Exam 2: Biochemistry

Passage The HIV-derived transactivator of transcription (Tat) is a short peptide with the sequence RKKRRQRRRPPQ.  When paired with liposomes (ie, synthetic vesicles) composed of cationic lipids, Tat can transport large molecules across cell membranes and has been studied as a vector for gene therapy.  However, Tat delivers cargo to cells with relatively low efficiency.  Researchers hypothesized that a Tat homodimer could yield stronger DNA binding than the monomer, which may improve the efficiency of DNA delivery.To study this hypothesis, Tat and two variant peptides (Variants 1 and 2) were produced by solid-phase peptide synthesis, in which the growing peptide chain is immobilized on polystyrene beads.  Unlike biological systems, solid-phase synthesis assembles peptides from the C-terminus to the N-terminus, as shown in Figure 1. Figure 1  Simplified schematic of one round of solid-phase peptide synthesis.  The protecting group is removed during deprotection.  The process is repeated until the peptide is complete.Variants 1 and 2 contained a C-terminal and an N-terminal cysteine residue, respectively.  Each variant was allowed to dimerize by disulfide bond formation.  Peptide-DNA binding interactions were then studied by mixing varying amounts of each peptide with plasmid DNA encoding luciferase protein (pLuc).  The resulting peptide-DNA complexes were run on agarose gels, with results displayed in Figure 2. Figure 2  Agarose gel results for pLuc plasmid exposed to varying amounts of each peptideEqual concentrations of each type of peptide-DNA complex were then incubated with cationic liposomes and exposed to cultured cells for 24 hours.  The cells were then assessed for luciferase activity.  Greater luciferase activity indicated more efficient delivery of the pLuc plasmid.  The results are shown in Figure 3. Figure 3  Plasmid delivery efficiency of three peptides, as measured by induced luciferase activity Adapted from Lee S-J, Yoon S-H, Doh K-O. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond. J Microbiol Biotechnol. 2011;21(8):802-807. -Which of the following best describes the reaction between amino acids during solid-phase peptide synthesis?

Passage Many cephalopods can change color to blend in with their surroundings by altering the conformations of particular proteins within their skin cells.  The reflectin proteins play a major role in this camouflage mechanism.  Reflectins are a group of intrinsically disordered proteins that do not adopt clearly defined secondary or tertiary structures.  Under certain conditions reflectins aggregate, allowing cells to reflect various wavelengths of light.Skin cells from the squid Doryteuthis opalescens were cultured and three reflectins-DoRA, DoRB, and DoRC-were purified and analyzed to determine amino acid composition.  They were found to contain an abundance of arginine and serine but relatively few cysteine, leucine, isoleucine, valine, phenylalanine, or tryptophan residues.  Figure 1 shows the relative hydrophobicity of each reflectin variant as a function of amino acid position. Figure 1  Hydrophobicity plots of three reflectins isolated from D. opalescens.  (Note: Negative numbers indicate hydrophilic regions.)In D. opalescens skin cells (pH ≈ 7.8), reflectins aggregate in response to acetylcholine (ACh), which causes a signal cascade that changes the phosphorylation state of the proteins.  Cells were incubated with or without ACh, and DoRA and DoRB were purified and assessed for phosphorylation by mass spectrometry.  DoRA became phosphorylated in the presence of ACh whereas DoRB became dephosphorylated.  After examining the isoelectric points of unmodified DoRA and DoRB, as shown in Figure 2, scientists proposed that reflectins aggregate upon charge neutralization. Figure 2  Isoelectric focusing of DoRA and DoRB Adapted from Demartini DG, Izumi M, Weaver AT, Pandolfi E, Morse DE.  Structures, Organization, and Function of Reflectin Proteins in Dynamically Tunable Reflective Cells.  J Biol Chem. 2015;290(24):15238-49. -To generate Figure 2, reflectins were loaded onto the low-pH side of a gel with a pH gradient, and a current was applied.  This resulted in:

