BY 422 Cell Biology Alvarez Chapter 4

Antibody

Protein produced by B lymphocytes in response to a foreign molecule or invading organism. Binds to the foreign molecule or cell extremely tightly, thereby inactivating it or marking it for destruction.

scaffold protein

Protein with multiple binding şitesfor other macromolecules, holding them in a way that speeds up their functional interactions.

intrinsically disordered sequence

Region in a polypeptide chain that lacks a definite structure.

Binding Site

Region on the surface of a protein, typically a cavity or groove, that interacts with another molecule (a ligand) through the formation of multiple noncovalent bonds.

Active Site

Region on the surface of an enzyme that binds to a substrate molecule and catalyzes its chemical transformation.

secondary structure

Regular local folding pattern of a polymeric molecule. In proteins, it refers to a helices and B sheets.

polypeptide backbone

Repeating sequence of the atoms (-N-C- C-) that form thecore of a protein molecule and to which the amino acid side chains are attached.

peptide bond

Covalent chemical bond between the carbonyl group of one amino acid and the amino group of a second amino acid.

disulfide bond

Covalent cross-link formed between the şulfhydryl groups on two cysteine side chains; often used to reinforce a secreted protein's structure or to join two different proteins together.

Ligand

General term for a small molecule that binds to a specific site on a macromolecule.

Protein

Macromolecule built from amino acids that provides cells with their shape and structure and performs most of their activities.

Assume you want to make and study fragments of a protein. Would you expect that any fragment of the polypeptide chain would fold the same way as it would in the intact protein? Consider the protein shown in Figure 4−20. Which fragments do you suppose are most likely to fold correctly?

Many secondary structural elements are not stable in isolation but are stabilized by other parts of the polypeptide chain. Hydrophobic regions of fragments, which would normally be hidden in the inside of a folded protein, would be exposed to water molecules in an aqueous solution; such fragments would tend to aggregate nonspecifically, and not have a defined structure, and they would be inactive for ligand binding, even if they contained all of the amino acids that would normally contribute to the ligand-binding site. A protein domain, in contrast, is considered a folding unit, and fragments of a polypeptide chain that correspond to intact domains are often able to fold correctly. Thus, separated protein domains often retain their activities, such as ligand binding, if the binding site is contained entirely within the domain. Thus the most likely place in which the polypeptide chain of the protein in Figure 4−20 could be severed to give rise to stable fragments is at the boundary between the two domains (i.e., at the loop between the two α helices at the bottom right of the structure shown).

Antigen

Molecule or fragment of a molecule that is recognized by an antibody.

quaternary structure

Complete structure formed by multiple, interacting polypeptide chains that form a larger protein molecule.

tertiary structure

Complete three-dimensional structure of a fully folded protein.

feedback inhibition

A form of metabolic control in which the end product of a chain of enzymatic reactions reduces the activity of an enzyme early in the pathway.

protein family

A group of polypeptides that share a similar amino acid sequence or three-dimensional structure, reflecting a common evolutionary origin. Individual members often have related but distinct functions, such as kinases that phosphorylate different target proteins.

intracellular condensate

A large aggregate of phase- separated macromolecules that creates a region with a special biochemistry without use of an encapsulating membrane.

Substrate

A molecule on which an enzyme acts to catalyze a chemical reaction.

Subunit

A monomer that forms part of a larger molecule, such as an amino acid residue in a protein or a nucleotide residue in a nucleic acid. Can also refer to a complete molecule that forms part of a larger molecule. Many proteins, for example, are called a protein subunit.

Enzyme

A protein that catalyzes a specific chemical reaction.

fibrous protein

A protein with an elongated, rodlike shape, such as collagen or a keratin filament.

