Superhelix Research Articles

Structural biology of plasmid partition: uncovering the molecular mechanisms of DNA segregation

DNA segregation or partition is an essential process that ensures stable genome transmission. In prokaryotes, partition is best understood for plasmids, which serve as tractable model systems to study the mechanistic underpinnings of DNA segregation at a detailed atomic level owing to their simplicity. Specifically, plasmid partition requires only three elements: a centromere-like DNA site and two proteins: a motor protein, generally an ATPase, and a centromere-binding protein. In the first step of the partition process, multiple centromere-binding proteins bind co-operatively to the centromere, which typically consists of several tandem repeats, to form a higher-order nucleoprotein complex called the partition complex. The partition complex recruits the ATPase to form the segrosome and somehow activates the ATPase for DNA separation. Two major families of plasmid par systems have been delineated based on whether they utilize ATPase proteins with deviant Walker-type motifs or actin-like folds. In contrast, the centromere-binding proteins show little sequence homology even within a given family. Recent structural studies, however, have revealed that these centromere-binding proteins appear to belong to one of two major structural groups: those that employ helix-turn-helix DNA-binding motifs or those with ribbon-helix-helix DNA-binding domains. The first structure of a higher-order partition complex was recently revealed by the structure of pSK41 centromere-binding protein, ParR, bound to its centromere site. This structure showed that multiple ParR ribbon-helix-helix motifs bind symmetrically to the tandem centromere repeats to form a large superhelical structure with dimensions suitable for capture of the filaments formed by the actinlike ATPases. Surprisingly, recent data indicate that the deviant Walker ATPase proteins also form polymer-like structures, suggesting that, although the par families harbour what initially appeared to be structurally and functionally divergent proteins, they actually utilize similar mechanisms of DNA segregation. Thus, in the present review, the known Par protein and Par-protein complex structures are discussed with regard to their functions in DNA segregation in an attempt to begin to define, at a detailed atomic level, the molecular mechanisms involved in plasmid segregation.

Rotational dynamics of a superhelix towed in a Stokes fluid

Motivated by the intriguing motility of spirochetes (helically shaped bacteria that screw through viscous fluids due to the action of internal periplasmic flagella), we examine the fundamental fluid dynamics of superhelices translating and rotating in a Stokes fluid. A superhelical structure may be thought of as a helix whose axial centerline is not straight, but also a helix. We examine the particular case in which these two superimposed helices have different handedness, and employ a combination of experimental, analytic, and computational methods to determine the rotational velocity of superhelical bodies being towed through a very viscous fluid. We find that the direction and rate of the rotation of the body is a result of competition between the two superimposed helices; for small axial helix amplitude, the body dynamics is controlled by the short-pitched helix, while there is a crossover at larger amplitude to control by the axial helix. We find far better, and excellent, agreement of our experimental results with numerical computations based upon the method of Regularized Stokeslets than upon the predictions of classical resistive force theory.

Superelasticity, energy dissipation and strain hardening of vimentin coiled-coil intermediate filaments: atomistic and continuum studies

Vimentin coiled-coil alpha-helical dimers are elementary protein building blocks of intermediate filaments, an important component of the cell’s cytoskeleton that has been shown to control the large-deformation behavior of eukaryotic cells. Here we use a combination of atomistic simulation and continuum theory to model tensile and bending deformation of single alpha-helices as well as coiled-coil double helices of the 2B segment of the vimentin dimer. We find that vimentin dimers can be extended to tensile strains up to 100% at forces below 50 pN, until strain hardening sets in with rapidly rising forces, approaching 8 nN at 200% strain. We systematically explore the differences between single alpha-helical structures and coiled-coil superhelical structures. Based on atomistic simulation, we discover a transition in deformation mechanism under varying pulling rates, resulting in different strength criteria for the unfolding force. Based on an extension of Bell’s theory that describes the dependence of the mechanical unfolding force on the pulling rate, we develop a fully atomistically informed continuum model of the mechanical properties of vimentin coiled-coils that is capable of predicting its nanomechanical behavior over a wide range of deformation rates that include experimental conditions. This model enables us to describe the mechanics of cyclic stretching experiments, suggesting a hysteresis in the force–strain response, leading to energy dissipation as the protein undergoes repeated tensile loading. We find that the dissipated energy increases continuously with increasing pulling rate. Our atomistic and continuum results help to interpret experimental studies that have provided evidence for the significnificance of vimentin intermediate filaments for the large-deformation regime of eukaryotic cells. We conclude that vimentin dimers are superelastic, highly dissipative protein assemblies.