Passage Synthesis of coenzyme A (CoA) requires vitamin B₅ as a substrate.  In bacteria, vitamin B₅ is synthesized by condensation of β-alanine and pantoic acid.  β-alanine is produced by decarboxylation of L-aspartate, mediated by the enzyme aspartate α-decarboxylase (ADC).  Active ADC is formed when its inactive precursor, PanD, binds to a regulatory complex composed of acetyl coenzyme A (AcCoA) and a protein known as PanZ.  Formation of the PanD-PanZ-AcCoA complex causes the activation loop of PanD to fold into a β-pleated sheet, resulting in PanD autocleavage.  Researchers hypothesize that this conformational change allows the Thr57 residue to cleave the N-terminal end of PanD to form ADC.The limited information on the role of PanZ in the synthesis of CoA has stimulated an investigation of its effect on ADC activity.  Escherichia coli cells were provided with varying concentrations of PanZ, and the rate of β-alanine production was measured in three different conditions (Figure 1).  Reaction vessels were maintained at pH 6 to ensure optimal decarboxylation activity. Figure 1  Catalytic activity of ADC in E. coli with increasing concentrations of L-aspartate Adapted from Monteiro DC, Patel V, Bartlett CP, et al. The structure of the PanD/PanZ protein complex reveals negative feedback regulation of pantothenate biosynthesis by coenzyme A. Chem Biol. 2015;22(4):492-503. -In E. coli, high levels of CoA lead to decreased β-alanine synthesis.  This is an example of:

Passage Dystroglycan (Dg) is a transmembrane protein that mediates interactions between the cytoskeleton and the extracellular matrix (ECM).  It binds the ECM protein laminin in an interaction that is required for intercellular communication and muscle membrane structural stability.  The Dg-laminin interaction is facilitated by a long carbohydrate chain called an O-mannosyl glycan, which is covalently linked to Dg (Figure 1). Figure 1  Schematic of interaction between Dg and laminin, mediated by the Dg-linked O-mannosyl glycanThe O-mannosyl glycan is added to Dg by a series of enzymes called glycosyltransferases.  The first enzyme in this series, protein O-mannosyltransferase (POMT), attaches D-mannose to the hydroxyl groups of serine and threonine residues in an α-linkage through the anomeric carbon.  The next sugar in the chain then forms a β-1,4 linkage to the mannose residue, followed by several more steps to produce the full glycan.  Genetic inactivation of any of the enzymes in the pathway results in an incomplete glycan and failure to bind laminin, and is the underlying cause of several severe forms of muscular dystrophy known as dystroglycanopathies.Researchers investigating dystroglycanopathies recently discovered the functions of two enzymes in the pathway:  protein O-mannose kinase (POMK) and fukutin.  POMK phosphorylates Dg-linked mannose at carbon 6, and fukutin adds the sugar ribitol 5-phosphate to the glycan chain.  To assess the roles of POMK and fukutin relative to each other, scientists conducted experiments in which lysates from muscle cells deficient in either POMK or fukutin were incubated with ³²P-labeled ATP or ³H-labeled activated ribitol 5-phosphate.  Combined lysates containing both fukutin and POMK were also tested.  Dg was then purified and detected by autoradiography and western blot using an antibody against Dg, as shown in Figure 2. Figure 2  Autoradiograms and western blots of Dg purified from lysates incubated with ³²P-labeled ATP or ³H-labeled ribitol 5-phosphate Adapted from Kanagawa M, Kobayashi K, Tajiri M, et al. Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy. Cell Rep. 2016;14(9):2209-23. -Which of the following structures could represent the completed O-mannosyl glycan?

Which factor decreases the stability of a DNA double helix during denaturation?