A. The reaction rates of the reaction S → P, catalyzed by enzyme E, were determined under conditions in which only very little product was formed. The data in the table below were measured, plot the data as a graph. Use this graph to estimate the KM and the Vmax for this enzyme. B. To determine the KM and Vmax values more precisely, a trick is generally used in which the Michaelis-Menten equation is transformed so that it is possible to plot the data as a straight line. A simple rearrangement yields 1/rate = (KM/Vmax) (1/[S]) + 1/Vmax which is an equation of the form y = ax + b. Calculate 1/rate and 1/[S] for the data given in part (A) and then plot1/rate versus 1/[S] as a new graph. Determine KM and Vmax from the intercept of the line with the axis, where 1/[S] = 0, combined with the slope of the line. Do your results agree with the estimates made from the first graph of the raw data? C. It is stated in part (A) that only very little product was formed under the reaction conditions. Why is this important? D. Assume the enzyme is regulated such that upon phosphorylation its KM increases by a factor of 3 without changing its Vmax. Is this an activation or inhibition? Plot the data you would expect for the phosphorylated enzyme in both the graph for (A) and the graph for (B).

A,B. The data in the boxes have been used to plot the red curve and red line in Figure A4-26. From the plotted data, the KM is 1 μM and the Vmax is 2 μmole/min. Note that the data are much easier to interpret in the linear plot, because the curve in (A) approaches, but never reaches, Vmax. C. It is important that only a small quantity of product is made, because otherwise the rate of reaction would decrease as the substrate was depleted and product accumulated. Thus the measured rates would be lower than they should be. D. If the KM increases, then the concentration of substrate needed to give a half-maximal rate is increased. As more substrate is needed to produce the same rate, the enzyme-catalyzed reaction has been inhibited by the phosphorylation. The expected data plots for the phosphorylated enzyme are the green curve and thegreen line in Figure A4-26.

Simple enzyme reactions often conform to the equation: E + S ↔ ES → EP ↔ E + P where E, S, and P are enzyme, substrate, and product, respectively. A. What does ES represent in this equation? B. Why is the first step shown with bidirectional arrows and the second step as a unidirectional arrow? C. Why does E appear at both ends of the equation? D. One often finds that high concentrations of P inhibit the enzyme. Suggest why this might occur. E. If compound X resembles S and binds to the active site of the enzyme but cannot undergo the reaction catalyzed by it, what effects would you expect the addition of X to the reaction to have? Compare the effects of X and of the accumulation of P.

A. ES represents the enzyme-substrate complex. B. Enzyme and substrate are in equilibrium between their free and bound states; once bound to the enzyme, a substrate molecule may either dissociate again (hence the bidirectional arrows) or be converted to product.As the substrate is converted to product (with the concomitant release of free energy), however, a reaction usually proceeds strongly in the forward direction, as indicated by the unidirectional arrow. C. The enzyme is a catalyst and is therefore liberated in an unchanged form after the reaction; thus, E appears at both ends of the equation. D. Often, the product of a reaction resembles the substrate sufficiently that it can also bind to the enzyme. Any enzyme molecules that are bound to the product (i.e., are part of an EP complex) are unavailable for catalysis; excess P therefore can inhibit the reaction by lowering the concentration of free E. E. Compound X would act as an inhibitor of the reaction and work similarly by forming an EX complex. However, since P has to be made before it can inhibit the reaction, it takes longer to act than X, which is present from the beginning of the reaction.

Consider the drawing in Figure 4−42. What will happen if, instead of the indicated feedback, A. feedback inhibition fromZ affects the step B → C only? B. feedback inhibition fromZ affects the step Y → Z only? C. Z is a positive regulator of the step B → X? D. Z is a positive regulator of the step B → C?For each case, discuss how useful these regulatory schemes would be for a cell.

A. Feedback inhibition from Z that affects the reaction B → C would increase the flow through the B → X → Y → Z pathway, because the conversion of B to C is inhibited. Thus, the more Z there is, the more production ECB5 EA4.05/A4.05 of Z would be stimulated. This is likely to result in an uncontrolled "runaway" amplification of this pathway. B. Feedback inhibition from Z affecting Y → Z would only inhibit the production of Z. In this scheme, however, X and Y would still be made at normal rates, even though both of these intermediates are no longer needed at this level. This pathway is therefore less efficient than the one shown in Figure 4−42. C. If Z is a positive regulator of the step B → X, then the more Z there is, the more B will be converted to X and therefore shunted into the pathway producing more Z. his would result in a runaway amplification similar to that described for (A). D. If Z is a positive regulator of the step B → C, then accumulation of Z leads to a redirection of the pathway to make more C. This is a second possible way, in addition to that shown in the figure, to balance the distribution of compounds into the two branches of the pathway.

