CiteULike: cactus's library [271 articles]

Monitoring Protein Conformation along the Pathway of Chaperonin-Assisted Folding

Shruti Sharma — 2008-04-10T09:33:33-00:00

Cell, Vol. 133, No. 1. (4 April 2008), pp. 142-153.

Summary The GroEL/GroES chaperonin system mediates protein folding in the bacterial cytosol. Newly synthesized proteins reach GroEL via transfer from upstream chaperones such as DnaK/DnaJ (Hsp70). Here we employed single molecule and ensemble FRET to monitor the conformational transitions of a model substrate as it proceeds along this chaperone pathway. We find that DnaK/DnaJ stabilizes the protein in collapsed states that fold exceedingly slowly. Transfer to GroEL results in unfolding, with a fraction of molecules reaching locally highly expanded conformations. ATP-induced domain movements in GroEL cause transient further unfolding and rapid mobilization of protein segments with moderate hydrophobicity, allowing partial compaction on the GroEL surface. The more hydrophobic regions are released upon subsequent protein encapsulation in the central GroEL cavity by GroES, completing compaction and allowing rapid folding. Segmental chain release and compaction may be important in avoiding misfolding by proteins that fail to fold efficiently through spontaneous hydrophobic collapse.

Kemp elimination catalysts by computational enzyme design

Daniela Röthlisberger — 2008-03-21T04:33:18-00:00

Nature (19 March 2008)

Dynamic binding orientations direct activity of HIV reverse transcriptase

Elio Abbondanzieri — 2008-05-08T07:42:20-00:00

Nature, Vol. 453, No. 7192. (May 2008), pp. 184-189.

Modeling transient collapsed states of an unfolded protein to provide insights into early folding events

Daniel Felitsky — 2008-05-07T07:23:14-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 17. (29 April 2008), pp. 6278-6283.

The primary driving force for protein folding is the sequestration of hydrophobic side chains from solvent water, but the means whereby the amino acid sequence directs the folding process to form the correct final folded state is not well understood. Measurements of NMR line broadening in spin-labeled samples of unfolded apomyoglobin at pH 2.3 have been used to derive a quantitative model for transient hydrophobic interactions between various sites in the polypeptide chain, as would occur during the initiation of protein folding. Local clusters of residues with high values for the parameter "average area buried upon folding" (AABUF) form foci not only for local contacts but for long-range interactions, the relative frequencies of which can be understood in terms of differences in the extent of reduction in chain configurational entropy that occurs upon formation of nonlocal contacts. These results complement the striking correlation previously observed between the kinetic folding process of apomyoglobin and the AABUF of its amino acid sequence [Nishimura C, Lietzow MA, Dyson HJ, Wright PE (2005) J Mol Biol 351:383-392]. For the acid-unfolded states of apomyoglobin, our approach identifies multiple distinct hydrophobic clusters of differing thermodynamic stability. The most structured of these clusters, although sparsely populated, have both native-like and nonnative character; the specificity of the transient long-range contacts observed in these states suggests that they play a key role in initiating chain collapse and folding. 10.1073/pnas.0710641105

Intrinsic noise, dissipation cost, and robustness of cellular networks: The underlying energy landscape of MAPK signal transduction

Saul Lapidus — 2008-04-18T18:35:19-00:00

Proceedings of the National Academy of Sciences (17 April 2008), 0708708105.

We develop a probabilistic method for analyzing global features of a cellular network under intrinsic statistical fluctuations, which is important when there are finite numbers of molecules. By making a self-consistent mean field approximation of splitting the variables in order to reduce the large number of degrees of freedom, which is reasonable for a not very strongly interacting network, we discovered that the underlying energy landscape of the mitogen-activated protein kinases (MAPKs) signal transduction network (with experimentally measured or inferred parameters such as chemical reaction rate coefficients in the network) is funneled toward a global minimum characterized by the nonequilibrium steady-state fixed point of the system at the end of the signal transduction process. For this system, we also show that the energy landscape is robust against intrinsic fluctuations and random perturbation to the inherent chemical reaction rates. The ratio of the slope versus the roughness of the energy landscape becomes a quantitative measure of robustness and stability of the network. Furthermore, we quantify the dissipation cost of this nonequilibrium system through entropy production, caused by the nonequilibrium flux in the system. We found that a lower dissipation cost corresponds to a more robust network. This least dissipation property might provide a design principle for robust and functional networks. Finally, we find the possibility of bistable and oscillatory-like solutions, which are important for cell fate decisions, upon perturbations. The method described here can be used in a variety of biological networks. 10.1073/pnas.0708708105

Conservation of the regulated structure of folded myosin 2 in species separated by at least 600 million years of independent evolution

Hyun Jung — 2008-05-07T06:28:28-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 16. (22 April 2008), pp. 6022-6026.

