MCAT Biochemistry Review

Chapter 3: Nonenzymatic Protein Function and Protein Analysis

Conclusion

In this chapter, we have explored the nonenzymatic aspects of proteins as well as the ways proteins can be analyzed. The cellular proteins and their functions that we discussed included structural proteins that play a role in cytoskeletal architecture, motor proteins involved in muscle contraction and movement along the cytoskeleton, and other proteins that play more complex roles such as binding, immunologic function, and biosignaling. The more complex proteins involved in biosignaling highlighted in this chapter included ion channels, enzyme-linked receptors, and G protein-coupled receptors. Finally, we determined how to isolate and identify a protein and its relevant properties.

In the next chapter, we'll turn our attention to another class of biomolecules: carbohydrates. As we transition from amino acids, peptides, and proteins to monosaccharides, oligosaccharides, and polysaccharides, look for key connections between the different types of macromolecules used by the body for structure and as fuel sources. In the end, all biomolecules are related to each other through metabolism, which we'll explore in Chapters 9 through 12 of MCAT Biochemistry Review.

Concept Summary

Cellular Functions

·        Structural proteins compose the cytoskeleton, anchoring proteins, and much of the extracellular matrix.

o   The most common structural proteins are collagenelastinkeratinactin, and tubulin.

o   They are generally fibrous in nature.

·        Motor proteins have one or more heads capable of force generation through a conformational change.

o   They have catalytic activity, acting as ATPases to power movement.

o   Muscle contraction, vesicle movement within cells, and cell motility are the most common applications of motor proteins.

o   Common examples include myosinkinesin, and dynein.

·        Binding proteins bind a specific substrate, either to sequester it in the body or hold its concentration at steady state.

·        Cell adhesion molecules (CAMs) allow cells to bind to other cells or surfaces.

o   Cadherins are calcium-dependent glycoproteins that hold similar cells together.

o   Integrins have two membrane-spanning chains and permit cells to adhere to proteins in the extracellular matrix. Some also have signaling capabilities.

o   Selectins allow cells to adhere to carbohydrates on the surfaces of other cells and are most commonly used in the immune system.

·        Antibodies (or immunoglobulinsIg) are used by the immune system to target a specific antigen, which may be a protein on the surface of a pathogen (invading organism) or a toxin.

o   Immunoglobulins contain a constant region and a variable region; the variable region is responsible for antigen binding.

o   Two identical heavy chains and two identical light chains form a single antibody; they are held together by disulfide linkages and noncovalent interactions.

Biosignaling

·        Ion channels can be used for regulating ion flow into or out of a cell. There are three main types of ion channels.

o   Ungated channels are always open.

o   Voltage-gated channels are open within a range of membrane potentials.

o   Ligand-gated channels open in the presence of a specific binding substance, usually a hormone or neurotransmitter.

·        Enzyme-linked receptors participate in cell signaling through extracellular ligand binding and initiation of second messenger cascades.

·        G protein-coupled receptors have a membrane-bound protein associated with a trimeric G protein. They also initiate second messenger systems.

o   Ligand binding engages the G protein.

o   GDP is replaced with GTP; the α subunit dissociates from the β and γ subunits.

o   The activated α subunit alters the activity of adenylate cyclase or phospholipase C.

o   GTP is dephosphorylated to GDP; the α subunit rebinds to the β and γ subunits.

Protein Isolation

·        Electrophoresis uses a gel matrix to observe the migration of proteins in response to an electric field.

o   Native PAGE maintains the protein's shape, but results are difficult to compare because the mass-to-charge ratio differs for each protein.

o   SDS-PAGE denatures the proteins and masks the native charge so that comparison of size is more accurate, but the functional protein cannot be recaptured from the gel.

o   Isoelectric focusing separates proteins by their isoelectric point (pI); the protein migrates toward an electrode until it reaches a region of the gel where pH = pI of the protein.

·        Chromatography separates protein mixtures on the basis of their affinity for a stationary phase or a mobile phase.

o   Column chromatography uses beads of a polar compound, like silica or alumina (stationary phase), with a nonpolar solvent (mobile phase).

o   Ion-exchange chromatography uses a charged column and a variably saline eluent.

o   Size-exclusion chromatography relies on porous beads. Larger molecules elute first because they are not trapped in the small pores.

o   Affinity chromatography uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of interest.

Protein Analysis

·        Protein structure is primarily determined through X-ray crystallography after the protein is isolated, though NMR can also be used.

·        Amino acid composition can be determined by simple hydrolysis, but amino acid sequencing requires sequential degradation, such as the Edman degradation.

·        Activity levels for enzymatic samples are determined by following the process of a known reaction, often accompanied by a color change.

·        Protein concentration is also determined colorimetrically, either by UV spectroscopy or through a color change reaction.

o   BCA assayLowry reagent assay, and Bradford protein assay each test for protein and have different advantages and disadvantages.

o   The Bradford protein assay, which uses a color change from brown-green to blue, is most common.

Answers to Concept Checks

·        3.1

1.    Cytoskeletal proteins tend to be fibrous with repeating domains, while motor proteins tend to have ATPase activity and binding heads. Both types of protein function in cellular motility.

2.    False. An enzyme is a protein or RNA molecule with catalytic activity, which motor proteins do have. Motor function is generally considered nonenzymatic, but the ATPase functionality of motor proteins indicates that these molecules do have catalytic activity.

3.    If the binding protein is present in sufficiently high quantities relative to the substrate, nearly all substrate will be bound despite a low affinity.

4.     

Cell Adhesion Molecule

Type of Adhesion

Cadherin

Two cells of the same or similar type using calcium

Integrin

One cell to proteins in the extracellular matrix

Selectin

One cell to carbohydrates, usually on the surface of other cells

5.    Antigen–antibody interactions can result in neutralization of the pathogen or toxin, opsonization (marking) of the antigen for destruction, or creation of insoluble antigen–antibody complexes that can be phagocytized and digested by macrophages (agglutination).

·        3.2

1.   

2.    Ungated channels are always open.

3.    Transport kinetics display both Km and vmax values. They also can be cooperative, like some binding proteins. However, transporters do not have analogous Keq values for reactions because there is no catalysis.

·        3.3

1.    Isoelectric focusing and ion-exchange chromatography both separate proteins based on charge; the charge of a protein in any given environment is determined by its isoelectric point (pI).

2.    Native PAGE allows a complete protein to be recovered after analysis; it also more accurately determines the relative globular size of proteins. SDS-PAGE can be used to eliminate conflation from mass-to-charge ratios.

3.    The protein of interest may not elute from the column because its affinity is too high or it may be permanently bound to the free receptor in the eluent.

4.    True. The small pores in size-exclusion chromatography trap smaller particles, retaining them in the column.

·        3.4

1.    Protein isolation is generally only the first step in an analysis. The protein identity must be confirmed by amino acid analysis or activity. With unknown proteins, classification of their features is generally desired.

2.    Contamination of the sample with detergent or SDS could yield an artificially increased protein level. Alternatively, the enzyme could have been denatured during isolation and analysis.

3.    False. The Edman degradation proceeds from the amino (N-) terminus.

Equations to Remember

(3.1) Migration velocity

Shared Concepts

·        Biochemistry Chapter 1

o   Amino Acids, Peptides, and Proteins

·        Biology Chapter 1

o   The Cell

·        Biology Chapter 8

o   The Immune System

·        Biology Chapter 11

o   The Musculoskeletal System

·        General Chemistry Chapter 12

o   Electrochemistry

·        Organic Chemistry Chapter 12

o   Separations and Purifications