Which of the following statements are correct? Explain your answers. A. The active site of an enzyme usually occupies only a small fraction of the enzyme surface. B. Catalysis by some enzymes involves the formation of a covalent bond between an amino acid side chain and a substrate molecule. C. A β sheet can contain up to five strands, but no more. D. The specificity of an antibody molecule is contained exclusively in loops on the surface of the folded light-chain domain. E. The possible linear arrangements of amino acids are so vast that new proteins almost never evolve by alteration of old ones. F. Allosteric enzymes have two or more binding sites. G. Noncovalent bonds are too weak to influence the three- dimensional structure of macromolecules. H. Affinity chromatography separates molecules according to their intrinsic charge. I. Upon centrifugation of a cell homogenate, smaller organelles experience less friction and thereby sediment faster than larger ones.

A. True. Only a few amino acid side chains contribute to the active site. The rest of the protein is required to maintain the polypeptide chain in the correct conformation, provide additional binding sites for regulatory purposes, and localize the protein in the cell. B. True. Some enzymes form covalent intermediates with their substrates (see middle panels of Figure 4−39); however, in all cases, the enzyme is restored to its original structure after the reaction. C. False. β sheets can, in principle, contain any number of strands because the two strands that form the rims of the sheet are available for hydrogen-bonding to other strands. (β sheets in known proteins contain from 2 to 16 strands.) D. False. It is true that the specificity of an antibody molecule is exclusively contained in polypeptide loops on its surface; however, these loops are contributed by both the folded light and heavy chains (see Figure 4−33). E. False. The possible linear arrangements of amino acids that lead to a stably folded protein domain are so few that most new proteins evolve by alteration of old ones. F. True. Allosteric enzymes generally bind one or more molecules that function as regulators at sites that are distinct from the active site. G. False. Although single noncovalent bonds are weak, many such bonds acting together are major contributors to the three-dimensional structure of macromolecules. H. False. Affinity chromatography separates specific macromolecules because of their interactions with specific ligands, not because of their charge. I. False. The larger an organelle is, the more centrifugal force it experiences and the faster it sediments, despite an increased frictional resistance from the fluid through which it moves.

The curve shown in Figure 4−35 is described by the Michaelis-Menten equation: rate (v) = Vmax [S]/(KM + [S]) Can you convince yourself that the features qualitatively described in the text are accurately represented by this equation? In particular, how can the equation be simplified when the substrate concentration [S] is in one of the following ranges: (A) [S] is much smaller than the KM (B) [S] equals the KM (C) [S] is much larger than the KM?

A. When [S] << KM, the term (KM + [S]) approaches KM. Therefore, the equation is simplified to rate = Vmax[S]/KM. Therefore, the rate is proportional to [S]. B. When [S] = KM, the term [S]/(KM + [S]) equals 1⁄2. Therefore, the reaction rate is half of the maximal rate Vmax. C. If [S] >> KM, the term (KM + [S]) approaches [S]. Therefore, [S]/(KM + [S]) equals 1 and the reaction occurs at its maximal rate Vmax.

Helix

An elongated structure whose subunits twist in a regular fashion around a central axis, like a spiral staircase.

globular protein

Any protein in which the polypeptide chain folds into a compact, rounded shape. Includes most enzymes.

Consider the following protein sequence as an α helix: Leu-Lys-Arg-Ile-Val-Asp-Ile-Leu-Ser-Arg-Leu-Phe-Lys-Val. How many turns does this helix make? Do you find anything remarkable about the arrangement of the amino acids in this sequence when folded into an α helix? (Hint: consult the properties of the amino acids in Figure 4−3.)

