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Analytical Ultracentrifugation (analytical + ultracentrifugation)

Distribution by Scientific Domains

Chemistry	78%
Polymers and Materials Science	13%

Selected Abstracts

Analytical Ultracentrifugation in the Former USSR: The MOM Ultracentrifuges

MACROMOLECULAR BIOSCIENCE, Issue 7 2010
Georges M. Pavlov
No abstract is available for this article. [source]

pyr RNA binding to the Bacillus caldolyticus PyrR attenuation protein , characterization and regulation by uridine and guanosine nucleotides

FEBS JOURNAL, Issue 4 2008
Casper M. Jørgensen
The PyrR protein regulates expression of pyrimidine biosynthetic (pyr) genes in many bacteria. PyrR binds to specific sites in the 5, leader RNA of target operons and favors attenuation of transcription. Filter binding and gel mobility assays were used to characterize the binding of PyrR from Bacillus caldolyticus to RNA sequences (binding loops) from the three attenuation regions of the B. caldolyticus pyr operon. Binding of PyrR to the three binding loops and modulation of RNA binding by nucleotides was similar for all three RNAs. The apparent dissociation constants at 0 °C were in the range 0.13,0.87 nm in the absence of effectors; dissociation constants were decreased by three- to 12-fold by uridine nucleotides and increased by 40- to 200-fold by guanosine nucleotides. The binding data suggest that pyr operon expression is regulated by the ratio of intracellular uridine nucleotides to guanosine nucleotides; the effects of nucleoside addition to the growth medium on aspartate transcarbamylase (pyrB) levels in B. subtilis cells in vivo supported this conclusion. Analytical ultracentrifugation established that RNA binds to dimeric PyrR, even though the tetrameric form of unbound PyrR predominates in solution at the concentrations studied. [source]

Modern analytical ultracentrifugation in protein science: A tutorial review

PROTEIN SCIENCE, Issue 9 2002
Jacob Lebowitz
Abstract Analytical ultracentrifugation (AU) is reemerging as a versatile tool for the study of proteins. Monitoring the sedimentation of macromolecules in the centrifugal field allows their hydrodynamic and thermodynamic characterization in solution, without interaction with any matrix or surface. The combination of new instrumentation and powerful computational software for data analysis has led to major advances in the characterization of proteins and protein complexes. The pace of new advancements makes it difficult for protein scientists to gain sufficient expertise to apply modern AU to their research problems. To address this problem, this review builds from the basic concepts to advanced approaches for the characterization of protein systems, and key computational and internet resources are provided. We will first explore the characterization of proteins by sedimentation velocity (SV). Determination of sedimentation coefficients allows for the modeling of the hydrodynamic shape of proteins and protein complexes. The computational treatment of SV data to resolve sedimenting components has been achieved. Hence, SV can be very useful in the identification of the oligomeric state and the stoichiometry of heterogeneous interactions. The second major part of the review covers sedimentation equilibrium (SE) of proteins, including membrane proteins and glycoproteins. This is the method of choice for molar mass determinations and the study of self-association and heterogeneous interactions, such as protein,protein, protein,nucleic acid, and protein,small molecule binding. [source]

Expression, purification and crystallization of Swi5 and the Swi5,Sfr1 complex from fission yeast

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010
Naoyuki Kuwabara
The assembly of the presynaptic filament of recombinases represents the most important step in homologous recombination. The formation of the filament requires assistance from mediator proteins. Swi5 and Sfr1 have been identified as mediators in fission yeast and these proteins form a complex that stimulates strand exchange. Here, the expression, purification and crystallization of Swi5 and its complex with an N-terminally truncated form of Sfr1 (,N180Sfr1) are presented. Analytical ultracentrifugation of the purified samples showed that Swi5 and the protein complex exist as tetramers and heterodimers in solution, respectively. Swi5 was crystallized in two forms belonging to space groups C2 and R3 and the crystals diffracted to 2.7,Å resolution. Swi5,,N180Sfr1 was crystallized in space group P21212 and the crystals diffracted to 2.3,Å resolution. The crystals of Swi5 and Swi5,,N180Sfr1 are likely to contain one tetramer and two heterodimers in the asymmetric unit, respectively. [source]

Structure of Staphylococcus aureus 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB) in complex with acetoacetyl-CoA