Passage Myalgic encephalopathy (ME), or chronic fatigue syndrome (CFS), is a physiological disease that affects 0.15% of the human population.  Compared with healthy individuals, persons with ME experience reduced phospholipid levels, muscular degeneration, urinary urgency, and other symptoms that contribute to a lower quality of life.  Studies in resting subjects have yielded inconsistent data and failed to identify a unique metabolic defect in patients with ME.Analyzing the blood plasma of male and female patients with ME, physicians have observed that male patients display elevated levels of 3-methylhistidine, a marker of protein catabolism.  Researchers expanded this finding by measuring the serum concentration of amino acids in blood samples obtained from nonfasting ME patients and healthy controls.  Amino acids were classified into separate categories according to their metabolic intermediates.Table 1  Classification of Amino Acids in Blood Plasma Figure 1  Serum concentrations of amino acids in patients with ME and healthy controls (HC)Next, researchers hypothesized that the inhibition of pyruvate dehydrogenase (PDH) and impairment of fatty acid oxidation were responsible for the fluctuations in amino acid levels.  PDH function and lipid metabolism were studied in peripheral blood mononuclear cells (PBMCs) by measuring the gene expression of proteins within regulatory pathways.  Researchers monitored the mRNA levels of the inhibitor pyruvate dehydrogenase kinase 1 (PDK1) and of sirtuin 4 (SIRT4), which removes lipoic acid groups from the PDH complex.  They also examined lipid metabolism by measuring the mRNA levels of peroxisome proliferator-activated receptor-α (PPARα), a transcription factor that upregulates the uptake of fatty acids.  The mRNA level of each protein in PBMCs of patients with ME was evaluated against healthy controls, as shown in Figure 2. Figure 2  Gene expression of PDK1, SIRT4, and PPARα in PBMCs Adapted from Fluge Ø, Mella O, Bruland O, et al.. JCI Insight. 2016;1(21):e89376. -Based on Figure 2, the availability of ATP in PBMCs is most likely affected by which process?

An enzyme was assessed for its ability to cleave three substrates with similar structures, Compounds 1-3, as shown. Based on the graph, which statement best describes the interaction of the enzyme with the compounds?  The enzyme:

Passage The sirtuins are a class of enzymes that regulate various metabolic processes by removing post-translational modifications from enzymes.  SIRT4 is an autosomally encoded sirtuin protein that colocalizes with the pyruvate dehydrogenase complex.  It deacylates lysine residues on certain metabolic proteins by removing methylglutaryl and hydroxymethylglutaryl units.  Methylglutaryl-CoA and hydroxymethylglutaryl-CoA are intermediates in leucine catabolism, and scientists hypothesized that SIRT4 regulates this pathway.  To test their hypothesis, livers from wild-type (WT) and SIRT4 knockout (KO) mice were treated with α-keto acid derivatives of several amino acids, including the derivative of leucine, α-ketoisocaproate (αKIC).  Figures 1 and 2 show the rate of oxidation of α-keto acids and oxygen consumption in both WT and KO livers, respectively. Figure 1  Box plot showing the rate of oxidation of α-ketoisocaproate (αKIC), α-ketoglutarate (αKG), and pyruvate in SIRT4 knockout livers (Note: Asterisk indicates p < 0.05 relative to WT level) Figure 2  Oxygen consumption in WT and KO livers in the presence and absence of succinate and reduced cytochrome C (Cyt-C)Because leucine is known to regulate insulin secretion, scientists injected WT and KO mice with leucine and measured plasma insulin levels over time.  They then injected mice with insulin and monitored blood glucose levels (Figure 3). Figure 3  (A) Plasma insulin levels after injection with leucine and (B) blood glucose levels after injection with insulin in both WT and KO mice Adapted from Anderson KA, Huynh FK, Fisher-Wellman K, et al. SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion. Cell Metab. 2017;25(4):838-855.e15. -Under aerobic conditions, which of the following molecules would be expected to be present in significantly decreased levels in the livers of KO mice?