As it takes 3.6 amino acids to complete a turn of an α helix, this sequence of 14 amino acids would make close to 4 full turns. It is remarkable because its polar and hydrophobic amino acids are spaced so that all the polar ones are on one side of the α helix and all the hydrophobic ones are on the other. It is therefore likely that such an amphipathic α helix is exposed on the protein surface with its hydrophobic side facing the protein's interior.

protein machine

Assembly of protein molecules that operates as a cooperative unit to perform a complex series of biological activities, such as replicating DNA.

Neurofilament proteins assemble into long, intermediate filaments (discussed in Chapter 17), found in abundance running along the length of nerve cell axons. The C-terminal region of these proteins is an unstructured polypeptide, hundreds of amino acids long and heavily modified by the addition of phosphate groups. The term "polymer brush" has been applied to this part of the neurofilament. Can you suggest why?

Because of the lack of secondary structure, the C-terminal region of neurofilament proteins undergoes continual Brownian motion. The high density of negatively charged phosphate groups means that the C-terminals also experience repulsive interactions, which cause them to stand out from the surface of the neurofilament like the bristles of a brush. In electron micrographs ofa cross section of an axon, the region occupied by the extended C-terminals appears as a clear zone around each neurofilament, from which organelles and other neurofilaments are excluded.

Either protein phosphorylation or the binding of a nucleotide (such as ATP or GTP) can be used to regulate a protein's activity. What do you suppose are the advantages of each form of regulation?

Both nucleotide binding and phosphorylation can induce allosteric changes in proteins. These can have a multitude of consequences, such as altered enzyme activity, drastic shape changes, and changes in affinity for other proteins or small molecules. Both mechanisms are quite versatile. An advantage of nucleotide binding is the fast rate with which a small nucleotidecan diffuse to the protein; the shape changes that accompany the function of motor proteins, for example, require quick nucleotide replenishment. If the different conformational states of a motor protein were controlledby phosphorylation, for example, a protein kinase would either need to diffuse into position at each step, a much slower process, or be associated permanently with each motor protein. One advantage of phosphorylation is that it requires only a single amino acid on the protein's surface, rather than a specific binding site. Phosphates can therefore be added to many different side chains on the same protein (as long as protein kinases with the proper specificities exist), thereby vastly increasing the complexity of regulation that can be achieved for a single protein.

Allosteric

Describes a protein that can exist in multiple conformations depending on the binding of a molecule (ligand) at a site other than the catalytic site; such changes from one conformation to another often alter the protein's activity or ligand affinity.

protein domain

a polypeptide chain that can fold into a compact, stable structure and that often carries out a specific function.

protein phosphatase

Enzyme that catalyzes the removal of a phosphate group from a protein, often with high specificity for the phosphorylated site.

protein kinase

Enzyme that catalyzes the transfer of a phosphate group from ATP to a specific amino acid side chain on a target protein.

Lysozyme

Enzyme that severs the polysaccharide chains that form the cell walls of bacteria; found in many secretions including saliva and tears, where it serves as an antibiotic.

Beta Sheet (B sheet)

Folding pattern found in many proteins in which neighboring regions of the polypeptide chain associate side-by-side with each other through hydrogen bonds to give a rigid, flattened structure.

alpha helix (a helix)

Folding pattern, common in many proteins, in which a single polypeptide chain twists around itself to form a rigid cylinder stabilized by hydrogen bonds between every fourth amino acid.

Select the correct options in the following and explain your choices. If [S] is very much smaller than KM, the active site of the enzyme is mostly occupied/unoccupied. If [S] is very much greater than KM, the reaction rate is limited by the enzyme/substrate concentration.

If [S] is very much smaller than KM, the active site of the enzyme is mostly unoccupied. If [S] is very much greater than KM, the reaction rate is limited by the enzyme concentration (because most of the catalytic sites are fully occupied).

What common feature of α helices and β sheets makes them universal building blocks for proteins?

In an α helix and in the central strands of a β sheet, all of the N-H and C=O groups in the polypeptide backbone are engaged in hydrogen bonds. This gives considerable stability to these secondary structural elements, and it allows them to form in many different proteins.