Structure and stability of designed TPR protein superhelices: unusual crystal packing and implications for natural TPR proteins

The structure and stability of repeat proteins has been little studied in comparison to the properties of the more familiar globular proteins. Here, the structure and stability of designed tetratricopeptide-repeat (TPR) proteins is described. The TPR is a 34-amino-acid motif which adopts a helix-turn-helix structure and occurs as tandem repeats. The design of a consensus TPR motif (CTPR) has previously been described. Here, the crystal structures and stabilities of proteins that contain eight or 20 identical tandem repeats of the CTPR motif (CTPR8 and CTPR20) are presented. Both CTPR8 and CTPR20 adopt a superhelical overall structure. The structures of the different-length CTPR proteins are compared with each other and with the structures of natural TPR domains. Also, the unusual and perhaps unique crystal-packing interactions resulting in pseudo-infinite crystalline superhelices observed in the different crystal forms of CTPR8 and CTPR20 are discussed. Finally, it is shown that the thermodynamic behavior of CTPR8 and CTPR20 can be predicted from the behavior of other TPRs in this series using an Ising model-based analysis. The designed protein series CTPR2-CTPR20 covers the natural size repertoire of TPR domains and as such is an excellent model system for natural TPR proteins.

Crystal Structure of the P Pilus Rod Subunit PapA

P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone–usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor–strand exchange (DSE) mechanism, whereby the chaperone's G1 β-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor–strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it.

Pentatricopeptide repeat proteins and their emerging roles in plants

Several protein families with tandem repeat motifs play a very important role in plant development and defense. The pentatricopeptide repeat (PPR) protein family, one of the largest families, is the most perplexing one in plants. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. PPR motifs are degenerate motifs, each with 35-amino-acid sequences and are present in tandem arrays of 2–27 repeats per protein. Although PPR proteins are found in other eukaryotes, their large number is probably required in plants to meet the specific needs of organellar gene expression. The repeats of PPR proteins form a superhelical structure to bind a specific ligand, probably a single-stranded RNA molecule, and modulate its expression. Functional studies on different PPR proteins have revealed their role in organellar RNA processing, fertility restoration in CMS plants, embryogenesis, and plant development. Functional genomic techniques can help identify the diverse roles of the PPR family of proteins in nucleus-organelle interaction and in plant development.

A designed curved DNA segment that is a remarkable activator of eukaryotic transcription

To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly, T20 was found to activate transcription in all the stable transformants, irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes, which is of importance in many fields, and may also be useful for the construction of nonviral vectors for gene therapy.

Identification of pentatricopeptide repeat proteins in Trypanosoma brucei

A new class of organellar proteins, characterized by pentatricopeptide repeat (PPR) motifs, has been identified in plants. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding a strand of RNA. All PPR proteins characterized to date appear to be involved in RNA processing pathways in organelles. Twenty-three PPR proteins have been identified in Trypanosoma brucei and database research indicates that most of these proteins are predicted to contain the traditional mitochondrial target sequence. Orthologues of each of the 23 proteins have also been identified in Leishmania major and Trypanosoma cruzi, indicating that these proteins represent a highly conserved class of proteins within the kinetoplastid family. Preliminary experiments using RNAi to specifically silence one identified PPR gene (TbPPRl- Tb927.2.3180), indicate that cells depleted of TbPPRl transcripts show a slow growth phenotype and altered mitochondrial maxicircle RNA profiles. This initial characterization suggests that PPR proteins will play important roles in the complex RNA processing required for mitochondrial gene expression in trypanosomes.

