Conformational Transitions of the Cross-linking Domains of Elastin during Self-assembly

Elastin is the intrinsically disordered polymeric protein imparting the exceptional properties of extension and elastic recoil to the extracellular matrix of most vertebrates. The monomeric precursor of elastin, tropoelastin, as well as polypeptides containing smaller subsets of the tropoelastin sequence, can self-assemble through a colloidal phase separation process called coacervation. Present understanding suggests that self-assembly is promoted by association of hydrophobic domains contained within the tropoelastin sequence, whereas polymerization is achieved by covalent joining of lysine side chains within distinct alanine-rich, α-helical cross-linking domains. In this study, model elastin polypeptides were used to determine the structure of cross-linking domains during the assembly process and the effect of sequence alterations in these domains on assembly and structure. CD temperature melts indicated that partial α-helical structure in cross-linking domains at lower temperatures was absent at physiological temperature. Solid-state NMR demonstrated that β-strand structure of the cross-linking domains dominated in the coacervate state, although α-helix was predominant after subsequent cross-linking of lysine side chains with genipin. Mutation of lysine residues to hydrophobic amino acids, tyrosine or alanine, leads to increased propensity for β-structure and the formation of amyloid-like fibrils, characterized by thioflavin-T binding and transmission electron microscopy. These findings indicate that cross-linking domains are structurally labile during assembly, adapting to changes in their environment and aggregated state. Furthermore, the sequence of cross-linking domains has a dramatic effect on self-assembly properties of elastin-like polypeptides, and the presence of lysine residues in these domains may serve to prevent inappropriate ordered aggregation.     

Recovery and fine structure variability of RGII sub-domains in wine (Vitis vinifera Merlot)

Background and Aims

Rhamnogalacturonan II (RGII) is a structurally complex pectic sub-domain composed of more than 12 different sugars and 20 different linkages distributed in five side chains along a homogalacturonan backbone. Although RGII has long been described as highly conserved over plant evolution, recent studies have revealed variations in the structure of the polysaccharide. This study examines the fine structure variability of RGII in wine, focusing on the side chains A and B obtained after sequential mild acid hydrolysis. Specifically, this study aims to differentiate intrinsic structural variations in these RGII side chains from structural variations due to acid hydrolysis.

Methods

RGII from wine (Vitis vinifera Merlot) was sequentially hydrolysed with trifluoroacetic acid (TFA) and the hydrolysis products were separated by anion-exchange chromatography (AEC). AEC fractions or total hydrolysates were analysed by MALDI-TOF mass spectrometry.

Key Results

The optimal conditions to recover non-degraded side chain B, side chain A and RGII backbone were 0·1 m TFA at 40 °C for 16 h, 0·48 m TFA at 40 °C for 16 h (or 0·1 m TFA at 60 °C for 8 h) and 0·1 m TFA at 60 °C for 16 h, respectively. Side chain B was particularly prone to acid degradation. Side chain A and the RGII GalA backbone were partly degraded by 0·1 m TFA at 80 °C for 1–4 h. AEC allowed separation of side chain B, methyl-esterified side chain A and non-methyl-esterified side chain A. The structure of side chain A and the GalA backbone were highly variable.

Conclusions

Several modifications to the RGII structure of wine were identified. The observed dearabinosylation and deacetylation were primarily the consequence of acidic treatment, while variation in methyl-esterification, methyl-ether linkages and oxidation reflect natural diversity. The physiological significance of this variability, however, remains to be determined.     

Interdomain Hydrophobic Interactions Modulate the Thermostability of Microbial Esterases from the Hormone-Sensitive Lipase Family

Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp²⁰² and Phe²⁰³ in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability.     

Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability

Structural calcium sites control protein thermostability and activity by stabilizing native folds and changing local conformations. Alicyclobacillus acidocaldarius survives in thermal-acidic conditions and produces an endoglucanase Cel9A (AaCel9A) which contains a calcium-binding site (Ser465 to Val470) near the catalytic cleft. By superimposing the Ca²⁺-free and Ca²⁺-bounded conformations of the calcium site, we found that Ca²⁺ induces hydrophobic interactions between the calcium site and its nearby region by driving a conformational change. The hydrophobic interactions at the high-B-factor region could be enhanced further by replacing the surrounding polar residues with hydrophobic residues to affect enzyme thermostability and activity. Therefore, the calcium-binding residue Asp468 (whose side chain directly ligates Ca²⁺), Asp469, and Asp471 of AaCel9A were separately replaced by alanine and valine. Mutants D468A and D468V showed increased activity compared with those of the wild type with 0 mM or 10 mM Ca²⁺ added, whereas the Asp469 or Asp471 substitution resulted in decreased activity. The D468A crystal structure revealed that mutation D468A triggered a conformational change similar to that induced by Ca²⁺ in the wild type and developed a hydrophobic interaction network between the calcium site and the neighboring hydrophobic region (Ala113 to Ala117). Mutations D468V and D468A increased 4.5°C and 5.9°C, respectively, in melting temperature, and enzyme half-life at 75°C increased approximately 13 times. Structural comparisons between AaCel9A and other endoglucanases of the GH9 family suggested that the stability of the regions corresponding to the AaCel9A calcium site plays an important role in GH9 endoglucanase catalysis at high temperature.     

Dissecting the Energies that Stabilize Sickle Hemoglobin Polymers

Sickle hemoglobin forms long, multistranded polymers that account for the pathophysiology of the disease. The molecules in these polymers make significant contacts along the polymer axis (i.e., axial contacts) as well as making diagonally directed contacts (i.e., lateral contacts). The axial contacts do not engage the mutant β6 Val and its nonmutant receptor region on an adjacent molecule, in contrast to the lateral contacts which do involve the mutation site. We have studied the association process by elastic light scattering measurements as a function of temperature, concentration, and primary and quaternary structure, employing an instrument of our own construction. Even well below the solubility for polymer formation, we find a difference between the association behavior of deoxy sickle hemoglobin molecules (HbS), which can polymerize at higher concentration, in comparison to COHbS, COHbA, or deoxygenated Hemoglobin A (HbA), none of which have the capacity to form polymers. The nonpolymerizable species are all quite similar to one another, and show much less association than deoxy HbS. We conclude that axial contacts are significantly weaker than the lateral ones. All the associations are entropically favored, and enthalpically disfavored, typical of hydrophobic interactions. For nonpolymerizable Hemoglobin, ΔH_o was 35 ± 4 kcal/mol, and ΔS was 102.7 ± 0.5 cal/(mol−K). For deoxyHbS, ΔH_o was 19 ± 2 kcal/mol, and ΔS was 56.9 ± 0.5 cal/(mol−K). The results are quantitatively consistent with the thermodynamics of polymer assembly, suggesting that the dimer contacts and polymer contacts are very similar, and they explain a previously documented significant anisotropy between bending and torsional moduli. Unexpectedly, the results also imply that a substantial fraction of the hemoglobin has associated into dimeric species at physiological conditions.     

Deciphering the Hidden Informational Content of Protein Sequences: FOLDABILITY OF PROINSULIN HINGES ON A FLEXIBLE ARM THAT IS DISPENSABLE IN THE MATURE HORMONE*

Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (Phe^B1 in placental mammals) is variably positioned within crystal structures and exhibits ¹H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1–B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with β-cell dysfunction and neonatal diabetes mellitus.     

Comparison of the NMR solution structure with the X-ray crystal structure of the activation domain from procarboxypeptidase B

Summary The NMR solution structure of the activation domain isolated from porcine procarboxypeptidase B is compared with the X-ray crystal structure of the corresponding segment in the intact proenzyme. For the region of the polypeptide chain that has a well-defined three-dimensional structure in solution, i.e., the backbone atoms of residues 11–76 and 25 amino acid side chains in this segment that form a hydrophobic core in the activation domain, the root-mean-square distance between the two structures is 1.1 Å. There are no significant differences in average atom positions between the two structures, but only the NMR structure shows increased structural disorder in three outlying loops located along the same edge of the activation domain. These regions of increased structural disorder in the free domain coincide only partially with the interface to the enzyme domain in the proenzyme.     

