residues.

14. What are the differences between homo-oligomers and hetero-oligomers in protein complexes? Provide examples of each.

14. Homo-oligomers: Composed of identical subunits (e.g., Cro repressor in bacteriophages).

    • Hetero-oligomers: Composed of different subunits (e.g., hemoglobin, which has α-globin and β-globin subunits).

15. How does hemoglobin function as a hetero-oligomer, and what is the significance of its subunit composition?

15. Hemoglobin is a hetero-oligomer made of two α-globin and two β-globin subunits. This composition allows hemoglobin to efficiently bind and release oxygen in the lungs and tissues through cooperative binding.

16. Describe how actin filaments are formed and how they contribute to cell structure and movement.

16. Actin filaments form by the polymerization of actin monomers into long, thin fibers. They provide structural support and are involved in cell movement, division, and shape changes.

17. What is the significance of intermediate filaments, and how do they differ from other types of protein filaments like actin?

17. Intermediate filaments provide mechanical strength to cells and resist tension. Unlike actin filaments (which are involved in cell movement), intermediate filaments provide structural integrity and form the nuclear lamina.

18. How do protein complexes such as the proteasome function in cellular processes?

18. The proteasome is a large complex responsible for degrading damaged or unneeded proteins. It breaks down proteins into small peptides, which are further processed into amino acids for recycling.

19. How does SDS-PAGE separate proteins, and what role does SDS play in this technique?

19. SDS-PAGE separates proteins based on their size. SDS (a detergent) denatures proteins by coating them with a negative charge, so they move through the gel based on their size, with smaller proteins migrating faster.

20. Explain the principle behind affinity chromatography and how it can be used to purify proteins based on their specific binding interactions.

20. Affinity chromatography uses a resin with a specific ligand attached. The target protein, which binds to this ligand, is retained on the column, while other proteins are washed away. The target protein can then be eluted using a solution that competes for binding.

21. What is the purpose of Western blotting, and how does it allow researchers to detect specific proteins in a sample?

21. Western blotting is used to detect specific proteins after separation by electrophoresis. Proteins are transferred to a membrane, where they are probed with antibodies specific to the target protein

22. Describe the process of mass spectrometry in identifying proteins. What is the role of the mass analyzer in this technique?

22. Mass spectrometry identifies proteins by analyzing the mass-to-charge ratio of their ions. The mass analyzer separates ions based on their mass, allowing researchers to deduce the protein’s sequence and structure.

23. How does two-dimensional gel electrophoresis provide a more effective separation of proteins compared to one-dimensional electrophoresis?

23. Two-dimensional gel electrophoresis separates proteins first by their charge (isoelectric focusing) and then by their size (SDS-PAGE). This provides better resolution and allows the identification of more proteins.

24. What are the principles behind fluorescence microscopy, and how do fluorescence filters work to visualize fluorescently labeled proteins?

24. Fluorescence microscopy uses fluorescent molecules that emit light at a longer wavelength when excited by light at a shorter wavelength. Excitation filters allow only the specific wavelength to pass through, while emission filters capture the emitted light from the sample.

25. Explain the concept of super-resolution microscopy. How do techniques like STED and PALM improve upon traditional fluorescence microscopy?

25. Super-resolution microscopy techniques, such as STED and PALM, allow imaging beyond the diffraction limit of light by using specialized methods to capture finer details of fluorescent molecules, providing higher resolution images than conventional microscopes.

26. What is the principle of FRET (Fluorescence Resonance Energy Transfer), and how is it used to study protein-protein interactions?

26. FRET measures energy transfer between two fluorescent molecules when they are in close proximity, indicating protein-protein interactions. A donor fluorophore transfers energy to an acceptor fluorophore when they are less than 10 nm apart.

27. How does photoactivation in fluorescence microscopy allow the study of dynamic processes in live cells?

27. Photoactivation involves using light to activate a fluorescent molecule in a specific area of a cell. The movement and behavior of these activated molecules can then be tracked over time.

28. Describe the process of Fluorescence Recovery After Photobleaching (FRAP) and how it helps in studying protein dynamics within cells.

28. FRAP involves bleaching a fluorescently labeled region of a cell with intense light and measuring how the fluorescence recovers as unbleached molecules move into the area. This provides insights into protein mobility and dynamics.