Differential Effects of Alcohols on Conformational Switchovers in α-Helical and β-Sheet Protein Models

Organic solvents may induce non-native structures of proteins that mimic folding intermediates and/or conformations that occur in proximity to biological membranes. Here we systematically investigate the effects of simple (i.e., MeOH and EtOH) and fluorinated (i.e., trifluoroethanol, TFE) alcohols on the secondary structure and thermodynamic stability of two complementary model proteins using a combination of circular dichroism, fluorescence, and Fourier transform infrared (FTIR) detection methods. The selected proteins are alpha-helical Borrelia burgdorferi VlsE and beta-sheet human mitochondrial co-chaperonin protein 10 (cpn10). We find that switches between VlsE's native and non-native superhelical and beta-sheet structures readily occur (pH 7, 20 degrees C). The pathway depends on the alcohol: addition of MeOH induces a transition to a superhelical structure that is followed by conversion to beta-structure, whereas EtOH only unfolds the protein. TFE unfolds VlsE at low percentages but promotes the formation of a superhelical state upon further additions. For cpn10, both MeOH and TFE additions govern initial unfolding; however, further additions of MeOH result in the formation of a non-native beta-structure, whereas subsequent additions of TFE induce a superhelical structure. EtOH additions promptly unfold and precipitate cpn10. Both VlsE's and cpn10's non-native structures exhibit high stability toward chemical and thermal perturbations. This study demonstrates that in response to different alcohols, polypeptides can readily adopt both alpha- and beta-enriched conformations. The biological significance of these findings is discussed.

Expression and Characterization of Full-length Human Huntingtin, an Elongated HEAT Repeat Protein

Huntington disease is an inherited neurodegenerative disorder that is caused by expanded CAG trinucleotide repeats, resulting in a polyglutamine stretch of >37 on the N terminus of the protein huntingtin (htt). htt is a large (347 kDa), ubiquitously expressed protein. The precise functions of htt are not clear, but its importance is underscored by the embryonic lethal phenotype in htt knock-out mice. Despite the fact that the htt gene was cloned 13 years ago, little is known about the properties of the full-length protein. Here we report the expression and preliminary characterization of recombinant full-length wild-type human htt. Our results support a model of htt composed entirely of HEAT repeats that stack to form an elongated superhelix.

Protein SRP68 of human signal recognition particle: Identification of the RNA and SRP72 binding domains

The signal recognition particle (SRP) plays an important role in the delivery of secretory proteins to cellular membranes. Mammalian SRP is composed of six polypeptides among which SRP68 and SRP72 form a heterodimer that has been notoriously difficult to investigate. Human SRP68 was purified from overexpressing Escherichia coli cells and was found to bind to recombinant SRP72 as well as in vitro-transcribed human SRP RNA. Polypeptide fragments covering essentially the entire SRP68 molecule were generated recombinantly or by proteolytic digestion. The RNA binding domain of SRP68 included residues from positions 52 to 252. Ninety-four amino acids near the C terminus of SRP68 mediated the binding to SRP72. The SRP68-SRP72 interaction remained stable at elevated salt concentrations and engaged approximately 150 amino acids from the N-terminal region of SRP72. This portion of SRP72 was located within a predicted tandem array of four tetratricopeptide (TPR)-like motifs suggested to form a superhelical structure with a groove to accommodate the C-terminal region of SRP68.

Deoxyhypusine hydroxylase: a novel HEAT‐repeat‐containing metalloenzyme

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the formation of the hypusine residue, uniquely found in eukaryotic translation initiation factor 5A (eIF5A). Although we have studied properties of DOHH in cells and partially purified from rat testis, its identity has been elusive. Recently we cloned the DOHH gene by screening a S. cerevisiae GST-ORF library (constructed by Dr. Eric M. Phizicky, University of Rochester), identified the human DOHH gene and expressed catalytically active recombinant enzymes. A homology-based model indicates that the protein has 8 distinct HEAT-repeat motifs arranged in tandem to form a superhelical solonoid structure. The essentially alpha-helical nature of the protein is confirmed by its circular dichroism UV spectrum (80% alpha-helix). DOHH contains two potential metal binding sites, each made up of a strictly conserved His-Glu pair. The purified DOHH displayed a distinctive blue color and indeed Fe was identified as the DOHH-bound metal. The role of the His-Glu motif was investigated by site-directed mutagenesis. Alanine substitution of H56, H89, E90, H207, H240 and E241 caused a marked reduction in Fe content and alanine substitution of any residues of the HisGlu motifs abolished DOHH activity, indicating their critical role in metal coordination and catalysis. This research was supported by the Intramural Research Program of the NIH (NIDCR, NCBI/NLM)