Cooling intact and demembranated trabeculae from rat heart releases myosin motors from their inhibited conformation

Myosin filament–based regulation supplements actin filament–based regulation to control the strength and speed of contraction in heart muscle. In diastole, myosin motors form a folded helical array that inhibits actin interaction; during contraction, they are released from that array. A similar structural transition has been observed in mammalian skeletal muscle, in which cooling below physiological temperature has been shown to reproduce some of the structural features of the activation of myosin filaments during active contraction. Here, we used small-angle x-ray diffraction to characterize the structural changes in the myosin filaments associated with cooling of resting and relaxed trabeculae from the right ventricle of rat hearts from 39°C to 7°C. In intact quiescent trabeculae, cooling disrupted the folded helical conformation of the myosin motors and induced extension of the filament backbone, as observed in the transition from diastole to peak systolic force at 27°C. Demembranation of trabeculae in relaxing conditions induced expansion of the filament lattice, but the structure of the myosin filaments was mostly preserved at 39°C. Cooling of relaxed demembranated trabeculae induced changes in motor conformation and filament structure similar to those observed in intact quiescent trabeculae. Osmotic compression of the filament lattice to restore its spacing to that of intact trabeculae at 39°C stabilized the helical folded state against disruption by cooling. The myosin filament structure and motor conformation of intact trabeculae at 39°C were largely preserved in demembranated trabeculae at 27°C or above in the presence of Dextran, allowing the physiological mechanisms of myosin filament–based regulation to be studied in those conditions.     

Thermotoga neapolitana Homotetrameric Xylose Isomerase Is Expressed as a Catalytically Active and Thermostable Dimer in Escherichia coli

The xylA gene from Thermotoga neapolitana 5068 was expressed in Escherichia coli. Gel filtration chromatography showed that the recombinant enzyme was both a homodimer and a homotetramer, with the dimer being the more abundant form. The purified native enzyme, however, has been shown to be exclusively tetrameric. The two enzyme forms had comparable stabilities when they were thermoinactivated at 95°C. Differential scanning calorimetry revealed thermal transitions at 99 and 109.5°C for both forms, with an additional shoulder at 91°C for the tetramer. These results suggest that the association of the subunits into the tetrameric form may have little impact on the stability and biocatalytic properties of the enzyme.     

α-Hemoglobin-stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket of Oxy-α-hemoglobin and Weakens the Iron-Oxygen Bond

α-Hemoglobin (αHb)-stabilizing protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in αHb heme coordination, but how these changes are facilitated by interactions at the αHb·AHSP interface is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe αHb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-αHb and CO-αHb·AHSP indicated that the seven helical elements of the native αHb structure are retained and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. However, chemical shift differences revealed alterations of the F, G, and H helices and the heme pocket of CO-αHb bound to AHSP. Comparisons of iron-ligand geometry using extended x-ray absorption fine structure spectroscopy showed that AHSP binding induces a small 0.03 Å lengthening of the Fe-O₂ bond, explaining previous reports that AHSP decreases αHb O₂ affinity roughly 4-fold and promotes autooxidation due primarily to a 3–4-fold increase in the rate of O₂ dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O₂ dissociation rate and equilibrium constants, and reduced O₂-αHb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote αHb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of αHb into Hb when βHb is abundant but diverts αHb to a redox resistant holding state when βHb is limiting.     