Explain why the hypothetical enzymes in Figure 4−51 have a great advantage in opening the safe if they work together in a protein complex, as opposed to working individually in an unlinked, sequential manner.

In working together in a complex, all three proteins contribute to the specificity (by binding to the safe and key directly). They help position one another correctly, and provide the mechanical bracing that allows them to perform a task that they could not perform individually (the key is grasped by two of the proteins, for example). Moreover, their functions are generally coordinated in time (for instance, the binding of ATP to one subunit is likely to require that ATP has already been hydrolyzed to ADP by another).

GTP-binding protein

Intracellular signaling protein whose activity is determined by its association with either GTP or GDP. Includes both trimeric G proteins and monomeric GTPases, such as Ras.

polypeptide, polypeptide chain

Linear polymer composed of multiple amino acids. Proteins are composed of one or more long polypeptide chains.

Random mutations only very rarely result in changes that improve a protein's usefulness for the cell, yet useful mutations are selected in evolution. Because these changes are so rare, for each useful mutation there are innumerable mutations that lead to either no improvement or inactive proteins. Why, then, do cells not contain millions of proteins that are of no use?

Mutations that are beneficial to an organism are selected in evolution because they confera reproductive or survival advantage to the organism. Examples might be a more efficient utilization of a food source, enhanced resistance to environmental insults, or an improved ability to attract a mate for sexual reproduction. In contrast, useless proteins are detrimental to organisms, as the metabolic energy required to make them is a wasted cost. If such mutant proteins were made in excess, the synthesis of normal proteins would suffer because the synthetic capacity of the cell is limited. In more severe cases, a mutant protein could interfere with the normal workings of the cell; a mutant enzyme that still binds an activated carrier molecule but does not catalyze a reaction, for example, may compete for a limited amount of this carrier and therefore inhibit normal processes. Natural selection therefore provides a strong driving force that eliminates both useless and harmful proteins.

Protein structure is determined solely by a protein's amino acid sequence. Should a genetically engineered protein in which the original order of all amino acids is reversed have the same structure as the original protein?

No. It would not have the same or even a similar structure, because the peptide bond has a polarity. Looking at two sequential amino acids in a polypeptide chain, the amino acid that is closer to the N-terminal end contributes the carboxyl group and the other amino acid contributes the amino group to the peptide bond that links the two amino acids. Changing their order would put the side chains into different positions with respect to the peptide backbone and therefore change the way the polypeptide folds.

side chain

Portion of an amino acid not involved in forming peptide bonds; its chemical identity gives each amino acid unique properties.

Conformation

Precise, three-dimensional shape of a protein or other macromolecule, based on the spatial location of its atoms in relation to one another.

mass spectrometry

Sensitive technique that enables the determination of the exact mass of all of the molecules in a complex mixture.

Coenzyme

Small molecule that binds tightly to an enzyme and helps it to catalyze a reaction.

coiled-coil

Stable, rodlike protein structure formed when two or more a helices twist repeatedly around each other.

Hair is composed largely of fibers of the protein keratin. Individual keratin fibers are covalently cross- linked to one another by many disulfide (S-S) bonds. If curly hair is treated with mild reducing agents that break a few of the cross-links, pulled straight, and then oxidized again, it remains straight. Draw a diagram that illustrates the three different stages of this chemical and mechanical process at the level of the keratin filaments, focusing on the disulfide bonds. What do you think would happen if hair were treated with strong reducing agents that break all the disulfide bonds?

Strong reducing agents that break all of the S-S bonds would cause all of the keratin filaments to separate. Individual hairs would be weakened and fragment. Indeed, strong reducing agents are used commercially in hair-removal creams sold by your local pharmacist. However, mild reducing agents are used in treatments that either straighten or curl hair, the latter requiring hair curlers.

cryoelectron microscopy (cryo-EM)

Technique for observing the detailed structure of a macromolecule at very low temperatures after freezing native structures in ice.