The Post-Translational Synthesis of a Polyamine-Derived Amino Acid, Hypusine, in the Eukaryotic Translation Initiation Factor 5A (eIF5A)

The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unique polyamine-derived amino acid, hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed in eIF5A by a novel post-translational modification reaction that involves two enzymatic steps. In the first step, deoxyhypusine synthase catalyzes the cleavage of the polyamine spermidine and transfer of its 4-aminobutyl moiety to the epsilon-amino group of one specific lysine residue of the eIF5A precursor to form a deoxyhypusine intermediate. In the second step, deoxyhypusine hydroxylase converts the deoxyhypusine-containing intermediate to the hypusine-containing mature eIF5A. The structure and mechanism of deoxyhypusine synthase have been extensively characterized. Deoxyhypusine hydroxylase is a HEAT-repeat protein with a symmetrical superhelical structure consisting of 8 helical hairpins (HEAT motifs). It is a novel metalloenzyme containing tightly bound iron at the active sites. Four strictly conserved His-Glu pairs were identified as iron coordination sites. The structural fold of deoxyhypusine hydroxylase is entirely different from those of the other known protein hydroxylases such as prolyl 4-hydroxylase and lysyl hydroxylases. The eIF5A protein and deoxyhypusine/hypusine modification are essential for eukaryotic cell proliferation. Thus, hypusine synthesis represents the most specific protein modification known to date, and presents a novel target for intervention in mammalian cell proliferation.

Complete Assignment and Secondary Structure of the Brazil Nut Allergen Ber e 1

In the first part of the thesis, protein-ligand interactions were investigated using the chaperone LcrH, from Yersinia as target protein. The structure of a peptide encompassing the amphipathic domain (residue 278-300) of the protein YopD from Yersinia was determined by NMR in 40% TFE. The structure of YopD278-300 is a well defined α-helix with a β-turn at the C-terminus of the helix capping the structure. This turn is crucial for the structure as peptides lacking the residues involved in the turn are unstructured. NMR relaxation indicates that the peptide is not monomeric. This is supported by intermolecular NOEs found from residue Phe280 to Ile288 and Val292 indicative of a multimeric structure with the helical structures oriented in an antiparallel manner with hydrophobic residues forming the oligomer. The interaction with the chaperone LcrH was confirmed by 1H relaxation experiments and induced chemical shift changes in the peptide Protein-ligand interactions were investigated further in the second paper using a different approach. A wide range of substances were used in screening for affinity against the chaperones PapD and FimC from uropathogenic Escherichia coli using 1H relaxation NMR experiments, surface plasmon resonance and 19F NMR. Fluorine NMR proved to be advantageous as compared to proton NMR as it is straight forward to identify binding ligands due to the well resolved 19F NMR spectra. Several compounds were found to interact with PapD and FimC through induced line-broadening and chemical shift changes for the ligands. Data corroborate well with surface plasmon resonance and proton NMR experiments. However, our results indicate the substances used in this study to have poor specificity for PapD and FimC as the induced chemical shift is minor and hardly no competitive binding is observed. Paper III and IV is an investigation of the structural features of the allergenic 2S albumin Ber e 1 from Brazil nut. Ber e 1 is a 2S albumin previously identified as the major allergen of Brazil nut. Recent studies have demonstrated that endogenous Brazil nut lipids are required for an immune response to occur in vivo. The structure was obtained from 3D heteronuclear NMR experiments followed by simulated annealing using the software ARIA. Interestingly, the common fold of the 2S albumin family, described as a right-handed super helix with the core composed of a helix bundle, is not found in Ber e 1. Instead the C-terminal region is participating in the formation of the core between helix 3, 4 and 5. The dynamic properties of Ber e 1 were investigated using 15N relaxation experiments and data was analyzed using the model-free approach. The analysis showed that a few residues in the loop between helix 2 and 3 experience decreased mobility, compared to the rest of the loop. This is consistent with NOE data as long range NOEs were found from the loop to the core region of the protein. The anchoring of this loop is a unique feature of Ber e 1, as it is not found in any other structures of 2S albumins. Chemical shift mapping of Ber e 1 upon the addition of lipid extract from Brazil nut identified 4 regions in the protein where chemical shift perturbations were detected. Interestingly, all four structural clusters align along a cleft in the structure formed by helix 1-3 on one side and helix 4-5 on the other. This cleft is big enough to encompass a lipid molecule. It is therefore tempting to speculate whether this cleft is the lipid binding epitope in Ber e 1.