Human Lutropin (hLH) and Choriogonadotropin (CG) Are Assembled by Different Pathways: A MODEL OF hLH ASSEMBLY

The glycoprotein hormones are all structurally related heterodimers consisting of an α-subunit and a ligand-specific β-subunit that confers their unique biological activity. Crystal structures showed how the β-subunit surrounds a part of the α-subunit, and we showed the existence of the two mechanisms responsible for that assembly. In human choriogonadotropin, the β-subunit is folded before the subunits dock, and the α-subunit becomes incorporated into the dimer by a mechanism we termed “threading,” passing between parts of the preassembled β-subunit. Here, we show that the human lutropin β-subunit is not folded completely prior to its interaction with the α-subunit and show that docking of the subunits enables the α-subunit to serve as a chaperone to the β-subunit. Based on data described here, we propose that the α-subunit facilitates formation of the human lutropin β-subunit by two mechanisms. First, the cystine knot of the α-subunit potentiates formation of the β-subunit cystine knot, and second, contacts between α-subunit loop 2 and a hydrophobic tail in the β-subunit facilitate formation of the seatbelt latch disulfide, which stabilizes the heterodimer. The primary influence of the α-subunit was seen when the hydrophobic tail was present or absent, but the secondary mechanism was required only when the hydrophobic tail of the β-subunit was present. During the evolution of human choriogonadotropin, neither of these α-subunit roles was necessary for folding of the β-subunit. The complex mechanism for lutropin assembly may be required to provide an additional control on its positive feedback function in vertebrate reproduction.     

The Subfamily-specific Assembly of Eag and Erg K+ Channels Is Determined by Both the Amino and the Carboxyl Recognition Domains

A functional voltage-gated K⁺ (Kv) channel comprises four pore-forming α-subunits, and only members of the same Kv channel subfamily may co-assemble to form heterotetramers. The ether-à-go-go family of Kv channels (KCNH) encompasses three distinct subfamilies: Eag (Kv10), Erg (Kv11), and Elk (Kv12). Members of different ether-à-go-go subfamilies, such as Eag and Erg, fail to form heterotetramers. Although a short stretch of amino acid sequences in the distal C-terminal section has been implicated in subfamily-specific subunit assembly, it remains unclear whether this region serves as the sole and/or principal subfamily recognition domain for Eag and Erg. Here we aim to ascertain the structural basis underlying the subfamily specificity of ether-à-go-go channels by generating various chimeric constructs between rat Eag1 and human Erg subunits. Biochemical and electrophysiological characterizations of the subunit interaction properties of a series of different chimeric and truncation constructs over the C terminus suggested that the putative C-terminal recognition domain is dispensable for subfamily-specific assembly. Further chimeric analyses over the N terminus revealed that the N-terminal region may also harbor a subfamily recognition domain. Importantly, exchanging either the N-terminal or the C-terminal domain alone led to a virtual loss of the intersubfamily assembly boundary. By contrast, simultaneously swapping both recognition domains resulted in a reversal of subfamily specificity. Our observations are consistent with the notion that both the N-terminal and the C-terminal recognition domains are required to sustain the subfamily-specific assembly of rat Eag1 and human Erg.     

Conformational Changes and Slow Dynamics through Microsecond Polarized Atomistic Molecular Simulation of an Integral Kv1.2 Ion Channel

Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how transitions occur in membrane proteins—not to mention numerous applications in drug design. Here, we present a full 1 µs atomic-detail molecular dynamics simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements, including up to 120° rotation of the S4 segment, changes in hydrogen-bonding patterns, but only low amounts of translation. A smaller rotation (∼35°) of the extracellular end of all S4 segments is present also in a reference 0.5 µs simulation without applied field, which indicates that the crystal structure might be slightly different from the natural state of the voltage sensor. The conformation change upon hyperpolarization is closely coupled to an increase in 3₁₀ helix contents in S4, starting from the intracellular side. This could support a model for transition from the crystal structure where the hyperpolarization destabilizes S4–lipid hydrogen bonds, which leads to the helix rotating to keep the arginine side chains away from the hydrophobic phase, and the driving force for final relaxation by downward translation is partly entropic, which would explain the slow process. The coordinates of the transmembrane part of the simulated channel actually stay closer to the recently determined higher-resolution Kv1.2 chimera channel than the starting structure for the entire second half of the simulation (0.5–1 µs). Together with lipids binding in matching positions and significant thinning of the membrane also observed in experiments, this provides additional support for the predictive power of microsecond-scale membrane protein simulations.     