Passage Diabetic nephropathy (kidney disease) is characterized in part by a significant decrease in the glomerular filtration rate.  Podocytes are differentiated cells that surround the glomerular capillaries and counteract the pressure of blood filtration.  Hyperglycemia (elevated blood glucose) and capillary hypertension (high blood pressure), both of which are common in diabetic patients, can subject podocytes to mechanical stress.  This mechanical stress has been shown to regulate glucose uptake by podocytes, possibly by altering the production of glucose transport (GLUT) proteins.Researchers investigating GLUT2 and GLUT4 expression in rat podocytes measured the impact of mechanical stress on glucose uptake.  Cells were grown for 4 hours on membranes exposed to atmospheric (atm) or high air pressure (HP) to mimic mechanical stress.  Antibodies specific to GLUT2 and GLUT4 were used to visualize the expression of each under normal glucose (NG) and hyperglycemic (HG) conditions, and to assess the effect of mechanical stress on expression.  The uptake of a radiolabeled glucose analogue, [³H]-2-deoxy-D-glucose (3H-2DG), was then determined.  The quantity of 3H-2DG transported by each GLUT protein was directly proportional to the cell surface expression. Figure 1  Cell surface expression of GLUT transporters in response to mechanical stress and varying glucose concentration.In vivo studies have shown that transport via GLUT2 is noninducible and depends only on extracellular glucose concentrations.  However, GLUT4-mediated uptake is induced by insulin binding and occurs under high glucose conditions when insulin is released.  The standard K_m values for GLUT2 and GLUT4 are 19.4 and 4.3 mM, respectively, at normal blood glucose levels after a meal (~7.8 mM).  The data shown in Figure 2 represent the combined kinetic properties of both transporters. Figure 2  Plot of glucose uptake in podocytes with and without mechanical stress. Adapted from Lewko, B., Bryl, E., Witkowski, J. M., Latawiec, E., Angielski, S., & Stepinski, J. (2005). Mechanical stress and glucose concentration modulate glucose transport in cultured rat podocytes. Nephrology Dialysis Transplantation, 20(2), 306-311. -The drug indinavir inhibits GLUT isoforms noncompetitively.  Researchers predict that GLUT4 is significantly more susceptible to indinavir than GLUT2.  Which of the following observations would best support their hypothesis?

A fatty acid undergoes complete β-oxidation to yield nine molecules of acetyl-CoA.  No isomerization reactions occur.  The structure of the original fatty acid was:

Passage Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is one of the most abundant enzymes on Earth.  In carbon-fixing organisms, it consumes CO₂ to carboxylate the sugar ribulose-1,5-bisphosphate (Ru1,5BP), and forms two molecules of 3-phosphoglycerate (3PG) as a product.  The functional form of rubisco from the bacterium Rhodospirillum rubrum is a noncovalent homodimer composed of two 51 kDa monomers.  Each subunit requires a magnesium ion as a prosthetic group in the active site.Rubisco is often used as a model for protein folding studies.  On their own, rubisco monomers typically cannot fold to completion and instead are trapped in a form known as a "kinetic intermediate."  These kinetically trapped monomers cannot dimerize correctly and are prone to aggregation.Scientists interested in studying protein folding dynamics labeled the rubisco kinetic intermediate with a set of fluorophores known as a FRET (fluorescence resonance energy transfer) pair, with one fluorophore at the N-terminus and the other at the C-terminus.  A FRET signal occurs when the emission spectrum of one fluorophore (the donor) overlaps with the excitation spectrum of the other (the acceptor).  When the two fluorophores are sufficiently close to each other, the donor can transfer energy from the light it absorbs directly to the acceptor, causing the acceptor to emit light when the donor absorbs light.The GroEL/ES complex is a chaperone protein that binds and releases misfolded proteins, hydrolyzing ATP each time the protein is released.  A misfolded protein may undergo multiple rounds of binding and release before adopting its correct conformation, or it may never fold correctly and instead be targeted for destruction (Figure 1). Figure 1  Representation of GroEL/ES-mediated protein foldingResearchers diluted the labeled kinetic intermediate into a refolding buffer in the presence or absence of 200 nM GroEL/ES and then monitored the FRET signal over time.  The results are shown in Figure 2. Figure 2  FRET signal of labeled rubisco with and without GroEL Lin Z, Rye HS. Expansion and compression of a protein folding intermediate by GroEL. Mol Cell. 2004;16(1):23-34. -Which of the following experiments, performed in a closed system, could confirm that GroEL/ES successfully facilitated proper rubisco folding?

