Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α₂β₂ tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α₂β₂ tetramer. Each β subunit in the α₂β₂ tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.     

Long-range effects of tag sequence on marginally stabilized structure in HIV-1 p24 capsid protein monitored using NMR

N-terminal domain of HIV-1 p24 capsid protein is a globular fold composed of seven helices and two β-strands with a flexible structure including the α4–5 loop and both N- and C-terminal ends. However, the protein shows a high tendency (48%) for an intrinsically disordered structure based on the PONDR VL-XT prediction from the primary sequence. To assess the possibility of marginally stabilized structure under physiological conditions, the N-terminal domain of p24 was destabilized by the addition of an artificial flexible tag to either N- or C-terminal ends, and it was analyzed using T₁, T₂, hetero-nuclear NOE, and amide-proton exchange experiments. When the C-terminal tag (12 residues) was attached, the regions of the α3–4 loop and helix 6 as well as the α4–5 loop attained the flexible structures. Furthermore, in the protein containing the N-terminal tag (27 residues), helix 4 in addition to the above-mentioned area including α3–4 and α4–5 loops as well as helix 6 exhibited highly disordered structures. Thus, the long-range effects of the existence of tag sequence was observed in the stepwise manner of the appearance of disordered structures (step 1: α4–5 loop, step 2: α3–4 loop and helix 6, and step 3: helix 4). Furthermore, the disordered regions in tagged proteins were consistent with the PONDR VL-XT disordered prediction. The dynamic structure located in the middle part (α3–4 loop to helix 6) of the protein shown in this study may be related to the assembly of the viral particle.     

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s

The major histocompatibility complex (MHC) class I antigens contain a light chain β₂-microglobulin, non-covalently associated to the transmembrane heavy α-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with β₂-microglobulin, and we recently found that activated C1s complement was capable of cleaving β₂-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22 000 and 24 000 g/ mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the α₂-andα₃-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-A^d). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the α₁andα₂-domains which represent the binding site for atnigenic peptide.     

Molecular dynamics modeling of tubulin C-terminal tail interactions with the microtubule surface

Tubulin, an α/β heterodimer, has had most of its 3D structure analyzed; however, the carboxy (C)-termini remain elusive. Importantly, the C-termini play critical roles in regulating microtubule structure and function. They are sites of most of the post-translational modifications of tubulin and interaction sites with molecular motors and microtubule-associated proteins. Simulated annealing was used in our molecular dynamics modeling to predict the interactions of the C-terminal tails with the tubulin dimer. We examined differences in their flexibility, interactions with the body of tubulin, and the existence of structural motifs. We found that the α-tubulin tail interacts with the H11 helix of β-tubulin, and the β-tubulin tail interacts with the H11 helix of α-tubulin. Tail domains and H10/B9 loops interact with each other and compete for interactions with positively-charged residues of the H11 helix on the neighboring monomer. In a simulation in which α-tubulin's H10/B9 loop switches on sub-nanosecond intervals between interactions with the C-terminal tail of α-tubulin and the H11 helix of β-tubulin, the intermediate domain of α-tubulin showed more fluctuations compared to those in the other simulations, indicating that tail domains may cause shifts in the position of this domain. This suggests that C-termini may affect the conformation of the tubulin dimer which may explain their essential function in microtubule formation and effects on ligand binding to microtubules. Our modeling also provides evidence for a disordered-helical/helical double-state system of the T3/H3 region of the microtubule, which could be linked to depolymerization following GTP hydrolysis.     

Structure of the integrin alpha2beta1-binding collagen peptide

We have determined the 1.8 Å crystal structure of a triple helical integrin-binding collagen peptide (IBP) with sequence (Gly-Pro-Hyp)₂-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp)₃. The central GFOGER hexapeptide is recognised specifically by the integrins α2β1, α1β1, α10β1 and α11β1. These integrin/collagen interactions are implicated in a number of key physiological processes including cell adhesion, cell growth and differentiation, and pathological states such as thrombosis and tumour metastasis. Comparison of the IBP structure with the previously determined structure of an identical collagen peptide in complex with the integrin α2-I domain (IBP^c) allows the first detailed examination of collagen in a bound and an unbound state. The IBP structure shows a direct and a water-mediated electrostatic interaction between Glu and Arg side-chains from adjacent strands, but no intra-strand interactions. The interactions between IBP Glu and Arg side-chains are disrupted upon integrin binding. A comparison of IBP and IBP^c main-chain conformation reveals the flexible nature of the triple helix backbone in the imino-poor GFOGER region. This flexibility could be important to the integrin-collagen interaction and provides a possible explanation for the unique orientation of the three GFOGER strands observed in the integrin-IBP^c complex crystal structure.     