The myosin 2 family of molecular motors includes isoforms regulated in different ways. Vertebrate smooth-muscle myosin is activated by phosphorylation of the regulatory light chain, whereas scallop striated adductor-muscle myosin is activated by direct calcium binding to its essential light chain. The paired heads of inhibited molecules from myosins regulated by phosphorylation have an asymmetric arrangement with motor-motor interactions. It was unknown whether such interactions were a common motif for inactivation used in other forms of myosin-linked regulation. Using electron microscopy and single-particle image processing, we show that indistinguishable structures are indeed found in myosins and heavy meromyosins isolated from scallop striated adductor muscle and turkey gizzard smooth muscle. The similarities extend beyond the shapes of the heads and interactions between them: In both myosins, the tail folds into three segments, apparently at identical sites; all three segments are in close association outside the head region; and two segments are associated in the same way with one head in the asymmetric arrangement. Thus, these organisms, which have different regulatory mechanisms and diverged from a common ancestor >600 Myr ago, have the same quaternary structure. Conservation across such a large evolutionary distance suggests that this conformation is of fundamental functional importance. 10.1073/pnas.0707846105

The lattice as allosteric effector: Structural studies of alphabeta- and gamma-tubulin clarify the role of GTP in microtubule assembly

Luke Rice — 2008-05-02T10:24:15-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 14. (8 April 2008), pp. 5378-5383.

GTP-dependent microtubule polymerization dynamics are required for cell division and are accompanied by domain rearrangements in the polymerizing subunit, alpha-tubulin. Two opposing models describe the role of GTP and its relationship to conformational change in alpha-tubulin. The allosteric model posits that unpolymerized alpha-tubulin adopts a more polymerization-competent conformation upon GTP binding. The lattice model posits that conformational changes occur only upon recruitment into the growing lattice. Published data support a lattice model, but are largely indirect and so the allosteric model has prevailed. We present two independent solution probes of the conformation of alpha-tubulin, the 2.3 A crystal structure of gamma-tubulin bound to GDP, and kinetic simulations to interpret the functional consequences of the structural data. These results (with our previous gamma-tubulin:GTPgammaS structure) support the lattice model by demonstrating that major domain rearrangements do not occur in eukaryotic tubulins in response to GTP binding, and that the unpolymerized conformation of alpha-tubulin differs significantly from the polymerized one. Thus, geometric constraints of lateral self-assembly must drive alpha-tubulin conformational changes, whereas GTP plays a secondary role to tune the strength of longitudinal contacts within the microtubule lattice. alpha-Tubulin behaves like a bent spring, resisting straightening until forced to do so by GTP-mediated interactions with the growing microtubule. Kinetic simulations demonstrate that resistance to straightening opposes microtubule initiation by specifically destabilizing early assembly intermediates that are especially sensitive to the strength of lateral interactions. These data provide new insights into the molecular origins of dynamic microtubule behavior. 10.1073/pnas.0801155105

The thermal impulse response of Escherichia coli

Eli Paster — 2008-05-02T10:19:05-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 14. (8 April 2008), pp. 5373-5377.

Swimming Escherichia coli responds to changes in temperature by modifying its motor behavior. Previous studies using populations of cells have shown that E. coli accumulate in spatial thermal gradients, but these experiments did not cleanly separate thermal responses from chemotactic responses. Here we have isolated the thermal response by studying the behavior of single, tethered cells. The motor output of cells grown at 33degreesC was measured at constant temperature, from 10degrees to 40degreesC, and in response to small, impulsive increases in temperature, from 23degrees to 43degreesC. The thermal impulse response at temperatures < 31degreesC is similar to the chemotactic impulse response: Both follow a similar time course, share the same directionality, and show biphasic characteristics. At temperatures > 31degreesC, some cells show an inverted response, switching from warm- to cold-seeking behavior. The fraction of inverted responses increases nonlinearly with temperature, switching steeply at the preferred temperature of 37degreesC. 10.1073/pnas.0709903105

Hierarchical structure and the prediction of missing links in networks

Aaron Clauset — 2008-04-30T19:31:59-00:00

Nature, Vol. 453, No. 7191., pp. 98-101.

