Sedimentation Equilibrium Centrifugation Research Articles

The Fusion Protein of Hendra Virus Contains a Leucine‐Isoleucine Zipper in the Transmembrane Domain that is Important for Association

Viral fusion proteins promote entry of enveloped viruses by fusion of the viral envelope and target cell membrane. Upon triggering, the fusion protein undergoes a large conformational change, resulting in membrane fusion. Although the pre‐ and post‐fusion conformations for paramyxovirus fusion proteins (F) are known, little is currently known about the transmembrane domain (TMD). To clarify its role, the Hendra virus (HeV) TMD was examined. Directly upstream of the TMD, a heptad repeat domain contains a leucine zipper (LIZ) that continues in frame into the TMD. LIZ mutants with alanine substitutions were expressed in Vero cells to analyze folding and fusion activity. Preliminary data suggested that replacing four residues in the TMD LIZ with alanine significantly decreased surface expression and fusion activity. Using a technique to directly analyze TMD interactions using sedimentation equilibrium centrifugation, we showed that the wild type HeV TMD associates in a monomer‐trimer equilibrium. However, the TMD four alanine mutant of the LIZ ablates trimerization, implicating the LIZ in TMD‐TMD interactions. This work implicates that stability of the pre‐fusion F requires TMD‐TMD interactions, so analysis of the role of LIZ in TMD association could offer new insights into fusion and potentially identify targets for antivirals. This work was supported by the National Institutes of Health grant R01AI051517.

Divalent metal activation of a GH43 β-xylosidase

Depolymerization of xylan, a major fraction of lignocellulosic biomass, releases xylose which can be converted into transportation fuels and chemical feedstocks. A requisite enzyme for the breakdown of xylan is β-xylosidase. A gene encoding the 324-amino acid β-xylosidase, RS223-BX, was cloned from an anaerobic mixed microbial culture. This glycoside hydrolase belongs to family 43. Unlike other GH43 enzymes, RS223-BX can be strongly activated by exogenously supplied Ca2+, Co2+, Fe2+, Mg2+, Mn2+ and Ni2+ (e.g., 28-fold by Mg2+) and it is inhibited by Cu2+ or Zn2+. Sedimentation equilibrium centrifugation experiments indicated that the divalent metal cations mediate multimerization of the enzyme from a dimeric to a tetrameric state, which have equal catalytic activity on an active-site basis. Compared to the determined active sites of other GH43 β-xylosidases, the predicted active site of RS223-BX contains two additional amino acids with carboxylated side chains that provide potential sites for divalent metal cations to reside. Thus, the divalent metal cations likely occupy the active site and participate in the catalytic mechanism. RS223-BX accepts as substrate xylobiose, arabinobiose, 4-nitrophenyl-β-d-xylopyranoside, and 4-nitrophenyl-α-l-arabinofuranoside. Additionally, the enzyme has good pH and temperature stabilities and a large Ki for d-glucose (1.3M), favorable properties for performance in saccharification reactors.

Complex formation and kinetics of filament assembly exhibited by the simple epithelial keratins K8 and K18

We have generated human recombinant keratins K8 and K18 and describe conditions to quantitatively follow their assembly into filaments. When renatured individually from 8M urea into a low ionic strength/high pH-buffer, K8 was present in a dimeric to tetrameric form as revealed by analytical ultracentrifugation. In contrast, K18 sedimented as a monomer. When mixed in 8 M urea and renatured together, K8 and K18 exhibited s-value profiles compatible with homogeneous tetrameric complexes. This finding was confirmed by sedimentation equilibrium centrifugation. Subsequently, these tetrameric starter units were subjected to assembly experiments at various protein concentrations. At low values such as 0.0025 g/l, unit-length filaments were abundantly present after 2s of assembly. During the following 5 min, filaments grew rapidly and by measuring the length of individual filaments we were able to generate time-dependent length profiles. These data revealed that keratins K8/K18 assemble several times faster than vimentin and desmin. In addition, we determined the persistence length l(p) of K8/K18 filaments to be in the range of 300 nm. Addition of 1 mM MgCl(2) increases l(p) to 480 nm indicating that magnesium ions affect the interaction of keratin subunits within the filament during assembly to some extent.