Passage Aldehyde dehydrogenase 2 (ALDH2) is essential for the oxidation of toxic aldehydes, and uses NAD⁺ as the oxidizing agent.  Mitochondrial ALDH2 exists as a 220-kD homotetrameric protein containing a catalytic, nucleotide-binding, and coenzyme-binding domain.  The most prevalent ALDH2 mutation (ALDH2*2) involves an E to K (E478K) substitution in the catalytic domain that leads to decreased enzymatic activity.Scientists hypothesize that Asian populations have higher rates of ALDH2*2 incidence.  The properties of ALDH2 variants in Caucasian and Japanese populations were analyzed in the following experiments.Experiment 1Mitochondrial extracts from both populations were combined in a single mixture and separated by native gel electrophoresis.  Proteins 210-230 kD in size were removed from the agarose gel and further isolated using affinity columns with antibodies designed to separate wild-type ALDH2 from ALDH2*2 by binding the catalytic domain of the wild-type but not the mutant protein.  Concentrated eluates E1 and E2 were produced by washing the affinity columns with buffers that remove unbound proteins and dislodge bound proteins from antibodies, respectively (Figure 1). Figure 1  Purification of ALDH2 proteinsALDH2 activity in each eluate was quantified by adding acetaldehyde and NAD⁺, and then monitoring the rate of NADH production.  The results are shown in Figure 2. Figure 2  Enzymatic activity of E1 and E2 eluatesExperiment 2Researchers predict that mutations at conserved amino acid sites containing S or T may prevent ALDH2 from being phosphorylated by protein kinase C epsilon (εPKC).  The activity of wild-type ALDH2 (WT) and ALDH2 mutants with substitutions at positions T185 and S279 in the presence or absence of εPKC are summarized in Table 1.Table 1  Effect of εPKC on ALDH2 Variants Adapted from Chen CH, Ferreira JC, Gross ER, Mochly-rosen D. Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiol Rev. 2014;94(1):1-34. -Replacing native gel electrophoresis with nonreducing sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) during the purification step results in eluates with no enzymatic activity.  The best explanation of this observation would be:

Passage The K-ras gene codes for a small guanine nucleotide-binding protein, which acts as a molecular activator in numerous signaling pathways that promote DNA replication along with cell growth and survival.  K-ras mutations at codons 12 and 13 function as biological markers for diagnosis of tumors.  The DNA of SW-480 cells, a line originating from a colon adenocarcinoma, is the standard positive control used to identify codon 12 mutations in K-ras testing. Figure 1  Structural comparison of K-ras codon 12 in wild-type cells and SW-480 cellsPCR sequence analysis has been the preferred method for studying genetic mutations such as those in K-ras.  However, challenges in analyzing PCR amplicons have prompted the development of DNA melting curve analysis (MCA).  In the MCA technique, DNA is extracted from clinical samples and amplified by PCR.  The amplified DNA is then melted (30-70°C) and analyzed by assessing the fluorescence (at 640 nm) of LC Green Plus, an intercalating dye that fluoresces when bound to double-stranded DNA (Figure 2). Figure 2  Melting curves of wild-type (WT) and SW-480 K-ras DNA.To study K-ras activity in colorectal cancer cells, short tandem repeats (STRs) were introduced into the K-ras gene.  STRs are short stretches of DNA with repeating nucleotides, and four different STR sequences were inserted into the K-ras gene and analyzed via MCA under different experimental conditions (Table 1).Table 1  Sequences Studied in K-ras DNA MCA Analysis. Liu YP, Wu HY, Yang X, et al. Diagnostic accuracy of high resolution melting analysis for detection of KRAS mutations: a systematic review and meta-analysis. Sci Rep. 2014;4:7521. -What are the physical states of wild-type (WT) and SW-480 K-ras DNA at 55°C?

Which of the following molecules could participate in cell signaling without a second messenger?

Passage The innate immune system relies heavily on the phagocytic action of neutrophils, mobile white blood cells that engulf and degrade bacteria and viruses as the first line of host defense against invading pathogens.  One mechanism for eliminating viruses engulfed in the phagosome involves the degradation of viral proteins by neutrophil serine proteases (NSPs).NSPs, which form part of the chymotrypsin family of serine proteases, cleave viral proteins using a catalytic triad relay system.  Originally discovered in granules released by neutrophils, NSP4 is a novel monomeric serine protease that has been studied to elucidate the catalytic mechanism of NSPs.  Prior experimental data suggest that NSP4 alters its tertiary structure upon substrate binding and cleaves viral proteins using the acylation reaction shown in Figure 1. Figure 1  Degradation of viral proteins by acylation of NSP4Computational methods were used to identify the energetic parameters of reactions between NSP4 and synthetic viral proteins.  To ensure that simulated reactions resembled physiological conditions, algorithms were adjusted to account for the enzyme being in an aqueous environment.  The rate-determining step in the acylation reaction was determined by calculating the changes in free energy in both the enzyme and substrate (Figure 2). Figure 2  Free energy profiles of synthetic viral protein cleavageKnowledge of amino acid frequency distribution at an enzymatic active site may facilitate the design of novel active sites and enzyme-specific inhibitors.  Researchers analyzed the composition of amino acids that form the NSP4 active site during protein folding to study the characteristics of residues involved in active site formation (Figure 3). Figure 3  Frequency distribution of amino acid residues at the NSP4 active site Adapted from Stapels DA, Geisbrecht BV, Rooijakkers SH. Neutrophil serine proteases in antibacterial defense. Curr Opin Microbiol. 2015;23:42-8. -What is the activation energy for the uncatalyzed reaction in Figure 2?