Evolvability and hierarchy in rewired bacterial gene networks

Mark Isalan — 2008-04-16T19:45:06-00:00

Nature, Vol. 452, No. 7189. (17 April 2008), pp. 840-845.

Following translation by single ribosomes one codon at a time

Jin-Der Wen — 2008-03-10T16:18:13-00:00

Nature (09 March 2008)

Minimalist Protein Model as a Diagnostic Tool for Misfolding and Aggregation

Silvina Matysiak — 2008-04-18T11:25:17-00:00

Journal of Molecular Biology, Vol. 363, No. 1. (13 October 2006), pp. 297-308.

We propose a realistic coarse-grained protein model and a technique to "anchor" the model to available experimental data. We apply this procedure to characterize the effect of multiple mutations on the folding mechanism of protein S6. We show that the mutation of a few "gatekeeper" residues triggers significant changes on the folding landscape of S6. These results suggest that gatekeeper residues control the flexibility of critical regions of S6, that in turn regulates the delicate balance between folding and aggregation. Although obtained with a minimalist protein model, these results are fully consistent with experimental evidence and offer a clue to understand the interplay between folding and aggregation in protein S6.

Allostery: Absence of a Change in Shape Does Not Imply that Allostery Is Not at Play.

Chung-Jung Tsai — 2008-03-26T10:26:57-00:00

J Mol Biol (29 February 2008)

Allostery is essential for controlled catalysis, signal transmission, receptor trafficking, turning genes on and off, and apoptosis. It governs the organism's response to environmental and metabolic cues, dictating transient partner interactions in the cellular network. Textbooks taught us that allostery is a change of shape at one site on the protein surface brought about by ligand binding to another. For several years, it has been broadly accepted that the change of shape is not induced; rather, it is observed simply because a larger protein population presents it. Current data indicate that while side chains can reorient and rewire, allostery may not even involve a change of (backbone) shape. Assuming that the enthalpy change does not reverse the free-energy change due to the change in entropy, entropy is mainly responsible for binding.

Nucleation of an Allosteric Response via Ligand-induced Loop Folding

Saranga Naganathan — 2008-04-18T09:05:02-00:00

Journal of Molecular Biology, Vol. 373, No. 1. (12 October 2007), pp. 96-111.

The Escherichia coli biotin repressor BirA is an allosteric transcription regulatory protein to which binding of the small ligand corepressor biotinyl-5'-AMP promotes homodimerization and subsequent DNA binding. Structural data indicate that the apo or unliganded repressor is characterized by four partially disordered loops that are ordered in the ligand-bound dimer. While three of these loops participate directly in the dimerization, the fourth, consisting of residues 212-234 is distal to the interface. This loop, which is ordered around the adenine ring of the adenylate moiety in the BirAadenylate structure, is referred to as the adenylate-binding loop (ABL). Although residues in the loop do not interact directly with the ligand, a hydrophobic cluster consisting of a tryptophan and two valine side-chains assembles over the adenine base. Results of previous measurements suggest that folding of the ABL is integral to the allosteric response. This idea and the role of the hydrophobic cluster in the process were investigated by systematic replacement of each side-chain in the cluster with alanine and analysis of the mutant proteins for small ligand binding and dimerization. Isothermal titration calorimetry measurements indicate defects in adenylate binding for all ABL variants. Additionally, sedimentation equilibrium measurements reveal that coupling between adenylate binding and dimerization is compromised in each mutant. Partial proteolysis measurements indicate that the mutants are defective in ligand-linked folding of the ABL. These results indicate that the hydrophobic cluster is critical to the ligand-induced disorder-to-order transition in the ABL and that this transition is integral to the allosteric response in the biotin repressor.

A Cytoskeletal Demolition Worker: Myosin II Acts as an Actin Depolymerization Agent

Lior Haviv — 2008-04-17T10:54:05-00:00

Journal of Molecular Biology, Vol. 375, No. 2. (11 January 2008), pp. 325-330.

Myosin II motors play several important roles in a variety of cellular processes, some of which involve active assembly/disassembly of cytoskeletal substructures. Myosin II motors have been shown to function in actin bundle turnover in neuronal growth cones and in the recycling of actin filaments during cytokinesis. Close examination had shown an intimate relationship between myosin II motor adenosine triphosphatase activity and actin turnover rate. However, the direct implication of myosin II in actin turnover is still not understood. Herein, we show, using high-resolution cryo-transmission electron microscopy, that myosin II motors control the turnover of actin bundles in a concentration-dependent manner in vitro. We demonstrate that disassembly of actin bundles occurs through two main stages: the first stage involves unbundling into individual filaments, and the second involves their subsequent depolymerization. These evidence suggest that, in addition to their "classical" contractile abilities, myosin II motors may be directly implicated in active actin depolymerization. We believe that myosin II motors may function similarly in vivo (e.g., in the disassembly of the contractile ring by fine tuning the local concentration/activity of myosin II motors).