S. aureus MscL Is a Pentamer In Vivo but of Variable Stoichiometries In Vitro: Implications for Detergent-Solubilized Membrane Proteins

Author SummaryThe ability to detect mechanical forces is at the basis of not only the senses of touch hearing and balance but also cardiovascular and osmotic regulation. One of the primary ways that organisms detect forces is through mechanosensitive channels, and mechanosensation is so vital that essentially all organisms have at least one such sensor. Indeed, the best-studied mechanosensitive channel is from bacteria, and because relatively little is known of mechanosensors from higher organisms, these channels are a model for how a protein can sense and respond to mechanical forces. Although the bacterial mechanosensitive channel MscL has been well studied, the simple issue of how many subunits it has is hotly debated. There are even two published crystal structures showing either tetrameric or pentameric complexes. Here we show that the channel is actually pentameric in vivo and that the detergent used to solubilize the protein can rearrange the complexes from pentamers to tetramers. The finding that detergents can have such a profound effect on structure may have broad implications for the study of other membrane proteins.

Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded theCENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.

Association Properties of βB1- and βA3-Crystallins: Ability To Form Heterotetramers

As major constituents of the mammalian lens, beta-crystallins associate into dimers, tetramers, and higher-order complexes to maintain lens transparency and refractivity. A previous study has shown that dimerization of betaB2- and betaA3-crystallins is energetically highly favored and entropically driven. While heterodimers further associate into higher-order complexes in vivo, a significant level of reversibly associated tetrameric crystallin has not been previously observed in vitro. To enhance our understanding of the interactions between beta-crystallins, we characterized the association of betaB1-crystallin, a major component of large beta-crystallin complexes (beta-high), with itself and with betaA3-crystallin. Mouse betaB1-crystallin and human betaA3-crystallin were expressed in Escherichia coli and purified chromatographically. Their association was then characterized using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and analytical sedimentation equilibrium centrifugation. When present alone, each beta-crystallin associates into homodimers; however, no tetramer formation is seen. Once mixing has taken place, formation of a heterocomplex between betaB1- and betaA3-crystallins is observed using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and sedimentation equilibrium. In contrast to results previously obtained after betaB2- and betaA3-crystallins had been mixed, mixed betaB1- and betaA3-crystallins show a dimer-tetramer equilibrium with a K d of 1.1 muM, indicating that these two beta-crystallins associate predominantly into heterotetramers in vitro. Thus, while each purified beta-crystallin associates only into homodimers and under the conditions studied mixed betaB2- and betaA3-crystallins form a mixture of homo- and heterodimers, mixed betaB1- and betaA3-crystallins associate predominantly into heterotetramers in equilibrium with heterodimers. These findings suggest a unique role for betaB1-crystallin in promoting higher-order crystallin association in the lens.

Static light scattering to characterize membrane proteins in detergent solution

Determination of the oligomeric state or the subunit stoichiometry of integral membrane proteins in detergent solution is notoriously difficult, because the amount of detergent (and lipid) associated with the proteins is usually not known. Only two classical methods (sedimentation equilibrium centrifugation and static light scattering) can measure directly the absolute molecular mass of a protein present in a protein/detergent micelle, without any assumption on the amount of detergent bound, or the shape of the proteins. Here the theoretical background and practical aspects of static light scattering analysis of membrane proteins are reviewed using a number of examples from our lab to highlight potential pitfalls. A brief comparison with sedimentation equilibrium centrifugation is given and a detailed protocol of how we perform light scattering analyses is provided.