Conformational dynamics of xylanase a from Streptomyces lividans: Implications for TIM‐barrel enzyme thermostability

The conformational dynamics of xylanase A from Streptomyces lividans (Sl‐XlnA) were studied using Molecular Dynamics (MD) simulation to identify the thermally sensitive regions. Sl‐XlnA begins to unfold at loop4 and this unfolding expands to the loops near the N‐terminus. The high flexibility of loop6 during the 300 K simulation is related to its function. The intense movements of the 3₁₀‐helices also affect the structural stability. The interaction between the α4β5‐loop and the neighboring α5β6‐loop plays a crucial role in stabilizing the region from the α4β5‐loop to α6. The most thermally sensitive region is from β3 to loop4. The high mobility of the long loop4 easily transfers to the adjacent β4 and α4 and causes β4 and α4 to fluctuate. And, salt bridges ASP124‐ARG79, ASP200‐ARG159, and ASP231‐LYS166 formed a “clamp” to stabilize the region including α4, β4, β5, β6, and β7. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 594–604, 2013.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

Conformational stabilities of the rat α- and β-parvalbumins

It is widely believed that β-parvalbumin (PV) isoforms are intrinsically less stable than α-parvalbumins, due to greater electrostatic repulsion and an abbreviated C-terminal helix. However, when examined by differential scanning calorimetry, the apo-form of the rat β-PV (i.e. oncomodulin) actually displays greater thermal stability than the α-PV. Whereas the melting temperature of the α isoform is 45.8°C at physiological pH and ionic strength, the T_m for the β isoform is more than 7° higher (53.6°C). This result suggests that factors besides net charge and C-terminal helix length strongly influence parvalbumin conformational stability. Extension of the F helix in the β-PV, by insertion of Ser-109, has a modest stabilizing effect, raising the T_m by 1.1°. Truncation of the α-PV F helix, by removal of Glu-108, has a more profound impact, lowering the T_m by 4.0°.     

Conformation of oligopeptides with L-leucyl-L-leucyl-L-alanyl and L-methionyl-L-methionyl-L-leucyl repeating units

The conformation of oligopeptides with hydrophobic side chains, Nps-(L -Leu-L -Leu-L -Ala)_n-OEt and Nps-(L -Met-L -Met-L -Leu)_n-OEt(n = 1–6), in the solid state, obtained either by evaporation of the solvent or by precipitation with diethyl ether from a 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solution, has been studied with ir spectroscopy and x-ray powder-diffraction measurements. The conformation of these peptides in the HFIP solution has been studied by CD spectroscopy. Due to a strong preference of the amino acids to form an α helix, the peptides begin forming α helices at the dodecapeptide in the HFIP solution, and in the solid state by evaporation. In the solid state, with precipitation, the α-helical conformation is first observed at the octadecapeptide and the lower peptides assume a β structure. The conformational change, from the α helix to the β structure of the peptides with 12 to 15 amino acid residues, during the precipitation process, is due to a strong tendency of the amino acids to form the β-structure in rather short peptide lengths.     