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2007
Venkatasubramanian Ulaganathan
Vitamin K2, or menaquinone, is an essential cofactor for many organisms and the enzymes involved in its biosynthesis are potential antimicrobial drug targets. One of these enzymes, 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB) from the pathogen Staphylococcus aureus, has been obtained in recombinant form and its quaternary structure has been analyzed in solution. Cubic crystals of the enzyme allowed a low-resolution structure (2.9,Å) to be determined. The asymmetric unit consists of two subunits and a crystallographic threefold axis of symmetry generates a hexamer consistent with size-exclusion chromatography. Analytical ultracentrifugation indicates the presence of six states in solution, monomeric through to hexameric, with the dimer noted as being particularly stable. MenB displays the crotonase-family fold with distinct N- and C-terminal domains and a flexible segment of structure around the active site. The smaller C-terminal domain plays an important role in oligomerization and also in substrate binding. The presence of acetoacetyl-CoA in one of the two active sites present in the asymmetric unit indicates how part of the substrate binds and facilitates comparisons with the structure of Mycobacterium tuberculosis MenB. [source]

Structure,activity relationships of wheat flavone O -methyltransferase , a homodimer of convenience

FEBS JOURNAL, Issue 9 2008
Jack A. Kornblatt
Wheat flavone O -methyltransferase catalyzes three sequential methylations of the flavone tricetin. Like other flavonoid O -methyltransferases, the protein is a homodimer. We demonstrate, using analytical ultracentrifugation, that perchlorate dissociates the dimer into monomers. The resulting monomers retain all their catalytic capacity, including the ability to catalyze the three successive methylations. We show, using isothermal titration calorimetry, that the binding constant for S -adenosyl- l -methionine does not change significantly as the protein dissociates. The second substrate, tricetin, binds to the dimers but could not be tested with the monomers. CD, UV and fluorescence spectroscopy show that there are substantial changes in the structure of the protein as it dissociates. The fact that there are differences between the monomers and dimers even as the monomers maintain activity may be the result of the very low catalytic capacity of this enzyme. Maximal turnover numbers for the dimers and monomers are only about 6,7 per minute. Even though the binding pockets for S -adenosyl- l -methionine, tricetin, selgin and tricin are intact, selection of a catalytically competent structure may be a very slow step during catalysis. [source]

Calcite-specific coupling protein in barnacle underwater cement

FEBS JOURNAL, Issue 24 2007
Youichi Mori
The barnacle relies for its attachment to underwater foreign substrata on the formation of a multiprotein complex called cement. The 20 kDa cement protein is a component of Megabalanus rosa cement, although its specific function in underwater attachment has not, until now, been known. The recombinant form of the protein expressed in bacteria was purified in soluble form under physiological conditions, and confirmed to retain almost the same structure as that of the native protein. Both the protein from the adhesive layer of the barnacle and the recombinant protein were characterized. This revealed that abundant Cys residues, which accounted for 17% of the total residues, were in the intramolecular disulfide form, and were essential for the proper folding of the monomeric protein structure. The recombinant protein was adsorbed to calcite and metal oxides in seawater, but not to glass and synthetic polymers. The adsorption isotherm for adsorption to calcite fitted the Langmuir model well, indicating that the protein is a calcite-specific adsorbent. An evaluation of the distribution of the molecular size in solution by analytical ultracentrifugation indicated that the recombinant protein exists as a monomer in 100 mm to 1 m NaCl solution; thus, the protein acts as a monomer when interacting with the calcite surface. cDNA encoding a homologous protein was isolated from Balanus albicostatus, and its derived amino acid sequence was compared with that from M. rosa. Calcite is the major constituent in both the shell of barnacle base and the periphery, which is also a possible target for the cement, due to the gregarious nature of the organisms. The specificity of the protein for calcite may be related to the fact that calcite is the most frequent material attached by the cement. [source]

Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin

FEBS JOURNAL, Issue 22 2007
Anastasia V. Pivovarova
Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548,1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27,3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin,Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of ,,16 nm, a sedimentation coefficient of 17,20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions. [source]

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna

FEBS JOURNAL, Issue 14 2006
Tobias Lamkemeyer
The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 ± 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 ± 11.1 kDa (hemoglobin-rich animals) and 597.5 ± 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 ± 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm. [source]

Physical characterization of plakophilin 1 reconstituted with and without zinc

FEBS JOURNAL, Issue 14 2000
Ilse Hofmann
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 , 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 × 10,7 cm2·s,1. The molecular mass of 2.44 × 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. [source]