Lanthanide Ion-Induced Folding of De Novo Designed Coiled-Coil Polypeptides

Abstract To construct a novel isoleucine zipper polypeptide that undergoes a random to coiled coil transition upon lanthanide ion binding, we designed a 37-residue polypeptide (Pep3) with a γ-carboxy glutamic acid (γ-Gla) as metal binding site. Pep3 was designed based on the sequence of the isoleucine zipper polypeptide (Pep1), forming a triple-stranded coiled coil. The coiled-coil structure was destabilized by ionic repulsions among γ-Gla residues at position 21 (hydrophobic 3a position) of one strand. On the other hand, a stable coiled coil formed in the presence of Eu3+ ions. This structural change in the designed polypeptide was selective toward Eu3+ ion from the circular dichroism (CD) measurements. Furthermore, we designed a lanthanide ion-induced heterotrimeric coiled-coil-assembled system by using two kinds of polypeptides, Pep4 and Pep5. CD spectroscopy, sedimentation equilibrium centrifugation, gel filtration, and HPLC analyses indicate that Pep4 and Pep5 could noncovalently assemble in a 1:2 ratio in the presence of Eu3+ ions.

The oligomeric structure of high molecular weight adiponectin

There is great interest in the structure of adiponectin as its oligomeric state may specify its biological activities. It occurs as a trimer, a hexamer and a high molecular weight complex. Epidemiological data indicate that the high molecular weight form is significant with low serum levels in type 2 diabetics but to date, has not been well-defined. To resolve this issue, characterization of this oligomer from bovine serum and 3T3-L1 adipocytes by sedimentation equilibrium centrifugation and gel electrophoresis respectively, was carried out, revealing that it is octadecameric. Further studies by dynamic light scattering and electron microscopy established that bovine and possibly mouse high molecular weight adiponectin is C1q-like in structure.

Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1.

The "mu loop," an 11-residue loop spanning amino acid residues 33-43, is a characteristic structural feature of the mu class of glutathione transferases. To assess the contribution of the mu loop to the structure and function of rat GST M1-1, amino acid residues 35-44 (35GDAPDYDRSQ44) were excised by deletion mutagenesis, resulting in the "Deletion Enzyme." Kinetic studies reveal that the Km values of the Deletion Enzyme are markedly increased compared with those of the wild-type enzyme: 32-fold for 1-chloro-2,4-dinitrobenzene, 99-fold for glutathione, and 880-fold for monobromobimane, while the Vmax value for each substrate is increased only modestly. Results from experiments probing the structure of the Deletion Enzyme, in comparison with that of the wild-type enzyme, suggest that the secondary and quaternary structures have not been appreciably perturbed. Thermostability studies indicate that the Deletion Enzyme is as stable as the wild-type enzyme at 4 degrees C and 10 degrees C, but it rapidly loses activity at 25 degrees C, unlike the wild-type enzyme. In the temperature range of 4 degrees C through 25 degrees C, the loss of activity of the Deletion Enzyme is not the result of a change in its structure, as determined by circular dichroism spectroscopy and sedimentation equilibrium centrifugation. Collectively, these results indicate that the mu loop is not essential for GST M1-1 to maintain its structure nor is it required for the enzyme to retain some catalytic activity. However, it is an important determinant of the enzyme's affinity for its substrates.

Soluble Mimetics of Human Immunodeficiency Virus Type 1 Viral Spikes Produced by Replacement of the Native Trimerization Domain with aHeterologous Trimerization Motif: Characterization and Ligand Binding Analysis