Kinetic Analysis of the Slow Skeletal Myosin MHC-1 Isoform from Bovine Masseter Muscle

MJ Bloemink — 2008-04-17T10:41:17-00:00

Journal of Molecular Biology, Vol. 373, No. 5. (9 November 2007), pp. 1184-1197.

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as [beta]-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin-ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant KD) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (KAD/KD ~ 5 instead of KAD/KD ~ 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s-1) than reported previously (138 s-1). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.

Predicting Allosteric Communication in Myosin via a Pathway ofConserved Residues

Susan Tang — 2007-09-04T11:46:14-00:00

Journal of Molecular Biology, Vol. In Press, Accepted Manuscript

Evidence for an Interaction between the SH3 Domain and the N-terminal Extension of the Essential Light Chain in Class II Myosins

Susan Lowey — 2008-04-17T08:30:26-00:00

Journal of Molecular Biology, Vol. 371, No. 4. (24 August 2007), pp. 902-913.

The function of the src-homology 3 (SH3) domain in class II myosins, a distinct [beta]-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the ~ 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.

Diversity of Structural Behavior in Vertebrate Conventional Myosins Complexed with Actin

Hiroyuki Iwamoto — 2008-04-17T06:29:24-00:00

Journal of Molecular Biology, Vol. 369, No. 1. (25 May 2007), pp. 249-264.

Low-resolution three-dimensional structures of acto-myosin subfragment-1 (S1) complexes were retrieved from X-ray fiber diffraction patterns, recorded either in the presence or absence of ADP. The S1 was obtained from various myosin-II isoforms from vertebrates, including rabbit fast-skeletal and cardiac, chicken smooth and human non-muscle IIA and IIB species, and was diffused into an array of overstretched, skinned skeletal muscle fibers. The S1 attached to the exposed actin filaments according to their helical symmetry. Upon addition of ADP, the diffraction patterns from acto-S1 showed an increasing magnitude of response in the order as listed above, with features of a lateral compression of the whole diffraction pattern (indicative of increased radius of the acto-S1 complex) and an enhancement of the fifth layer-line reflection. The structure retrieval indicates that these changes are mainly due to the swing of the light chain (LC) domain in the direction consistent with the cryo-electron microscopic results. In the non-muscle isoforms, the swing is large enough to affect the manner of quasi-crystal packing of the S1-decorated actin filaments and their lattice dimension, with a small change in the twist of actin filaments. Variations also exist in the behavior of the 50K-cleft, which apparently opens upon addition of ADP to the non-muscle isoforms but not to other isoforms. The fast-skeletal S1 remains as the only isoform that does not clearly exhibit either of the structural changes. The results indicate that the "conventional" myosin-II isoforms exhibit a wide variety of structural behavior, possibly depending on their functions and/or the history of molecular evolution.

Watching rocks grow

Veysey — 2008-04-02T09:32:48-00:00

Nat Phys, Vol. 4, No. 4. (April 2008), pp. 310-313.

GroEL stimulates protein folding through forced unfolding

Zong Lin — 2008-03-06T04:15:38-00:00

Nature Structural & Molecular Biology, Vol. 15, No. 3. (02 March 2008), pp. 303-311.

Stabilization of a beta-hairpin in monomeric Alzheimer's amyloid-beta peptide inhibits amyloid formation

Wolfgang Hoyer — 2008-04-02T09:13:28-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 13. (1 April 2008), pp. 5099-5104.

According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid- (A) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar A assemblies is accompanied by a conformational change toward a high content of -structure. Here, we report the solution structure of A(140) in complex with the phage-display selected affibody protein ZA3, a binding protein of nanomolar affinity. Bound A(140) features a -hairpin comprising residues 1736, providing the first high-resolution structure of A in conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar A. ZA3 stabilizes the -sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the A hairpin within a large hydrophobic tunnel-like cavity. Consequently, ZA3 acts as a stoichiometric inhibitor of A fibrillation. The selected A conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates. 10.1073/pnas.0711731105

Interconversion between two unrelated protein folds in the lymphotactin native state

Robbyn Tuinstra — 2008-04-02T09:10:15-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 13. (1 April 2008), pp. 5057-5062.