Structural basis of integrin transmembrane activation

Integrins are cell adhesion receptors that transmit bidirectional signals across plasma membrane and are crucial for many biological functions. Recent structural studies of integrin transmembrane (TM) and cytoplasmic domains have shed light on their conformational changes during integrin activation. A structure of the resting state was solved based on Rosetta computational modeling and experimental data using intact integrins on mammalian cell surface. In this structure, the α_IIb GXXXG motif and their β₃ counterparts of the TM domains associate with ridge‐in‐groove packing, and the α_IIb GFFKR motif and the β₃ Lys‐716 in the cytoplasmic segments play a critical role in the α/β association. Comparing this structure with the NMR structures of the monomeric α_IIb and β₃ (represented as active conformations), the α subunit helix remains similar after dissociation whereas β subunit helix is tilted by embedding additional 5–6 residues into the lipid bilayer. These conformational changes are critical for integrin activation and signaling across the plasma membrane. We thus propose a new model of integrin TM activation in which the recent NMR structure of the α_IIbβ₃ TM/cytoplasmic complex represents an intermediate or transient state, and the electrostatic interaction in the cytoplasmic region is important for priming the initial α/β association, but not absolutely necessary for the resting state. J. Cell. Biochem. 109: 447–452, 2010. © 2009 Wiley‐Liss, Inc.     

Starch- and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes

Sequence alignment and structure prediction are used to locate catalytic α-amylase-type (β/α)₈-barrel domains and the positions of their β-strands and α-helices in isoamylase, pullulanase, neopullulanase, α-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes—all enzymes involved in hydrolysis or synthesis of α-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of β ? α loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved β-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.     

Local secondary structure in ribonuclease A denatured by guanidine · HCl near 1 °C

Recent work has shown that a short α-helix can be stable in water near 1 °C when stabilized by specific interactions between side-chains, while earlier “host-guest” results with random copolymers have shown that a short α-helix is unstable in water at all temperatures in the absence of stabilizing side-chain interactions. As regards the mechanism of protein folding, it is now reasonable on energetic grounds to consider isolated α-helices and β-sheets as the first intermediates on the pathway of protein folding. Proton nuclear magnetic resonance is used here to detect isolated secondary structures in ribonuclease A denatured by guanidine · HCl (GuHCl). Temperatures near 1 °C are used because the low-temperature stability of the C-peptide helix may be a general property of isolated secondary structures in water.Our procedure is to titrate with GuHCl the C2H resonance lines of the four histidine residues of denatured ribonuclease A. Studies of model peptides (C-peptide (lactone) and C-peptide carboxylate, residues 1 to 13 of ribonuclease A; S-peptide, residues 1 to 20) show linear titration curves for the C2H resonance of His12 above 0.5 M-GuHCl, once helix unfolding is complete. Deviations from this line are used to monitor helix formation. The GuHCl titration curves of the other three histidine residues are also linear, once unfolding is complete. The results show that the helix found in C-peptide and S-peptide is also found in denatured ribonuclease A, where it behaves as an isolated helix not stabilized significantly by interactions with other chain segments. Studies of denatured S-protein show that the remaining three His residues, His48, His105 and His119, are involved in structure only below 1 m-GuHCl at 9 °C, pH 1.9. The nature of this structure is not known. The main conclusion from this work is that the His12 helix can be observed as a stable, isolated helix in denatured ribonuclease A near 1 °C, and that none of the other three His residues is involved in a comparably stable local structure. In native ribonuclease A, His12 is within an α-helix and the other three His residues are involved in a 3-stranded β-sheet structure.The helix-coil transition of C-peptide has also been studied for other side-chain resonances by GuHCl titration. Typically, but not always, the titration curves are linear after helix unfolding takes place and resonance lines from different residues of the same amino acid type can be resolved in GuHCl solutions. This is true of the four histidine residues of ribonuclease A although their pK values in 5 m-GuHCl are nearly the same. In C-peptide, the βCH₃ resonance of Ala6 is affected strongly by GuHCl while the lines of Ala4 and Ala5 are shifted only weakly by GuHCl. Evidently the interactions between GuHCl and side-chains in an unfolded peptide depend upon neighboring groups.     