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, mediates binding to the viral receptors and, along with the transmembrane glycoprotein gp41, is a major target for neutralizing antibodies. We asked whether replacing the gp41 fusion/trimerization domain with a stable trimerization motif might lead to a more stable gp120 trimer that would be amenable to structural and immunologic analysis. To obtain stable gp120 trimers, a heterologous trimerization motif, GCN4, was appended to the C terminus of YU2gp120. Biochemical analysis indicated that the gp120-GCN4 trimers were superior to gp140 molecules in their initial homogeneity, and trilobed structures were observable by electron microscopy. Biophysical analysis of gp120-GCN4 trimers by isothermal titration calorimetry (ITC) and ultracentrifugation analyses indicated that most likely two molecules of soluble CD4 could bind to one gp120-GCN4 trimer. To further examine restricted CD4 stoichiometric binding to the gp120-GCN4 trimers, we generated a low-affinity CD4 binding trimer by introducing a D457V change in the CD4 binding site of each gp120 monomeric subunit. The mutant trimers could definitively bind only one soluble CD4 molecule, as determined by ITC and sedimentation equilibrium centrifugation. These data indicate that there are weak interactions between the gp120 monomeric subunits of the GCN4-stabilized trimers that can be detected by low-affinity ligand sensing. By similar analysis, we also determined that removal of the variable loops V1, V2, and V3 in the context of the gp120-GCN4 proteins allowed the binding of three CD4 molecules per trimer. Interestingly, both the gp120-GCN4 variants displayed a restricted stoichiometry for the CD4-induced antibody 17b of one antibody molecule binding per trimer. This restriction was not evident upon removal of the variable loops V1 and V2 loops, consistent with conformational constraints in the wild-type gp120 trimers and similar to those inherent in the functional Env spike. Thus, the gp120-GCN4 trimers demonstrate several properties that are consistent with some of those anticipated for gp120 in the context of the viral spike.

Pex5p, the Peroxisomal Cycling Receptor, Is a Monomeric Non-globular Protein

In mammals, targeting of newly synthesized peroxisomal matrix proteins to the organelle requires Pex5p, the peroxisomal cycling receptor. Pex5p is a multidomain protein involved in a complex network of transient protein-protein interactions. Besides interacting directly with most peroxisomal proteins en route to the organelle, Pex5p has also binding domains for several components of the peroxisomal docking/translocation machinery. However, our knowledge of how binding of a cargo protein to Pex5p influences its properties is still rather limited. Here, we describe a protease assay particularly useful for identifying and characterizing protein-protein interactions involving human Pex5p. Binding of a PTS1-containing peptide/protein to Pex5p as well as the interaction of this peroxin with the Src homology domain 3 of Pex13p could be easily demonstrated using this assay. To address the possible effects of these Pex5p-interacting peptides/proteins on the assumed quaternary structure of Pex5p, we have analyzed the hydrodynamic properties of human Pex5p using size exclusion chromatography, sucrose gradient centrifugation, and sedimentation equilibrium centrifugation. Our results show that Pex5p is a monomeric protein with an abnormal shape. The implications of these findings on current models of protein translocation across the peroxisomal membrane are discussed.

Mechanistic Analysis of the Mitotic Kinesin Eg5

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k(cat) = 5.5 s(-1), K(1/2,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-1)s(-1), followed immediately by ATP hydrolysis at 15 s(-1). ATP-dependent Mt.Eg5 dissociation was relatively slow and rate-limiting at 8 s(-1) with mantADP release at 40 s(-1). Surprisingly, Eg5-367 binds microtubules more effectively (11 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,Mt). These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.

Characterization of recombinant soluble carcinoembryonic antigen cell adhesion molecule 1

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a type 1 transmembrane, homotypic cell adhesion protein expressed on epithelial and hematopoietic cells. CEACAM1 has four major isoforms with three or four immunoglobulin (Ig)-like ectodomains and either long or short cytoplasmic domains. In a 3D model of breast epithelial cell morphogenesis, CEACAM1 plays an essential role in lumen formation [J. Cell Sci. 112 (1999) 4193]. Two soluble ectodomain isoforms of CEACAM1 expressed in myeloma cells were immunologically active and highly glycosylated. The molecular weights of the 3-ecto- and 4-ectodomain isoforms were 90 and 110 kDa, respectively, and monomers by sedimentation equilibrium centrifugation. Both isoforms were prolate ellipsoids with axial ratios of 6 for the 3-ecto- and 8 for 4-ectodomain isoforms, respectively, by size exclusion chromatography and analytical ultracentrifugation. Both isoforms caused a significant reduction in lumen formation when tested in the 3D model culture system.