Proteins often have multiple functional states, which might not always be accommodated by a single fold. Lymphotactin (Ltn) adopts two distinct structures in equilibrium, one corresponding to the canonical chemokine fold consisting of a monomeric three-stranded -sheet and carboxyl-terminal helix. The second Ltn structure solved by NMR reveals a dimeric all--sheet arrangement with no similarity to other known proteins. In physiological solution conditions, both structures are significantly populated and interconvert rapidly. Interconversion replaces long-range interactions that stabilize the chemokine fold with an entirely new set of tertiary and quaternary contacts. The chemokine-like Ltn conformation is a functional XCR1 agonist, but fails to bind heparin. In contrast, the alternative structure binds glycosaminoglycans with high affinity but fails to activate XCR1. Because each structural species displays only one of the two functional properties essential for activity in vivo, the conformational equilibrium is likely to be essential for the biological activity of lymphotactin. These results demonstrate that the functional repertoire and regulation of a single naturally occurring amino acid sequence can be expanded by access to a set of highly dissimilar native-state structures. 10.1073/pnas.0709518105

Reassessing a sparse energetic network within a single protein domain

Celestine Chi — 2008-03-15T01:25:34-00:00

Proceedings of the National Academy of Sciences (13 March 2008), 0711732105.

Understanding the molecular principles that govern allosteric communication is an important goal in protein science. One way allostery could be transmitted is via sparse energetic networks of residues, and one such evolutionary conserved network was identified in the PDZ domain family of proteins by multiple sequence alignment [Lockless SW, Ranganathan R (1999) Science 286:295299]. We have reassessed the energetic coupling of these residues by double mutant cycles together with ligand binding and stability experiments and found that coupling is not a special property of the coevolved network of residues in PDZ domains. The observed coupling for ligand binding is better explained by a distance relationship, where residues close in space are more likely to couple than distal residues. Our study demonstrates that statistical coupling from sequence analysis is not necessarily a reporter of energetic coupling and allostery. 10.1073/pnas.0711732105

Myosin V and Kinesin act as tethers to enhance each others' processivity

Yusuf Ali — 2008-04-02T08:23:16-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 12. (25 March 2008), pp. 4691-4696.

Organelle transport to the periphery of the cell involves coordinated transport between the processive motors kinesin and myosin V. Long-range transport takes place on microtubule tracks, whereas final delivery involves shorter actin-based movements. The concept that motors only function on their appropriate track required further investigation with the recent observation that myosin V undergoes a diffusional search on microtubules. Here we show, using single-molecule techniques, that a functional consequence of myosin V's diffusion on microtubules is a significant enhancement of the processive run length of kinesin when both motors are present on the same cargo. The degree of run length enhancement correlated with the net positive charge in loop 2 of myosin V. On actin, myosin V also undergoes longer processive runs when kinesin is present on the same cargo. The process that causes run length enhancement on both cytoskeletal tracks is electrostatic. We propose that one motor acts as a tether for the other and prevents its diffusion away from the track, thus allowing more steps to be taken before dissociation. The resulting run length enhancement likely contributes to the successful delivery of cargo in the cell. 10.1073/pnas.0711531105

The kinetic mechanism of kinesin.

RA Cross — 2005-03-03T06:35:47-00:00

Trends Biochem Sci, Vol. 29, No. 6. (June 2004), pp. 301-309.

The chemical kinetic mechanism of kinesin (K) is considered by using a consensus scheme incorporating biochemically defined open, closed and trapped states. In the absence of microtubules, the dominant species is a trapped K*ADP state, which is defined by its ultra-slow release of ADP (off rate, k(off) approximately 0.002 s(-1)) and weak microtubule binding (dissociation constant, K(d) approximately 10-20 microM). Once bound, this trapped state equilibrates with a strongly binding open state that rapidly releases ADP (k(off) approximately 300 s(-1)). After ADP release, Mg*ATP binds (on rate, k(on) approximately 2 microM(-1)s(-1)) driving formation of a closed state that is defined by hydrolysis competence and by strong binding to microtubules. Hydrolysis (k(hyd) approximately 100-300 s(-1)) and phosphate release (k(off)>100 s(-1)) both occur in this microtubule-bound closed state. Phosphate release acts as a gate that controls reversion to the trapped K*ADP state, which detaches from the microtubule, completing the cycle.

Weak and Strong States of Kinesin and ncd

Isabelle Crevel — 2008-03-26T08:32:30-00:00

Journal of Molecular Biology, Vol. 257, No. 1. (22 March 1996), pp. 66-76.