Passage A newborn girl is diagnosed with a rare genetic disorder that causes premature lysis of red blood cells.  Through genome sequencing, physicians identified a specific mutation (R510Q) in both copies of her pyruvate kinase gene, which encodes the enzyme that catalyzes the final step of glycolysis.  Pyruvate kinase produces pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP.PEP + ADP ↔ Pyruvate + ATPReaction 1Because red blood cells lack mitochondria, pyruvate in erythrocytes does not enter the citric acid cycle and the electron transport chain.  Therefore, red blood cells rely almost exclusively on glycolysis to produce sufficient ATP for cellular functions.  In patients with pyruvate kinase deficiency, it is thought that a lack of ATP compromises the integrity of the red blood cell membrane, leading to premature lysis in the spleen.  Although these observations are well established, the exact mechanism by which low ATP alters the cell membrane is unclear.Scientists interested in analyzing this particular patient's gene variant expressed and purified recombinant R510Q pyruvate kinase.  They performed several biochemical tests to characterize the R510Q mutant with respect to wild-type (WT) pyruvate kinase.  In the first experiment, WT and R510Q pyruvate kinases were each incubated with a constant amount of ADP and varying amounts of PEP.  The activity of the enzyme was measured at each concentration of PEP and plotted.  They then repeated the experiment in the presence of ATP and found that ATP is a noncompetitive inhibitor of the WT enzyme, but a mixed inhibitor of the R510Q enzyme.  The results are shown in Figure 1.  All experiments were done at the same enzyme concentration. Figure 1  Pyruvate kinase activity with and without ATP as a function of [PEP].  Open markers are shown at ½V_max for each plot. Adapted from Wang C, Chiarelli LR, Bianchi P, et al. Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia. Blood. 2001;98(10):3113-20. -Scientists predicted that gene copy number is proportional to protein expression for pyruvate kinase.  If this hypothesis is correct, which kinetic parameter would be expected to double when the gene copy number doubles?

Passage Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is one of the most abundant enzymes on Earth.  In carbon-fixing organisms, it consumes CO₂ to carboxylate the sugar ribulose-1,5-bisphosphate (Ru1,5BP), and forms two molecules of 3-phosphoglycerate (3PG) as a product.  The functional form of rubisco from the bacterium Rhodospirillum rubrum is a noncovalent homodimer composed of two 51 kDa monomers.  Each subunit requires a magnesium ion as a prosthetic group in the active site.Rubisco is often used as a model for protein folding studies.  On their own, rubisco monomers typically cannot fold to completion and instead are trapped in a form known as a "kinetic intermediate."  These kinetically trapped monomers cannot dimerize correctly and are prone to aggregation.Scientists interested in studying protein folding dynamics labeled the rubisco kinetic intermediate with a set of fluorophores known as a FRET (fluorescence resonance energy transfer) pair, with one fluorophore at the N-terminus and the other at the C-terminus.  A FRET signal occurs when the emission spectrum of one fluorophore (the donor) overlaps with the excitation spectrum of the other (the acceptor).  When the two fluorophores are sufficiently close to each other, the donor can transfer energy from the light it absorbs directly to the acceptor, causing the acceptor to emit light when the donor absorbs light.The GroEL/ES complex is a chaperone protein that binds and releases misfolded proteins, hydrolyzing ATP each time the protein is released.  A misfolded protein may undergo multiple rounds of binding and release before adopting its correct conformation, or it may never fold correctly and instead be targeted for destruction (Figure 1). Figure 1  Representation of GroEL/ES-mediated protein foldingResearchers diluted the labeled kinetic intermediate into a refolding buffer in the presence or absence of 200 nM GroEL/ES and then monitored the FRET signal over time.  The results are shown in Figure 2. Figure 2  FRET signal of labeled rubisco with and without GroEL Lin Z, Rye HS. Expansion and compression of a protein folding intermediate by GroEL. Mol Cell. 2004;16(1):23-34. -Based on the molecular weight of a rubisco monomer, approximately how many amino acids are present in a rubisco dimer?