Conformational changes and activation of yeast aldehyde dehydrogenase by various agents

Activity assays are characterised by a slow activation phase lasting 1–2 min which arises from the conversion of the low activity/inactive form of the isolated yeast aldehyde dehydrogenase into a high activity form upon binding NADP +. When Mg 2+ is present as an activator, the binding of coenzyme is relatively strong and activation is effectively complete when the steady-state rates are measured. Lineweaver–Burk plots with 1/[NADP +] as variable are linear. In the absence of Mg 2+, coenzyme binding is relatively weak and the conversion of inactive to active enzyme is not complete when steady-state rates are measured. The balance of equilibrium is now finely balanced and the degree of activation depends on the NADP + concentration. Under these conditions, rate versus [NADP +] plots are sigmoid, characterised by a Hill coefficient of 1.8 because the active (as opposed to the total) enzyme concentration is different in assays with different [NADP +]. Kinetic studies indicate that the activation process involves two steps, but the data do not allow discrimination between alternative mechanisms. Gel filtration and sedimentation equilibrium centrifugation indicate that the activation process does not involve enzyme association–dissociation. Activation of the enzyme can also be achieved by replacing Mg 2+ by small amounts of protamine sulphate, poly- l-lysine or poly- l-arginine. The mechanism for these activations is unknown, but presumably involves the binding of the enzyme to these positively charged molecules.

Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments

BackgroundRiboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs.ResultsA cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg-1 min-1 with an apparent KM of 5.7 microM. 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins.ConclusionRiboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism.

Biosynthesis of riboflavin: 6,7-dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe.

A cDNA sequence from Schizosaccharomyces pombe with similarity to 6,7-dimethyl-8-ribityllumazine synthase was expressed in a recombinant Escherichia coli strain. The recombinant protein is a homopentamer of 17-kDa subunits with an apparent molecular mass of 87 kDa as determined by sedimentation equilibrium centrifugation (it sediments at an apparent velocity of 5.0 S at 20 degrees C). The protein has been crystallized in space group C2221. The crystals diffract to a resolution of 2.4 A. The enzyme catalyses the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy- 2-butanone 4-phosphate. Steady-state kinetic analysis afforded a vmax value of 13 000 nmol.mg-1.h-1 and Km values of 5 and 67 microm for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, respectively. The enzyme binds riboflavin with a Kd of 1.2 microm. The fluorescence quantum yield of enzyme-bound riboflavin is < 2% as compared with that of free riboflavin. The protein/riboflavin complex displays an optical transition centered around 530 nm as shown by absorbance and CD spectrometry which may indicate a charge transfer complex. Replacement of tryptophan 27 by tyrosine or phenylalanine had only minor effects on the kinetic properties, but complexes of the mutant proteins did not show the anomalous long wavelength absorbance of the wild-type protein. The replacement of tryptophan 27 by aliphatic amino acids substantially reduced the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.

ActA from Listeria monocytogenes Can Interact with Up to Four Ena/VASP Homology 1 Domains Simultaneously