Novel Low Voltage Piezoactuators for High Displacements

To improve the performance of piezoelectric actuators, new 3-D designs were developed to gain higher displacements or specific bending effects. Such 3-D actuators of e.g. helical structure have next to the standard actuator or sensor applications the potential to be used in Hi-Fi digital sound projectors. To lower the voltage of the power supply for these miniaturized devices, multilayer structures based on thin tapes must be used. Two processing routes to manufacture helical shaped multilayer PZT structures with specific electrode designs are tested. One route starts from tape cast PZT green tapes of 90 μ m or 50 μ m thickness, the other from extruded and stretched PZT filled thermoplastic films (Solufill™) of 9 μ m thickness. The properties of these two films are compared. The sheets are screen printed and laminated similar to standard planar multilayer processing. To manufacture helical three-dimensional multi-layers from these laminates new processing techniques are required. The most important difference compared to planar multilayer processing is the necessity to bend the laminated structures. The bending behavior depends on the layer thickness, the number of layers, the diameter of the helix and the flexibility of the tape. In addition, specific measures have to be taken to ensure that the bent multilayer keeps its shape and keeps the alignment of the electrode design during binder burnout and sintering. Finally, working products of super-helical structure suitable for miniaturized loudspeaker application are built and tested.

Solution Structure and Stability against Digestion of rproBnIb, a Recombinant 2S Albumin from Rapeseed: Relationship to Its Allergenic Properties,

NMR spectroscopy has been used to determine the solution structure of the precursor form of the recombinant napin BnIb, rproBnIb, a 2S albumin, 109-residue protein from the seeds of Brassica napus. More than 90% of the side-chain proton resonances were unambiguously assigned from the analysis of two-dimensional correlation (COSY), total correlation (TOCSY), and nuclear Overhauser effect (NOESY) spectra. The final structures were computed by using restrained molecular dynamics on the basis of 1316 upper-limit distance constraints derived from NOE cross-correlation intensities. The computed structures exhibited a root-mean-square deviation (RMSD) radius of 0.66 A for the backbone and 1.16 A for the side-chain heavy atoms of the structural core. The resulting structure consists of five amphipathic helices arranged in a right-handed super helix, a folding motif found in other proteins of the prolamin superfamily. As in the case of the mature protein, the recombinant precursor behaves as a plant food allergen. To trace out the origin and characteristics of its allergenic properties, rproBnIb was assayed against simulated gastric fluid and found to be very resistant to proteolysis. Also, heat treatment of the protein followed up to 85 degrees C by circular dichroism showed a very limited unfolding, which was recovered after cooling to 20 degrees C, indicating a high thermal stability. These results suggest that rproBnIb, as other 2S albumins, may be able to reach the gut immune system intact. A comparison of the putative epitopes against IgE antibodies of the three members of the prolamine family [2S albumins, nonspecific lipid transfer proteins (nsLTPs), and alpha-amylase/trypsin inhibitors] indicates that there are not common surfaces of interaction with IgE. Though the epitopes appear to be located in different regions of the proteins, they do comply with the requirements of being solvent-exposed and flexible.

The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix

Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an approximately 4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChE(T) tetramer.

Structure of the Cand1-Cul1-Roc1 Complex Reveals Regulatory Mechanisms for the Assembly of the Multisubunit Cullin-Dependent Ubiquitin Ligases

The SCF ubiquitin ligase complex regulates diverse cellular functions by ubiquitinating numerous protein substrates. Cand1, a 120 kDa HEAT repeat protein, forms a tight complex with the Cul1-Roc1 SCF catalytic core, inhibiting the assembly of the multisubunit E3 complex. The crystal structure of the Cand1-Cul1-Roc1 complex shows that Cand1 adopts a highly sinuous superhelical structure, clamping around the elongated SCF scaffold protein Cul1. At one end, a Cand1 β hairpin protrusion partially occupies the adaptor binding site on Cul1, inhibiting its interactions with the Skp1 adaptor and the substrate-recruiting F box protein subunits. At the other end, two Cand1 HEAT repeats pack against a conserved Cul1 surface cleft and bury a Cul1 lysine residue, whose modification by the ubiquitin-like protein, Nedd8, is able to block Cand1-Cul1 association. Together with biochemical evidence, these structural results elucidate the mechanisms by which Cand1 and Nedd8 regulate the assembly-disassembly cycles of SCF and other cullin-dependent E3 complexes.