Passage Many cephalopods can change color to blend in with their surroundings by altering the conformations of particular proteins within their skin cells.  The reflectin proteins play a major role in this camouflage mechanism.  Reflectins are a group of intrinsically disordered proteins that do not adopt clearly defined secondary or tertiary structures.  Under certain conditions reflectins aggregate, allowing cells to reflect various wavelengths of light.Skin cells from the squid Doryteuthis opalescens were cultured and three reflectins-DoRA, DoRB, and DoRC-were purified and analyzed to determine amino acid composition.  They were found to contain an abundance of arginine and serine but relatively few cysteine, leucine, isoleucine, valine, phenylalanine, or tryptophan residues.  Figure 1 shows the relative hydrophobicity of each reflectin variant as a function of amino acid position. Figure 1  Hydrophobicity plots of three reflectins isolated from D. opalescens.  (Note: Negative numbers indicate hydrophilic regions.)In D. opalescens skin cells (pH ≈ 7.8), reflectins aggregate in response to acetylcholine (ACh), which causes a signal cascade that changes the phosphorylation state of the proteins.  Cells were incubated with or without ACh, and DoRA and DoRB were purified and assessed for phosphorylation by mass spectrometry.  DoRA became phosphorylated in the presence of ACh whereas DoRB became dephosphorylated.  After examining the isoelectric points of unmodified DoRA and DoRB, as shown in Figure 2, scientists proposed that reflectins aggregate upon charge neutralization. Figure 2  Isoelectric focusing of DoRA and DoRB Adapted from Demartini DG, Izumi M, Weaver AT, Pandolfi E, Morse DE.  Structures, Organization, and Function of Reflectin Proteins in Dynamically Tunable Reflective Cells.  J Biol Chem. 2015;290(24):15238-49. -If DoRA aggregation is caused by charge neutralization, then aggregates should form as pH:

Which of the following structures represents the Fischer projection of a β-D-glucose unit found in cellulose?

Passage The influenza A virus is an enveloped RNA virus with potential for zoonosis (transmission from animals to humans).  It obtains its plasma membrane from its host cell, but the composition of the viral envelope and vertebrate plasma membrane are distinct.  The envelope of H6-G225D consists mostly of phosphatidylethanolamine as well as some negatively charged glycerophospholipids.  The outer membrane contains hemagglutinin (HA) proteins that bind to the carbohydrate known as sialic acid (SA) on host cell receptor proteins, facilitating viral entry.  It also contains neuraminidase enzymes that break the bond between SA and galactose in the host membrane glycans, allowing new virions to be released.Minute changes in the genes that code for HA proteins can alter the virulence, host, and attachment mechanism.  In general, influenza A infects avian species by attaching to tracheal tissue, which displays SA groups bound to receptors through an α-2,3-glycosidic linkage to galactose (Figure 1).  Avian viruses generally cannot attack human cells, in which SA is linked to galactose through an α-2,6-glycosidic linkage instead of an α-2,3 linkage. Figure 1  Structure of the influenza A receptor in avian cell membranesIn a recent study, however, a G225D substitution in HA, caused by a GGC to GAU codon mutation, enabled avian virions to infect human cells.  The stability of G225D HA proteins was compared to that of H6-P186L, which is created by a single CCC to CUC mutation and exclusively infects avian cells, and H1N1, which targets only humans.  The viral RNA of H6-P186L, H6-G225D, and H1N1 was cloned using cDNA and expressed in embryonic kidney cells.  The melting curve of each purified HA protein was determined using differential scanning calorimetry, with peaks representing melting temperatures (Figure 2). Figure 2  Hemagglutinin melting curves for H6N1 and H1N1 influenza A strains Adapted from De vries RP, Tzarum N, Peng W, et al.  A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors.  EMBO Mol Med. 2017;9(9):1314-1325. -Which of the following correctly identifies a reaction involved in the formation of the P186L mutation in H6N1 RNA?