The facultative intracellular human pathogenic bacterium Listeria monocytogenes actively recruits host actin to its surface to achieve motility within infected cells. The bacterial surface protein ActA is solely responsible for this process by mimicking fundamental steps of host cell actin dynamics. ActA, a modular protein, contains an N-terminal actin nucleation site and a central proline-rich motif of the 4-fold repeated consensus sequence FPPPP (FP(4)). This motif is specifically recognized by members of the Ena/VASP protein family. These proteins additionally recruit the profilin-G-actin complex increasing the local concentration of G-actin close to the bacterial surface. By using analytical ultracentrifugation, we show that a single ActA molecule can simultaneously interact with four Ena/VASP homology 1 (EVH1) domains. The four FP(4) sites have roughly equivalent affinities with dissociation constants of about 4 microm. Mutational analysis of the FP(4) motifs indicate that the phenylalanine is mandatory for ActA-EVH1 interaction, whereas in each case exchange of the third proline was tolerated. Finally, by using sedimentation equilibrium centrifugation techniques, we demonstrate that ActA is a monomeric protein. By combining these results, we formulate a stoichiometric model to describe how ActA enables Listeria to utilize efficiently resources of the host cell microfilament for its own intracellular motility.

Quaternary Structure of the Lactose Transport Protein ofStreptococcus thermophilus in the Detergent-solubilized and Membrane-reconstituted State

The quaternary structure of LacS, the lactose transporter of Streptococcus thermophilus, has been determined for the detergent-solubilized and the membrane-reconstituted state of the protein. The quaternary structure of the n-dodecyl-beta-d-maltoside-solubilized state was studied using a combination of sedimentation velocity and equilibrium centrifugation analysis. From these measurements it followed that the detergent-solubilized LacS undergoes reversible self-association with a monomer to dimer mode of association. The association constants were 5.4 +/- 3.6 and 4.4 +/- 1.0 ml mg(-1) as determined from the velocity and equilibrium sedimentation measurements, respectively. The experiments did not indicate significant changes in the shape of the protein-detergent complex or the amount of detergent bound in going from the monomeric to dimeric state of LacS. Importantly, a single Cys mutant of LacS is labeled by 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid in a substrate-dependent manner, indicating that the detergent-solubilized protein exhibits ligand binding activity. The quaternary structure of membrane-reconstituted LacS was determined by freeze-fracture electron microscopy analysis. Recent developments in the analysis of freeze-fracture images (Eskandari, S. P., Wright, E. M., Freman, M., Starace, D. M., and Zampighi, G. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11235-11240) allowed us to directly correlate the cross-sectional area of the transmembrane segment to a dimeric state of the functionally membrane-reconstituted LacS protein. The cross-sectional area of the LacS protein was calibrated using the membrane-reconstituted transmembrane domain of the mannitol transporter enzyme II, an intramembrane particle for which the cross-sectional area was obtained from maps of two-dimensional crystals. The consequences of the determined quaternary structure for the transport function and regulation of LacS are discussed.

Physical characterization of plakophilin 1 reconstituted with and without zinc.

Plakophilin 1 (PKP1) belongs to the arm-repeat protein family which is characterized by the presence of a conserved 42-amino-acid motif. Despite individual members of the family containing a similar type of structural domain, they exhibit diverse cellular functions. PKP1 is ubiquitously expressed in human tissues and, depending on the type of cell, found prominently in the karyoplasm and/or in desmosomes. In surface plasmon resonance detection experiments, we noticed that PKP1 specifically bound zinc but not calcium or magnesium. Therefore we have used circular dichroism spectroscopy, limited proteolysis, analytical ultracentrifugation, electron microscopy and dynamic light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc. The alpha helix content of PKP1 was considerably higher when reconstituted with zinc than without. By atomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly associated with one molecule of wild-type PKP1. The zinc-reconstituted protein formed globular particles of 21.9 +/- 8.4 nm diameter, as measured by electron microscopy after glycerol spraying/rotary metal shadowing. In parallel, the average sedimentation coefficient (s20, w) for zinc-containing PKP1 was 41S and its diffusion coefficient, as obtained by dynamic light scattering, 1.48 x 10-7 cm2.s-1. The molecular mass of 2.44 x 106 obtained from s and D yields an average stoichiometry of 30 for the PKP1 oligomer. In contrast, PKP1, reconstituted without zinc, contained no significant amount of zinc, sedimented with 4.6S, and was present in monomeric form as determined by sedimentation equilibrium centrifugation.