Kinesin superfamily molecular motors step along microtubules (MTs)viaa cycle of conformational changes which is coupled to ATP turnover. To probe the coupling mechanism, we titrated the effects of various nucleotides on MT binding by two superfamily members; MT plus-end-directed kinesin and MT minus-end-directed non claret disjunctional (ncd). For both motors, the nucleotide-free state induced by apyrase was the strongest binding (Kkind~0.003 [mu]M,Kncdd~0.24 [mu]M), whilst the ADP state was the weakest binding (Kkind~11.32 [mu]M,Kncdd~12.02 [mu]M). In ATP, the motor.ADP state dominates and the binding is accordingly ADP-like, but in the presence of the slowly hydrolysed analogue adenosine 5'-O-(3-thiotriphosphate) there is a shift towards tighter binding (Kkind~4.23 [mu]M,Kncdd~2.34 [mu]M), consistent with a tight-binding motor.ATP-like state being enriched. In the presence of non-hydrolysable analogue [beta],[gamma]- imidoadenosine 5'-triphosphate the binding is still tighter (Kkind~<0.27 [mu]M,Kncdd~0.21 [mu]M), close to the values obtained with apyrase. For both kinesin and ncd, ADP has the unique quality that it traps the motor in a weak binding state. MT tight binding catalyses escape from this state, changing the active site conformation such that both ADP release and ADP binding are accelerated. The data are consistent with a simple two-state scheme in which both kinesin and ncd switch from weak to strong bindingviaADP release, and back againviaADP trapping. In a two-state model, the transition from weak to strong binding is force-generating.

X-ray Structure and Microtubule Interaction of the Motor Domain of Neurospora crassa NcKin3, a Kinesin with Unusual Processivity(,).

Alexander Marx — 2008-01-22T20:23:46-00:00

Biochemistry (19 January 2008)

Neurospora crassa kinesin NcKin3 belongs to a unique fungal-specific subgroup of small Kinesin-3-related motor proteins. One of its functions appears to be the transport of mitochondria along microtubules. Here, we present the X-ray structure of a C-terminally truncated monomeric construct of NcKin3 comprising the motor domain and the neck linker, and a 3-D image reconstruction of this motor domain bound to microtubules, by cryoelectron microscopy. The protein contains Mg.ADP bound to the active site, yet the structure resembles an ATP-bound state. By comparison with structures of the Kinesin-3 motor Kif1A in different nucleotide states (Kikkawa, M. et al. (2001) Nature (London, U.K.) 411, 439-445), the NcKin3 structure corresponds to the AMPPCP complex of Kif1A rather than the AMPPNP complex. NcKin3-specific differences in the coordination of the nucleotide and asymmetric interactions between adjacent molecules in the crystal are discussed in the context of the unusual kinetics of the dimeric wild-type motor and the monomeric construct used for crystal structure analysis. The NcKin3 motor decorates microtubules at a stoichiometry of one head per alphabeta-tubulin heterodimer, thereby forming an axial periodicity of 8 nm. In spite of unusual extensions at the N-terminus and within flexible loops L2, L8a, and L12 (corresponding to the K-loop of monomeric kinesins), the microtubule binding geometry is similar to that of other members of the kinesin family.

A cool look at the structural changes in kinesin motor domains

Linda Amos — 2008-02-28T11:40:50-00:00

J Cell Sci, Vol. 120, No. 22. (15 November 2007), pp. 3919-3927.

Recently, several 3D images of kinesin-family motor domains interacting with microtubules have been obtained by analysis of electron microscope images of frozen hydrated complexes at much higher resolutions (9-12 A) than in previous reports (15-30 A). The high-resolution maps show a complex interaction interface between kinesin and tubulin, in which kinesin's switch II helix alpha4 is a central feature. Differences due to the presence of ADP, as compared with ATP analogues, support previously determined crystal structures of kinesins alone in suggesting that alpha4 is part of a pathway linking the nucleotide-binding site and the neck that connects to cargo. A 3D structure of the microtubule-bound Kar3 motor domain in a nucleotide-free state has revealed dramatic changes not yet reported for any crystal structure, including melting of the switch II helix, that may be part of the mechanism by which information is transmitted. A nucleotide-dependent movement of helix alpha6, first seen in crystal structures of Kif1a, appears to bring it into contact with tubulin and may provide another communication link. A microtubule-induced movement of loop L7 and a related distortion of the central -sheet, detected only in the empty state, may also send a signal to the region of the motor core that interacts with the neck. Earlier images of a kinesin-1 dimer in the empty state, showing a close interaction between the two motor heads, can now be interpreted in terms of a communication route from the active site of the directly bound head via its central -sheet to the tethered head. 10.1242/jcs.016931

Free-energy landscape of kinesin by a realistic lattice model

Hiroo Kenzaki — 2008-02-28T11:19:18-00:00

Proteins: Structure, Function, and Bioinformatics, Vol. 71, No. 1. (2008), pp. 389-395.