Advances in membrane receptor screening and analysis

JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2004
Matthew A. Cooper
Abstract During the last decade there has been significant progress in the development of analytical techniques for the screening of ligand binding to membranes and membrane receptors. This review focuses on developments using label-free assays that facilitate ligand,membrane,receptor screening without the need for chemical-, biological- or radiological-labelled reagents. These assays include acoustic, optical surface plasmon resonance biosensing, sedimentation (analytical ultracentrifugation), chromatographic assays, isothermal titration calorimetry and differential scanning calorimetry. The merits and applications of cell-based screening systems and of different model membrane systems, including planar supported lipid layers, bead-supported membranes and lipid micro-arrays, are discussed. Recent advances involving more established techniques including intrinsic fluorescence, FRET spectroscopy, scintillation proximity assays and automated patch clamping are presented along with applications to peripheral membrane proteins, ion channels and G protein-coupled receptors. Novel high-throughput assays for determination of drug- and protein-partitioning in membranes are also highlighted. To aid the experimenter, a brief synopsis of the techniques commonly employed to purify and reconstitute membranes and membrane receptors is included. Copyright © 2004 John Wiley & Sons, Ltd [source]

Determination of particle heterogeneity and stability of recombinant adenovirus by analytical ultracentrifugation in CsCl gradients

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2008
Xiaoyu Yang
Abstract Recombinant adenoviruses (rAd), widely used as vectors for gene therapy, are generally purified by column chromatography and frequently contain empty capsids and other aberrant forms of virus particles. To determine particle heterogeneity we utilized analytical ultracentrifugation (AUC) in CsCl density gradients. Preparations of three different rAd vectors were assessed. AUC was able to resolve multiple density forms including two empty capsid types in various virus preparations. One unusual density form (form V), was noninfectious and lacked protein VI. AUC was able to quantify empty capsids and monitor their removal during process development. Their relative concentrations were reduced by either addition of an immobilized zinc affinity chromatography (IZAC) step or by extension of the infection time. The Adenovirus Reference Material (ARM), a wild-type Ad5, had 2.2% empty capsids and no other detectable minor particle forms. Finally, AUC was utilized to monitor the thermal instability of the three rAd vectors via the transformations of different density forms. The vector and empty capsids containing protein IX were more stable than those without IX. Together, these results exemplify AUC in CsCl density gradients as a valuable technique for evaluating product particle heterogeneity and stability. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:746,763, 2008 [source]

,-Conjugated Donor and Donor,Acceptor Metallo-Polymers

MACROMOLECULAR RAPID COMMUNICATIONS, Issue 9-10 2010
Andreas Wild
Abstract Two zinc(II)- and two ruthenium(II) containing ,-conjugated metallo-polymers were synthesized and characterized in detail. We could prove by SEC, analytical ultracentrifugation (AUC) and viscosimetry the ruthenium(II) metallo-polymers to be high molar mass materials (Mfs,=,20,000,g,·,mol,1Ru1-2; Mfs,=,34,000,g,·,mol,1Ru1) exhibiting intrinsic viscosities of up to [,],=,192,·,cm3,·,g,1. Applying spin-coating we produced homogeneous films of the polymers and could, subsequently, investigate the photophysical properties in the solid state. Introducing the Ru(II) metallo-polymers mixed with PCBM[60] as photoactive layer in bulk-heterojunction solar cells resulted in very low efficiencies due to morphology problems. [source]

Advances in the Synthesis and Characterization of Polypeptide-Based Hybrid Block Copolymers

MACROMOLECULAR SYMPOSIA, Issue 1 2004
Ivaylo Dimitrov
Abstract Linear polystyrene- block -poly(Z-L-lysine) copolymers with a very narrow molecular weight distribution (polydispersity index < 1.03) could be obtained via the ring-opening polymerization of Z-L-lysine- N -carboxyanhydride using ,-(primary amino hydrochloride)-polystyrenes as macroinitiators in N,N -dimethylformamide as the solvent at 40-80 °C. The block copolymer samples were analyzed by means of NMR, size exclusion chromatography, and analytical ultracentrifugation. [source]

A characteristic finite element method with local mesh refinements for the Lamm equation in analytical ultracentrifugation

NUMERICAL METHODS FOR PARTIAL DIFFERENTIAL EQUATIONS, Issue 2 2009
Jiangguo Liu
Abstract The Lamm equation is a fundamental differential equation in analytical ultracentrifugation, for describing the transport of solutes in an ultracentrifuge cell. In this article, we present a characteristic finite element method with local mesh refinements for solving the Lamm equation. The numerical method is mass-conservative by design and allows relatively large time steps to be used. Numerical experiments indicate that the numerical solutions are oscillation-free in the region near the cell bottom, where mass build up and large concentration gradients occur. Positivity of solutions is also well kept. © 2008 Wiley Periodicals, Inc. Numer Methods Partial Differential Eq, 2009 [source]