Electrophoresis

Technique for separating a mixture of proteins or DNA fragments by placing them on a polymer gel and subjecting them to an electric field. The molecules migrate through the gel at different speeds depending on their size and net charge.

nuclear magnetic resonance (NMR) spectroscopy

Technique used for determining the three-dimensional structure of a protein in solution.

Chromatography

Technique used to separate the individual molecules in a complex mixture on the basis of their size, charge, or their ability to bind to a particular chemical group. In a common form of the technique, the mixture is run through a column filled with a material that binds the desired molecule, and it is then eluted from the column with a solvent gradient.

Remembering that the aminoacid side chains projecting from each polypeptide backbone in aβ sheet point alternately above and below the plane of the sheet (see Figure 4−13A), considerthe following protein sequence: Leu-Lys-Val-Asp-Ile-Ser-Leu-Arg- Leu-Lys-Ile-Arg-Phe-Glu. Do you find anything remarkable about the arrangement of the amino acids in this sequence when incorporated into a β sheet? Can you make any predictions as to how the β sheet might be arranged in a protein? (Hint: consult the properties of the amino acids listed in Figure 4−3.)

The amino acid sequence consists of alternating nonpolar and charged or polar amino acids. The resulting strand in a β sheet would therefore be polar on one side and hydrophobic on the other. Such a strand would probably be surrounded on either side by similar strands that together form a β sheet with a hydrophobic face and a polar face. In a protein, such a β sheet (called "amphipathic," from the Greek amphi, "of both kinds," and pathos, "passion," because of its two surfaces with such different properties) would be positioned so that the hydrophobic side would face the protein's interior and the polar side would be on its surface, exposed to the water outside.

primary structure

The amino acid sequence of a protein.

How is it possible that a change in a single amino acid in a protein of 1000 amino acids can destroy protein function, even when that amino acid is far away from any ligand- binding site?

The atoms at the binding sites of proteins must be precisely located to fit the molecules that they bind. Their location in turn requires the precise positioning of many of the amino acids and their side chains in the core of the protein, distant from the binding site itself. Thus, even a small change in this core can disrupt protein function by altering the conformation at a binding site far away.

Michaelis constant (KM)

The concentration of substrate at which an enzyme works at half its maximum velocity; serves as a measure of how tightly the substrate is bound.

protein phosphorylation

The covalent addition of a phosphate group to a side chain of a protein, catalyzed by a protein kinase; serves as a form of regulation that usually alters the activity or properties of the target protein.

N-terminus

The end of a polypeptide chain that carries a free a-amino group.

C-terminus

The end of a polypeptide chain that carries a free carboxyl group (-COOH).

An enzyme isolated from a mutant bacterium grown at20°C works in a test tube at 20°C but not at 37°C (37°C is the temperature of the gut, where this bacterium normally lives). Furthermore, once the enzyme has been exposedto the higher temperature, it no longer works at the lower one. The same enzyme isolated from the normal bacterium works at both temperatures. Can you suggest what happens (at the molecular level) to the mutant enzyme as the temperature increases?

The heat-inactivation of the enzyme suggests that the mutation causes the enzyme to have a less stable structure. For example, a hydrogen bond that is normally formed between two amino acid side chains might no longer be formed because the mutation replaces one of these amino acids with a different one that cannot participate in the bond. Lacking such a bond that normally helps to keep the polypeptide chain folded properly, the protein partially or completely unfolds at a temperature at which it would normally be stable. Polypeptide chains that denature when the temperature is raised often aggregate, and they rarely refold into active proteins when the temperature is decreased.

turnover number

The maximum number of substrate molecules that an enzyme can convert into product per second.

Vmax

The maximum rate of an enzymatic reaction, reached when the active sites of all of the enzyme molecules in a sample are fully occupied by substrate.

A motor protein moves along protein filaments in the cell. Why are the elements shown in the illustration not sufficient to mediate directed movement (Figure Q4-19)? With reference to Figure 4−50, modify the illustration shown here to include other elements that are required to create a unidirectional motor, and justify each modification you make to the illustration.