Structural fluctuations in the thermal equilibrium of the kinesin motor domain are studied using a lattice protein model with G&omacr; interactions. By means of the multi-self-overlap ensemble Monte Carlo method and the principal component analysis, the free-energy landscape is obtained. It is shown that kinesins have two subdomains that exhibit partial folding/unfolding at functionally important regions: one is located around the nucleotide binding site and the other includes the main microtubule binding site. These subdomains are consistent with structural variability that was reported recently based on experimentally-obtained structures. On the other hand, such large structural fluctuations have not been captured by B-factor or normal mode analyses. Thus, they are beyond the elastic regime, and it is essential to take into account chain connectivity for studying the function of kinesins. Proteins 2008. © 2007 Wiley-Liss, Inc.

Subunits interactions in kinesin motors

Krzysztof Skowronek — 2008-02-28T11:10:21-00:00

European Journal of Cell Biology, Vol. 86, No. 9. (14 September 2007), pp. 559-568.

Kinesins form a large and diverse superfamily of proteins involved in numerous important cellular processes. The majority of them are molecular motors moving along microtubules. Conversion of chemical energy into mechanical work is accomplished in a sequence of events involving both biochemical and conformational alternation of the motor structure called the mechanochemical cycle. Different members of the kinesin superfamily can either perform their function in large groups or act as single molecules. Conventional kinesin, a member of the kinesin-1 subfamily, exemplifies the second type of motor which requires tight coordination of the mechanochemical cycle in two identical subunits to accomplish processive movement toward the microtubule plus end. Recent results strongly support an asymmetric hand-over-hand model of "walking" for this protein. Conformational strain between two subunits at the stage of the cycle where both heads are attached to the microtubule seems to be a major factor in intersubunit coordination, although molecular and kinetic details of this phenomenon are not yet deciphered. We discuss also current knowledge concerning intersubunit coordination in other kinesin subfamilies. Members of the kinesin-3 class use at least three different mechanisms of movement and can translocate in monomeric or dimeric forms. It is not known to what extent intersubunit coordination takes place in Ncd, a dimeric member of the kinesin-14 subfamily which, unlike conventional kinesin, exercises a power-stroke toward the microtubule minus end. Eg5, a member of the kinesin-5 subfamily is a homotetrameric protein with two kinesin-1-like dimeric halves controlled by their relative orientation on two microtubules. It seems that diversity of subunit organization, quaternary structures and cellular functions in the kinesin superfamily are reflected also by the divergent extent and mechanism of intersubunit coordination during kinesin movement along microtubules.

From the Cover: Detection of fractional steps in cargo movement by the collective operation of kinesin-1 motors

Cecile Leduc — 2008-02-28T11:09:30-00:00

Proceedings of the National Academy of Sciences, Vol. 104, No. 26. (26 June 2007), pp. 10847-10852.

The stepping behavior of single kinesin-1 motor proteins has been studied in great detail. However, in cells, these motors often do not work alone but rather function in small groups when they transport cellular cargo. Until now, the cooperative interactions between motors in such groups were poorly understood. A fundamental question is whether two or more motors that move the same cargo step in synchrony, producing the same step size as a single motor, or whether the step size of the cargo movement varies. To answer this question, we performed in vitro gliding motility assays, where microtubules coated with quantum dots were driven over a glass surface by a known number of kinesin-1 motors. The motion of individual microtubules was then tracked with nanometer precision. In the case of transport by two kinesin-1 motors, we found successive 4-nm steps, corresponding to half the step size of a single motor. Dwell-time analysis did not reveal any coordination, in the sense of alternate stepping, between the motors. When three motors interacted in collective transport, we identified distinct forward and backward jumps on the order of 10 nm. The existence of the fractional steps as well as the distinct jumps illustrate a lack of synchronization and has implications for the analysis of motor-driven organelle movement investigated in vivo. 10.1073/pnas.0701864104

Large conformational changes in a kinesin motor catalyzed by interaction with microtubules.

K Hirose — 2007-07-27T06:57:25-00:00

Mol Cell, Vol. 23, No. 6. (15 September 2006), pp. 913-923.