The folding state of the lumenal loop determines the thermal stability of light-harvesting chlorophyll a/b protein.

The major light-harvesting protein of photosystem II (LHCIIb) is the most abundant chlorophyll-binding protein in the thylakoid membrane. It contains three membrane-spanning alpha helices; the first and third one closely interact with each other to form a super helix, and all three helices bind most of the pigment cofactors. The protein loop domains connecting the alpha helices also play an important role in stabilizing the LHCIIb structure. Single amino acid exchanges in either loop were found to be sufficient to significantly destabilize the complex assembled in vitro [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093. Mick, V., Eggert, K., Heinemann, B., Geister, S., and Paulsen, H (2004) Biochemistry 43, 5467-5473]. This work presents an analysis of such point mutations in the lumenal loop with regard to the extent and nature of their effect on LHCIIb stability to obtain detailed information on the contribution of this loop to stabilizing the complex. Most of the mutant proteins yielded pigment-protein complexes if their reconstitution and/or isolation was performed under mild conditions; however, the yields were significantly different. Several mutations in the vicinity of W97 in the N-proximal section of the loop gave low reconstitution yields even under very mild conditions. This confirms our earlier notion that W97 may be of particular relevance in stabilizing LHCIIb. The same amino acid exchanges accelerated thermal complex dissociation in the absence of lithium dodecyl sulfate (LDS) and raised the accessibility of the lumenal loop to protease; both effects were well correlated with the reduction in reconstitution yields. We conclude that a detachment of the lumenal loop is a possible first step in the dissociation of LHCIIb. Dramatically reduced complex yields in the presence but not in the absence of LDS were observed for some but not all mutants, particularly those near the C-proximal end of the loop. We conclude that complex stabilities in the absence and in the presence of LDS do not correlate and most likely are determined by different structural characteristics, at least in LHCIIb but maybe also in other membrane proteins.

Clathrin-mediated transport: assembly required. Workshop on Molecular Mechanisms of Vesicle Selectivity.

Maintenance of the structural and functional organization of eukaryotic cells requires the correct targeting of proteins and lipids to their final destinations. This workshop focused on the mechanisms that mediate the formation of transport intermediaries, which carry cargo between intracellular compartments and the plasma membrane. The formation of transport vesicles is a highly complex process requiring the coordinated and selective recruitment of lipids, transmembrane proteins and their lumenal cargo, and the sorting machinery components that are required for vesicle transport and fusion with acceptor compartments. Although different types of transport vesicles have been described, clathrin‐coated vesicles (CCVs), which mediate endocytosis from the plasma membrane, are the best characterized and are therefore often considered a model for transport‐intermediary formation. Recently, the resolution of the structure of several coat‐component domains through X‐ray crystallography and the identification of new regulatory proteins has greatly advanced our understanding of CCV formation and function. Selected highlights from these and other recent studies that were presented at the meeting are the subject of this report. ### Formation and function of the clathrin scaffold In the mid‐1970s, the isolation of CCVs by density centrifugation allowed B. Pearse (Cambridge, UK) to purify clathrin, one of the main components of these structures (Pearse, 1976). The assembly unit of clathrin, commonly referred to as the ‘triskelion’, consists of three heavy chains (∼190 kDa) and three light chains (∼30 kDa) that oligomerize to form three‐dimensional (3D) polygonal lattices, or cage‐like structures (Fig 1). Clathrin heavy chains can be sub‐divided into five structurally and functionally distinct regions: an aminoterminal globular domain; a highly flexible linker region composed of short α‐helices; a distal segment; a proximal segment and a carboxy‐terminal domain, which mediates the trimerization of the clathrin chains (reviewed in Brodsky, 2001). The heavy‐chain proximal domain has a superhelical structure composed of clathrin heavy‐chain repeats of ten short α‐helices and contains …