29. How does TIRF (Total Internal Reflection Fluorescence) microscopy differ from traditional fluorescence microscopy, and what is its advantage in studying membrane-associated proteins?

29. TIRF microscopy uses total internal reflection to excite fluorescence only in a thin region near the surface of a sample, which is ideal for studying proteins at the cell membrane.

30. What are the differences between Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) in terms of resolution and the types of images they produce?

30. TEM provides high-resolution images of thin internal structures at the nanoscale, while SEM produces 3D images of surfaces with a lower resolution but greater depth of field.

30. TEM provides high-resolution

31. Describe the concept of cryo-electron microscopy (cryo-EM) and how it allows for the imaging of proteins in their near-native state.

31. Cryo-EM involves rapidly freezing proteins to preserve their structure close to their natural state. This allows for high-resolution imaging without the need for crystallization.

32. What are the benefits of combining cryo-EM with single-particle reconstruction for studying protein complexes?

32. Combining cryo-EM with single-particle reconstruction allows for the detailed study of protein complexes in their native, unaltered state, providing a more accurate view of their structure and function.

33. Explain the principle of scanning electron microscopy (SEM) and how it creates 3D images of cell structures.

33. SEM scans a sample with an electron beam and detects scattered electrons to create 3D images. It provides high resolution and depth of field for surface structures.

34. How do co-immunoprecipitation (Co-IP) techniques help researchers identify protein-protein interactions?

34. Co-IP allows researchers to isolate and identify interacting proteins by using an antibody that binds to one protein, pulling down the complex and revealing the other proteins that bind to it.

35. What is the principle behind the use of fluorescence anisotropy to measure protein interactions?

35. Fluorescence anisotropy measures changes in the rotational motion of a fluorescently tagged molecule, which changes when the molecule binds to another protein, providing insights into binding interactions.

36. How does mass spectrometry help identify the specific proteins that interact in a protein complex?

36. Mass spectrometry identifies proteins by measuring their mass-to-charge ratios. By analyzing peptides from protein complexes, it can determine the identity of interacting proteins.

37. What is the significance of the evolutionary trace method in studying protein families and their functions?

37. Evolutionary trace compares conserved regions of protein sequences across species to predict functions and identify critical residues necessary for the protein’s activity.

38. How do GTP-binding proteins function as molecular switches, and what is their role in cellular signaling?

38. GTP-binding proteins switch between active (GTP-bound) and inactive (GDP-bound) states, regulating cellular processes like signal transduction. They act as molecular switches that control various signaling pathways.

20. What is the significance of symmetry in protein assemblies?

20. Symmetry in protein assemblies ensures that the protein functions efficiently and stably. Symmetric arrangements help to coordinate interactions between subunits, enabling proteins to perform complex, cooperative functions, as seen in hemoglobin and viral capsids.

19. How do protein domains contribute to protein evolution?

19. Protein domains are evolutionary units that can be recombined in new ways to create proteins with different functions. This modular nature of domains allows proteins to evolve new functions by shuffling existing domains.

18. How does elastin contribute to tissue function?

18. Elastin is a protein that provides elasticity to tissues. It allows tissues like lungs, blood vessels, and skin to stretch and return to their original shape after deformation, ensuring that these tissues can resist stress and maintain functionality.

17. What is the role of collagen in tissue structure?

17. Collagen is a structural protein that provides strength and support to tissues, such as skin, tendons, ligaments, and cartilage. It forms a fibrous network that gives tissues structural integrity.

16. How do proteins serve as subunits for large functional assemblies?

16. Proteins often function as subunits that assemble into larger structures to perform complex tasks. For example, the ribosome is a large protein-RNA complex responsible for protein synthesis, while the proteasome degrades unwanted proteins to maintain cellular protein homeostasis.

15. How does phosphorylation regulate protein function?

15. Phosphorylation involves the addition of a phosphate group (usually from ATP) to specific amino acids (e.g., serine, threonine, or tyrosine) in a protein. This modification can induce a conformational change in the protein, altering its activity. Phosphorylation is a common mechanism in signal transduction and cellular regulation.