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

PROTEIN SCIENCE, Issue 9 2010
Heather Franey
Abstract Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti-streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti-streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8-Anilino-1-naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near-UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular ,-sheet and turn structures between the monomer and aggregate species. Free thiol determination showed ,2.4-fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4-fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions. [source]

Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis

PROTEIN SCIENCE, Issue 8 2007
Nicole A. Kruh
Abstract The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD+ (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein,protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer,dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present. [source]

Crystal structure of an enhancer of rudimentary homolog (ERH) at 2.1 Å resolution

PROTEIN SCIENCE, Issue 7 2005
Ryoichi Arai
Abstract The enhancer of rudimentary gene, e(r), of Drosophila melanogaster encodes an enhancer of rudimentary (ER) protein with functions implicated in pyrimidine biosynthesis and the cell cycle. The ER homolog (ERH) is highly conserved among vertebrates, invertebrates, and plants. Xenopus laevis ERH was reported to be a transcriptional repressor. Here we report the 2.1 Å crystal structure of murine ERH (Protein Data Bank ID 1WZ7), determined by the multiwavelength anomalous dispersion (MAD) method. The monomeric structure of ERH comprises a single domain consisting of three ,-helices and four ,-strands, which is a novel fold. In the crystal structure, ERH assumes a dimeric structure, through interactions between the ,-sheet regions. The formation of an ERH dimer is consistent with the results of analytical ultracentrifugation. The residues at the core region and at the dimer interface are highly conserved, suggesting the conservation of the dimer formation as well as the monomer fold. The long flexible loop (44,53) is also significantly conserved, suggesting that this loop region may be important for the functions of ERH. In addition, the putative phosphorylation sites are located at the start of the ,2-strand (Thr18) and at the start of the ,1-helix (Ser24), implying that the phosphorylation might cause some structural changes. [source]

Dioxane contributes to the altered conformation and oligomerization state of a designed engrailed homeodomain variant

PROTEIN SCIENCE, Issue 4 2005
Geoffrey K. Hom
Abstract Our goal was to compute a stable, full-sequence design of the Drosophila melanogaster engrailed homeodomain. Thermal and chemical denaturation data indicated the design was significantly more stable than was the wild-type protein. The data were also nearly identical to those for a similar, later full-sequence design, which was shown by NMR to adopt the homeodomain fold: a three-helix, globular monomer. However, a 1.65 Å crystal structure of the design described here turned out to be of a completely different fold: a four-helix, rodlike tetramer. The crystallization conditions included ,25% dioxane, and subsequent experiments by circular dichroism and sedimentation velocity analytical ultracentrifugation indicated that dioxane increases the helicity and oligomerization state of the designed protein. We attribute at least part of the discrepancy between the target fold and the crystal structure to the presence of a high concentration of dioxane. [source]

How do helix,helix interactions help determine the folds of membrane proteins?

PROTEIN SCIENCE, Issue 4 2003
Perspectives from the study of homo-oligomeric helical bundles
FRET, fluorescence resonance energy transfer; NBD, 7-nitrobenz-2-oxa-1,3-diazole; C-14 betaine, N -tetradecyl- N,N -dimethyl-3-ammonio-1-propanesulfonate; MF, mole fraction Abstract The final, structure-determining step in the folding of membrane proteins involves the coalescence of preformed transmembrane helices to form the native tertiary structure. Here, we review recent studies on small peptide and protein systems that are providing quantitative data on the interactions that drive this process. Gel electrophoresis, analytical ultracentrifugation, and fluorescence resonance energy transfer (FRET) are useful methods for examining the assembly of homo-oligomeric transmembrane helical proteins. These methods have been used to study the assembly of the M2 proton channel from influenza A virus, glycophorin, phospholamban, and several designed membrane proteins,all of which have a single transmembrane helix that is sufficient for association into a transmembrane helical bundle. These systems are being studied to determine the relative thermodynamic contributions of van der Waals interactions, conformational entropy, and polar interactions in the stabilization of membrane proteins. Although the database of thermodynamic information is not yet large, a few generalities are beginning to emerge concerning the energetic differences between membrane and water-soluble proteins: the packing of apolar side chains in the interior of helical membrane proteins plays a smaller, but nevertheless significant, role in stabilizing their structure. Polar, hydrogen-bonded interactions occur less frequently, but, nevertheless, they often provide a strong driving force for folding helix,helix pairs in membrane proteins. These studies are laying the groundwork for the design of sequence motifs that dictate the association of membrane helices. [source]