Protein conformational changes induced in human stratum corneum by organic sulfoxides: An infrared spectroscopic investigation

Formation of the antiparallel-chain β-sheet protein conformation is induced in in vitro human stratum corneum by three homologous organic sulfoxides known to enhance skin permeability: dimethylsulfoxide (Me₂SO), hexylmethylsulfoxide (HxMeSO), and decylmethylsulfoxide (DecMeSO). Me₂SO and HxMeSO apparently function by displacing water molecules bound to polar protein side-chains, whereas DecMeSO probably interacts hydrophobically with the protein. The conformational transition does not result from lipid removal. The β-sheet protein, most likely formed in normally α-helical portions of the intracellular keratin filaments, is reconverted to α-helix upon rehydration of the tissue. Though neat Me₂SO produces the most β-sheet of all treatments examined, the sequence of ability to promote β-sheet formation at the 1M level is HxMeSO > DecMeSO > Me₂SO. Spectroscopic evidence is presented regarding the dependence of β-sheet formation on sulfoxide concentration, treatment duration, pH, and tissue hydration. The relationship of this conformational change to the enhancement of skin permeability is briefly discussed. The result of sulfoxide treatment is different from results of sodium dodecylsulfate and heat treatments of stratum corneum.     

Conformational studies on substance P. C-terminal pentapeptide pGlu-Phe-Phe-Gly-Leu-Met NH2

The conformational behavior of the active C-terminal pentapeptide of substance P(SP), pGlu-Phe-Phe-Gly-Leu-Met NH₂ [pGlu-SP(7–11)] was investigated using empirical energy calculations. A sequential approach was used to display the specific contribution of each residue to induce stable conformations of the whole pentapeptide. The most stable conformations include the α_R helix and some partially helical structures; some conformations with glycyl residue in a C₇^eq and C₇^ax configurations (γ and $\text{γ}$ turns) are also favoured. Helical conformations provide a good accessibility of side-chains which play an important role in interacting with the receptor. Fully extended structures and β turns are not specially stable. Such helical stable structures would favour a “lock and key” model of binding.     

Engineered Cystine-Knot Peptides that Bind αvβ3 Integrin with Antibody-Like Affinities

The α_vβ₃ integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4-kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to α_vβ₃ integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a six amino acid loop of AgRP with a nine amino acid loop containing the Arg-Gly-Asp integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting to identify clones with high affinity to detergent-solubilized α_vβ₃ integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing α_vβ₃ integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown to bind specifically to α_vβ₃ integrins and had only minimal or no binding to α_vβ₅, α₅β₁, and α_iibβ₃ integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally occurring ligand for α_vβ₃ and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second-generation libraries by individually randomizing these loops in one of the high-affinity integrin-binding variants. Screening of these loop-randomized libraries against α_vβ₃ integrins resulted in peptides that retained high affinities for α_vβ₃ and had increased specificities for α_vβ₃ over α_iibβ₃ integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.     

Effects of mutations in the beta subunit hinge domain on ATP synthase F1 sector rotation: interaction between Ser 174 and Ile 163

A complex of γ, ε, and c subunits rotates in ATP synthase (F_oF₁) coupling with proton transport. Replacement of βSer174 by Phe in β-sheet4 of the β subunit (βS174F) caused slow γ subunit revolution of the F₁ sector, consistent with the decreased ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704]. Modeling of the domain including β-sheet4 and α-helixB predicted that the mutant βPhe174 residue undergoes strong and weak hydrophobic interactions with βIle163 and βIle166, respectively. Supporting this prediction, the replacement of βIle163 in α-helixB by Ala partially suppressed the βS174F mutation: in the double mutant, the revolution speed and ATPase activity recovered to about half of the levels in the wild-type. Replacement of βIle166 by Ala lowered the revolution speed and ATPase activity to the same levels as in βS174F. Consistent with the weak hydrophobic interaction, βIle166 to Ala mutation did not suppress βS174F. Importance of the hinge domain [phosphate-binding loop (P-loop)/α-helixB/loop/β-sheet4, βPhe148-βGly186] as to driving rotational catalysis is discussed.     