14. What are post-translational modifications (PTMs), and how do they regulate protein activity?

14. Post-translational modifications (PTMs) are chemical modifications made to proteins after synthesis. Common PTMs include phosphorylation, acetylation, and ubiquitination. These modifications can regulate protein activity, stability, localization, and interactions. For example, phosphorylation can activate or deactivate protein function, while ubiquitination often targets proteins for degradation.

13. What is co-translational folding, and why is it important?

13. Co-translational folding occurs as a protein is being synthesized by the ribosome. As the polypeptide chain emerges from the ribosome, it starts to fold into its final 3D structure. This process is important because it helps prevent misfolding and aggregation of proteins, ensuring that proteins attain their functional conformations.

12. What are some examples of protein assemblies in viruses?

12. In viruses, protein assemblies form the capsid, which protects the viral genetic material. For example, the Tobacco Mosaic Virus (TMV) has a helical capsid formed by repeating protein subunits that encapsulate viral RNA. This protein assembly ensures the virus’s stability and efficiency in infecting host cells.

11. What is the role of symmetry in protein assemblies?

11. Symmetry in protein assemblies is essential for functional efficiency. Symmetric arrangements allow proteins to interact in a coordinated manner. For instance, hemoglobin, a tetrameric protein, uses symmetry to effectively bind and release oxygen in a cooperative manner.

10. What are protein assemblies, and how do they contribute to cellular functions?

10. Protein assemblies are large, multi-subunit complexes that perform crucial functions in the cell. For example, the ribosome is a protein-RNA complex that synthesizes proteins by translating mRNA. Similarly, the proteasome is a protein complex responsible for protein degradation, maintaining protein homeostasis within the cell.

9. How do intrinsically disordered regions contribute to protein-protein interactions?

9. Intrinsically disordered regions (IDRs) are able to undergo conformational changes when binding to different molecules. This adaptability allows proteins to engage in dynamic interactions with various partners. For example, the p53 tumor suppressor protein has disordered regions that facilitate its binding to multiple regulatory molecules, enabling it to act as a master regulator in cell cycle control.

8. Why are intrinsically disordered regions important for protein function?

8. Disordered regions provide flexibility and versatility, allowing proteins to interact with many different molecular partners. These regions are involved in cell signaling, molecular recognition, and regulatory functions, where flexibility is essential for adapting to different conditions.

7. What are intrinsically disordered proteins (IDPs)?

7. Intrinsically disordered proteins (IDPs) are proteins or regions of proteins that lack a stable, fixed 3D structure. These regions are highly flexible and can interact with multiple binding partners, making IDPs crucial for regulation and signal transduction in cells.

6. How do protein families relate to domains?

6. Protein families are groups of proteins that share a similar structural domain or functional domain. Members of the same family typically perform related tasks in the cell. For instance, serine proteases share a similar catalytic domain that allows them to cleave peptide bonds in other proteins.

5. What is domain shuffling, and how does it contribute to protein evolution?

5. Domain shuffling refers to the process in which existing protein domains are recombined in new ways to create novel proteins with different functions. This allows for the evolution of proteins with diverse functionalities while maintaining the stability of the individual domains. For example, combining an EGF domain with a calcium-binding domain may result in a new protein that performs different tasks.

4. What are protein domains, and why are they important for protein function?

4. Protein domains are distinct, structural and functional regions of a protein that fold independently. Each domain usually performs a specific function, such as catalysis or binding to other molecules. For example, a kinase domain is involved in phosphorylation, a process important for cell signaling.

3. How do protein dimers function in a biological context?

3. Protein dimers serve important biological functions, especially in gene regulation and enzyme activity. For instance, the Cro repressor in bacteriophages is a homodimer that binds to DNA and regulates gene expression by blocking transcription. The symmetry of the dimer ensures that it interacts equally and effectively with the DNA.

2. What is the difference between a homodimer and a heterodimer?

2. A homodimer is a protein complex made of two identical subunits, whereas a heterodimer is composed of two different subunits. Symmetry plays a crucial role in the stability and functionality of these protein complexes. Homodimers tend to exhibit simpler functions, while heterodimers allow for more diversity in function.