Entropically-driven binding of mithramycin in the minor groove of C/G-rich DNA sequences

The antitumour antibiotic mithramycin A (MTA) is a DNA minor-groove binding ligand. It binds to C/G-rich tracts as a dimer that forms in the presence of divalent cations such as Mg²⁺. Differential scanning calorimetry, UV thermal denaturation, isothermal titration calorimetry and competition dialysis were used, together with computations of the hydrophobic free energy of binding, to determine the thermodynamic profile of MTA binding to DNA. The results were compared to those obtained in parallel using the structurally related mithramycin SK (MSK). The binding of MTA to salmon testes DNA determined by UV melting studies (K_obs = 1.2 (±0.3) × 10⁵M⁻¹) is tighter than that of MSK (2.9 (±1.0) × 10⁴M⁻¹) at 25°C. Competition dialysis studies showed a tighter MTA binding to both salmon testes DNA (42% C + G) and Micrococcus lysodeikticus DNA (72% C + G). The thermodynamic analysis of binding data at 25°C shows that the binding of MTA and MSK to DNA is entropically driven, dominated by the hydrophobic transfer of the antibiotics from solution to the DNA-binding site. Direct molecular recognition between MTA or MSK and DNA through hydrogen bonding and van der Waals contacts may also contribute significantly to complex formation.     

Formation and Growth of Oligomers: A Monte Carlo Study of an Amyloid Tau Fragment

Small oligomers formed early in the process of amyloid fibril formation may be the major toxic species in Alzheimer's disease. We investigate the early stages of amyloid aggregation for the tau fragment AcPHF6 (Ac-VQIVYK-NH2) using an implicit solvent all-atom model and extensive Monte Carlo simulations of 12, 24, and 36 chains. A variety of small metastable aggregates form and dissolve until an aggregate of a critical size and conformation arises. However, the stable oligomers, which are β-sheet-rich and feature many hydrophobic contacts, are not always growth-ready. The simulations indicate instead that these supercritical oligomers spend a lengthy period in equilibrium in which considerable reorganization takes place accompanied by exchange of chains with the solution. Growth competence of the stable oligomers correlates with the alignment of the strands in the β-sheets. The larger aggregates seen in our simulations are all composed of two twisted β-sheets, packed against each other with hydrophobic side chains at the sheet–sheet interface. These β-sandwiches show similarities with the proposed steric zipper structure for PHF6 fibrils but have a mixed parallel/antiparallel β-strand organization as opposed to the parallel organization found in experiments on fibrils. Interestingly, we find that the fraction of parallel β-sheet structure increases with aggregate size. We speculate that the reorganization of the β-sheets into parallel ones is an important rate-limiting step in the formation of PHF6 fibrils.     

Effect of Low Temperatures on Growth, Structure, and Metabolism of Campylobacter coli SP10

The effect of low temperatures on the survival, structure, and metabolism of Campylobacter coli SP10, a virulent strain, was investigated. C. coli became nonculturable rapidly at 20 and 10°C and slightly later at 4°C. Incubation in a microaerobic atmosphere improved survival, but after day 8, campylobacters were detectable by direct-count procedures only. The increase in the number of coccoid cells was most pronounced at 37°C but also was noticeable at 20 and 10°C. Two forms of coccoid cells were seen electron microscopically, but only one (20 and 10°C) seemed to be a degenerative form. The flagella were shorter at 20 and 10°C, a result which correlates well with the observed slight changes in the 62-kDa protein band. The fatty acid composition of bacterial cells was influenced significantly by low temperatures. An increase in the short-chain and unsaturated acids was noted; above all, a drastic increase in C_19:0 cyc at 20°C with a concomitant decrease in C_18:1 trans9,cis11 was seen. The concentrations of excreted metabolites were analyzed to obtain information on metabolic activity. Depending on the magnitude of the temperature downshift, the production of organic acids decreased, but it was always observable after a temperature-specific lag phase and regardless of ability to be cultured. Under optimal conditions, succinate, lactate, and acetate were the main metabolites, other acids being of less importance. The pattern changed significantly at lower temperatures. Succinate was never detected at 20°C and was only occasionally detected at 10 and 4°C. At the same time, fumarate concentrations, which are normally not detectable at 37°C, were highest at 20°C and reduced at 10 and 4°C. Inactivation of fumarate reductase was considered to be a possible explanation.     

Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils.     

A Residue-specific Shift in Stability and Amyloidogenicity of Antibody Variable Domains

Variable (V) domains of antibodies are essential for antigen recognition by our adaptive immune system. However, some variants of the light chain V domains (V_L) form pathogenic amyloid fibrils in patients. It is so far unclear which residues play a key role in governing these processes. Here, we show that the conserved residue 2 of V_L domains is crucial for controlling its thermodynamic stability and fibril formation. Hydrophobic side chains at position 2 stabilize the domain, whereas charged residues destabilize and lead to amyloid fibril formation. NMR experiments identified several segments within the core of the V_L domain to be affected by changes in residue 2. Furthermore, molecular dynamic simulations showed that hydrophobic side chains at position 2 remain buried in a hydrophobic pocket, and charged side chains show a high flexibility. This results in a predicted difference in the dissociation free energy of ∼10 kJ mol⁻¹, which is in excellent agreement with our experimental values. Interestingly, this switch point is found only in V_L domains of the κ family and not in V_Lλ or in V_H domains, despite a highly similar domain architecture. Our results reveal novel insight into the architecture of variable domains and the prerequisites for formation of amyloid fibrils. This might also contribute to the rational design of stable variable antibody domains.     

CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS : I. Location of the Polypeptides within Ribosomes

Free ribosomes containing nascent polypeptide chains labeled in vitro were submitted to proteolysis at 0° by a mixture of trypsin and chymotrypsin. Sucrose gradient analysis showed that polysome patterns are retained even after 24 hr of proteolysis in the cold, while messenger RNA-free ribosomes (generated progressively during in vitro incorporation) are, within 2 hr, completely dissociated into subunits by trypsin. Although ribosomes and subunits are not extensively degraded into smaller fragments during low temperature proteolysis, changes in the acrylamide gel electrophoresis pattern showed that most ribosomal proteins are accessible to and are partially degraded by the proteases. Ribosome-bound nascent polypeptides are partially resistant to proteolysis at 0°, although they are totally digested at 37° or when the ribosomal subunit structure is disrupted by other means. Radioactivity incorporated into nascent chains during incubation times shorter than 3 min was mostly resistant to digestion at 0°. A larger fraction of the initial radioactivity became degraded in ribosomes which incorporated for longer times. In these ribosomes, the amount of radioactivity which was resistant to proteolysis was constant and independent of the initial value, which reflects the labeled length of the nascent chains. These results suggest that the growing end of the nascent polypeptide is resistant to digestion and is protected from proteolytic attack by the ribosomal structure. A pulse and chase experiment confirmed this suggestion, showing that the protected segment is located at the carboxy-terminal end of the nascent chain. The protected segment was contained in the large ribosomal subunit and had a length of ~39 amino acid residues, as estimated by chromatography on Sephadex G-50.     

Three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at 2.9 A resolution

The three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 Å resolution by X-ray crystallographic methods. The MIR-electron density map was substantially improved by two-fold non-crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 Å. The enzyme subunit is a typical two-domain protein. The smaller, N-terminal domain consists of 137 amino acid residues and forms a central, mixed five-stranded β-sheet with α-helices on both sides of the sheet. The larger C-terminal domain consists of 329 amino acid residues. This domain has an eight-stranded parallel α/β barrel structure as found in triosephosphate isomerase and a number of other functionally non-related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy-end of the β-strands in the α/β barrel of the C-terminal domain. There are few domain–domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C-terminal domains, which build up the core of the molecule. There are also interactions between the N-terminal domain of one subunit and the C-terminal domain of the second subunit, close to the active site.