Structure,Function, and Regulation of the Three β-Adrenergic Receptors

Three β-adrenergic receptor subtypes are now known to be functionally expressed in mammals. All three belong to the R₇G family of receptors coupled to G-proteins, and characterized by an extracellular glycosylated N-terminal and an intracellular C-terminal region and seven transmembrane domains, linked by three exta- and three intracellular loops. The catecholamine ligand binding domain, studied using affinity-labeling and site-directed mutagenesis, is a pocket lined by residues belonging to the transmembrane domains. The region responsible for the interaction with the Gs protein which, when activated, stimulates adenylyl cyclase, is composed of residues belonging to the parts most proximal to the membrane of intracellular loop i₃ and the C-terminal region. The pharmacology of the three subtypes is quite distinct: in fact most of the potent β₁/β₂ antagonists (the well known β blockers) act as agonists on β₃. The subtype is resistant to short-term desensitization mediated by phosphorylation through PKA or βARK, in stark contrast to the β₁ or β₂ subtypes. Various compounds (dexamethasone, butyrate, insulin) up regulate β₁ or β₁ subtypes while down-regulating β₃ whose expression strictly correlates with differentiation of 3T3-F442A fibroblasts into adipocytes, thus confirming that the expression of the three subtypes may each be regulated independently to exert a specific physiologic role in different tissues or at different stages of development.     

Analysis of the Open and Closed Conformations of the β Subunits in Thermophilic F1-ATPase by Solution NMR

F₁-ATPase, composed of α, β, γ, δ, and ? subunits, is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this function is the α₃β₃γ complex. We have investigated the α₃β₃γ?^ΔC (?^ΔC, truncated ?) complex from thermophilic Bacillus PS3 (TF₁′, 360 kDa) in the solution state by using the combination of extensive deuteration, segmental-labeling, and CRINEPT (cross-correlated relaxation-enhanced polarization transfer) NMR. Well-resolved CRINEPT-HMQC (heteronuclear multiple-quantum correlation) spectra of partially ¹⁵N-labeled TF₁′ were obtained for this huge and asymmetric protein complex. The spectrum of the C-terminal domain of the β subunit revealed that the open form of the β subunit in the TF₁′ complex is similar to that of the free β monomer. The open β subunit in the TF₁′ complex does not exhibit high affinity for nucleotides unlike the monomer, but this is in agreement with the results of single-molecule analysis of TF₁α₃β₃γ. On the other hand, the closed form of the β subunit in the TF₁′ complex was shown to be distinct from that of the nucleotide-bound β monomer. This is consistent with a previous report that the closed form of the TF₁β monomer could be a catalytically activated state. The loop between the N-terminal β-barrel and the central domain is highly flexible in the TF₁′ complex, in contrast to that in the α₃β₃ hexamer, suggesting that it is affected by the presence of the γ subunit in this area.     

BIOSYNTHESIS OF HETEROGENEOUS FORMS OF MAMMALIAN BRAIN TUBULIN SUBUNITS BY MULTIPLE MESSENGER RNAs

—Heterogeneity among the primary translation products of rat brain tubulin messenger RNA was examined. On two-dimensional gels native cytoplasmic tubulin from randomly bred rats (PB21) consists of two groups of α tubulin subunits among which the most acidic forms, α₁ and α₂, are most abundant; and β tubulin consists of a minimum of two species, β₁ and β₂. In the same group of animals the primary translation products of rat brain tubulin mRNA consist of at least these four subunit forms (α₁α₂, β₁ and β₂); however, minor basic forms of α subunits were not synthesized. This same result was obtained from a homologous brain protein synthesizing system, a heterologous system prepared from brain polysomes and rabbit reticulocyte initiation factors, and a wheat germ lysate programmed with brain poly A mRNA. A variant form of brain tubulin was found in rats bred monogamously for over 30 generations (MB71 rats). MB71 brain polysomes synthesize overlapping a subunits which migrate in two-dimensional gels to the α₁ position, and the typical PB21 α₂ is not present. The addition of PB21 brain mRNA to a protein synthesizing system composed of MB71 polysomes plus reticulocyte initiation factors allowed synthesis of the typical α₂ tubulin in addition to the MB71 tubulin subunits. The structural relationship among subunits was examined by radioiodinated peptide mapping. The α subunits are structurally different from the β subunits; however, among the major tyrosine-containing tryptic peptides no prominent differences were observed between α₁ and α₂, or between β₁ and β₂ by the radioiodination procedure. The results provide evidence for heterogeneity among the primary translation products of brain tubulin mRNA, and for the existence of multiple functional tubulin genes in rat brain.