1. What is a protein complex, and how do subunits contribute to its formation?

1. A protein complex is formed when multiple protein subunits assemble together. These subunits can be identical (homodimers) or different (heterodimers). Each subunit contributes to the complex’s overall function, and the symmetry of these subunits ensures that the complex operates efficiently. For example, hemoglobin is a heterotetramer consisting of two α-globin and two β-globin subunits, working together to bind and release oxygen.

• Coiled-Coil:

• A structure where two or more alpha-helices twist around each other, often forming a supercoil.

• Oligomerization

• Many proteins form large structures by combining multiple subunits. These assemblies may be homo-oligomers (same subunits) or hetero-oligomers (different subunits). For example, hemoglobin is a hetero-oligomer composed of two α-globin and two β-globin subunits.

Enzyme Kinetics

• Vmax refers to the maximum rate of an enzyme-catalyzed reaction. The turnover number is the number of substrate molecules converted to product per enzyme molecule per second.

GTP-binding Proteins

• GTP-binding proteins (e.g., Ras) function as molecular switches, turning “on” when bound to GTP and “off” when bound to GDP.

• Negative Regulation:

• This refers to processes that inhibit or decrease the activity of a protein or enzyme. A common form of negative regulation is feedback inhibition, where the product of a biochemical pathway inhibits the activity of an enzyme that is involved in its own synthesis. This prevents overproduction of the product.
  

  • Example: In metabolic pathways, a product can bind to an enzyme, preventing further production of that product.

• Positive Regulation:

• This refers to processes that enhance or increase the activity of a protein or enzyme. In some cases, proteins are activated by other molecules or by modifications like phosphorylation.
  

  • Example: Certain enzymes can be activated by the binding of a cofactor or by the binding of another protein.

• Electrophoresis

• This technique is used to separate proteins by size and charge. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a common method for this, where proteins are coated with a detergent that gives them a uniform charge, and then they migrate through a gel matrix under an electric field.

• Western Blotting:

• After separating proteins by electrophoresis, Western blotting allows the detection of specific proteins using antibodies. This method is often used to quantify protein expression or to detect modifications like phosphorylation.

• Mass Spectrometry:

• This powerful tool is used to identify proteins by measuring the mass-to-charge ratio of ionized molecules. It helps determine the amino acid sequence of proteins and can also be used to identify modifications, such as phosphorylation.

• Protein-Protein Interaction Studies: Co-immunoprecipitation (Co-IP)

• and other methods allow scientists to study how proteins interact with each other. This is important for understanding signaling pathways and protein complexes in cells.

What is Fluorescence Microscopy?

Fluorescence microscopy is a technique that uses the fluorescence of molecules to visualize and study specific structures or proteins within cells.

• Fluorescent Dyes:

• These are chemicals that absorb light at one wavelength and emit it at a longer wavelength, usually in the visible spectrum. Proteins or other cellular components can be tagged with these fluorescent dyes to make them visible under a microscope.

• Fluorescence Microscopes:

• These microscopes are equipped with special filters that allow the excitation of fluorescent dyes and the collection of emitted light. Fluorescence microscopy is highly sensitive and allows researchers to study proteins, cellular organelles, and dynamic processes within living cells.

• Oligomerization

• Many proteins are made of more than one polypeptide chain. These chains are often referred to as subunits. Proteins like hemoglobin (which has four subunits) are examples of oligomeric proteins.

• Homo-oligomers:

• These are proteins made up of identical subunits, such as Cro repressor protein in bacteriophages.

• Hetero-oligomers:

• These consist of different subunits, like hemoglobin, which contains two α-globin subunits and two β-globin subunits

• Protein Domains:

• A protein domain is a part of the polypeptide chain that can fold into a stable, independent structure and often performs a specific function.
  

  • Domains can be reused in different proteins, which allows for the evolution of proteins with new functions by combining pre-existing domains. This process is known as domain shuffling.

• Modular Nature of Domains:

• Domains can be combined in various ways to form proteins with different functions. For example, immunoglobulin domains (found in antibodies) are used to form antigen-binding sites.

Examples of Domain Shuffling

• Chymotrypsin: A serine protease with multiple domains that can cleave peptide bonds in proteins.

• Epidermal Growth Factor (EGF): This protein contains multiple domains that help in cell signaling and interaction with growth factor receptors.