The motor protein in the illustration can move just as easily to the left as to the right and so will not move steadily in one direction. However, if just one of the steps is coupled to ATP hydrolysis (for example, by making detachment of one foot dependent on binding of ATP and coupling the reattachment to hydrolysis of the bound ATP), then the protein will show unidirectional movement that requires the continued consumption of ATP. Note that, in principle, it does not matter which step is coupled to ATP hydrolysis.

Amino Acid Sequence

The order of the amino acid subunits in a protein chain. Sometimes called the primary structure of a protein.

Which of the following amino acids would you expectto find more often near the center of a folded globular protein? Which ones would you expect to find more often exposed to the outside? Explain your answers. Ser, Ser-P (a Ser residue that is phosphorylated), Leu, Lys, Gln, His, Phe, Val, Ile, Met, Cys-S-S-Cys (two cysteines that are disulfide- bonded), and Glu. Where would you expect to find the most N-terminal amino acid and the most C-terminal amino acid?

The polar amino acids Ser, Ser-P, Lys, Gln, His, and Glu are more likely to be found on a protein's surface, and the hydrophobic amino acids Leu, Phe, Val,Ile, and Met are more likely to be found in its interior. The oxidation of two cysteine side chains to form a disulfide bond eliminates their potential to form hydrogen bonds and therefore makes them even more hydrophobic; thus disulfide bonds are usually found in the interior of proteins. Irrespective of the nature of their side chains, the most N-terminal amino acid and the most C-terminal amino acid each contain a charged group (the amino and carboxyl groups, respectively, that mark the ends of the polypeptide chain) and hence are usually found on the protein's surface.

Gel-filtration chromatography separates molecules according to their size (see Panel 4−4, p. 166). Smaller molecules diffuse faster in solution than larger ones, yet smaller molecules migrate more slowly through a gel- filtration column than larger ones. Explain this paradox. What should happen at very rapid flow rates?

The slower migration of small molecules through a gel-filtration column occurs because smaller molecules have access to many more spaces in the porous beads that are packed into the column than do larger molecules. However, it is important that the flow rate through the column is slow enough to give the smaller molecules sufficient time to diffuse into the spaces inside the beads. At very rapid flow rates, all molecules will move rapidly around the beads, so that large and small molecules will now tend to exit together from the column.

As shown in Figure 4−16, both α helices and the coiled-coil structures that can form from them are helical structures, but do they have the same handedness in the figure? Explain why?

The α helix in the figure is right-handed, whereas the coiled-coil is left-handed. The reversal occurs because of the staggered positions of hydrophobic side chains in the α helix.

Look at the models of the protein in Figure 4−11. Is thered α helix right- or left-handed? Are the three strands that form the large β sheet parallel or antiparallel? Starting at the N-terminus (the purple end), trace your finger along the peptide backbone. Are there any knots? Why, or why not?

The α helix is right-handed. The three strands that form the large β sheet are antiparallel. There are no knots in the polypeptide chain, presumably because a knot would interfere with the folding of the protein into its three-dimensional conformation after protein synthesis.

transition state

Transient structure that forms during the course of a chemical reaction; in this configuration, a molecule has the highest free energy; it is no longer the substrate, but is not yet the product.

Urea, used in the experiment shown in Figure 4−7, is a molecule that disrupts the hydrogen-bonded network of water molecules. Why might high concentrations of urea unfold proteins? The structure of urea is shown here.

Urea is a very small organic molecule that functions both as an efficient hydrogen-bond donor (through its NH2 groups) and as an efficient hydrogen-bond acceptor (through its C=O group). As such, it can squeeze between hydrogen bonds that stabilize protein molecules and thus destabilize protein structures. In addition, the nonpolar side chains of a protein are held together in the interior of the folded structure because they would disrupt the structure of water if they were exposed. At high concentrations of urea, the hydrogen-bonded network of water molecules becomes disrupted so that these hydrophobic forces are significantly diminished. Proteins unfold in urea as a consequence of its effect on these two forces.

motor protein

myosin or kinesin that uses energy derived from the hydrolysis of a tightly bound ATP molecule to propel itself along a protein filament or polymeric molecule.