Kinesin motor proteins release nucleotide upon interaction with microtubules (MTs), then bind and hydrolyze ATP to move along the MT. Although crystal structures of kinesin motors bound to nucleotides have been solved, nucleotide-free structures have not. Here, using cryomicroscopy and three-dimensional (3D) reconstruction, we report the structure of MTs decorated with a Kinesin-14 motor, Kar3, in the nucleotide-free state, as well as with ADP and AMPPNP, with resolution sufficient to show alpha helices. We find large structural changes in the empty motor, including melting of the switch II helix alpha4, closure of the nucleotide binding pocket, and changes in the central beta sheet reminiscent of those reported for nucleotide-free myosin crystal structures. We propose that the switch II region of the motor controls docking of the Kar3 neck by conformational changes in the central beta sheet, similar to myosin, rather than by rotation of the motor domain, as proposed for the Kif1A kinesin motor.

Myosin II isoforms in smooth muscle: heterogeneity and function

Thomas Eddinger — 2008-02-28T10:12:33-00:00

Am J Physiol Cell Physiol, Vol. 293, No. 2. (1 August 2007), pp. C493-508.

Both smooth muscle (SM) and nonmuscle class II myosin molecules are expressed in SM tissues comprising hollow organ systems. Individual SM cells may express one or more of multiple myosin II isoforms that differ in myosin heavy chain (MHC) and myosin light chain (MLC) subunits. Although much has been learned, the expression profiles, organization within contractile filaments, localization within cells, and precise roles in various contractile functions of these different myosin molecules are still not well understood. However, data supporting unique physiological roles for certain isoforms continues to build. Isoform differences located in the S1 head region of the MHC can alter actin binding and rates of ATP hydrolysis. Differences located in the MHC tail can alter the formation, stability, and size of the myosin thick filament. In these distinct ways, both head and tail isoform differences can alter force generation and muscle shortening velocities. The MLCs that are associated with the lever arm of the S1 head can affect the flexibility and range of motion of this domain and possibly the motion of the S2 and motor domains. Phosphorylation of MLC20 has been associated with conformational changes in the S1 and/or S2 fragments regulating enzymatic activity of the entire myosin molecule. A challenge for the future will be delineation of the physiological significance of the heterogeneous expression of these isoforms in developmental, tissue-specific, and species-specific patterns and or the intra- and intercellular heterogeneity of myosin isoform expression in SM cells of a given organ. 10.1152/ajpcell.00131.2007

Kinetic Characterization of the Function of Myosin Loop 4 in the Actin-Myosin Interaction

M Gyimesi — 2008-02-28T09:48:50-00:00

Biochemistry, Vol. 47, No. 1. (8 January 2008), pp. 283-291.

Abstract: Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin's ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (AL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states.

Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles

Anthony Cammarato — 2008-02-28T08:39:49-00:00

Mol. Biol. Cell, Vol. 19, No. 2. (1 February 2008), pp. 553-562.

Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc5 affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc5 (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc5 myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc5 mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders. 10.1091/mbc.E07-09-0890

Engineering the Processive Run Length of Myosin V

Alex Hodges — 2008-02-28T07:54:30-00:00

J. Biol. Chem., Vol. 282, No. 37. (14 September 2007), pp. 27192-27197.

The processive motor myosin V has a high affinity for actin in the weak binding states when compared with non-processive myosins. Here we test whether this feature is essential for myosin V to walk processively along an actin filament. The net charge of loop 2, a surface loop implicated in the initial weak binding between myosin and actin, was increased or decreased to correspondingly change the affinity of myosin V for actin in the weak binding state, without changing the velocity of movement. Processive run lengths of single molecules were determined by total internal reflection fluorescence microscopy. Reducing the net positive charge of loop 2 significantly decreased both the affinity of myosin V for actin and the processive run length. Conversely, the addition of positive charge to loop 2 increased actin affinity and processive run length. We hypothesize that a high affinity for actin allows the detached head of a stepping myosin V to find its next actin binding site more quickly, thus decreasing the probability of run termination. 10.1074/jbc.M703968200

The post-rigor structure of myosin VI and implications for the recovery stroke

Julie Menetrey — 2007-11-30T17:38:26-00:00

The EMBO Journal, Vol. aop, No. current. (29 November 2007)

Kinetic Mechanism of the Fastest Motor Protein, Chara Myosin

Kohji Ito — 2008-02-27T07:21:43-00:00

J. Biol. Chem., Vol. 282, No. 27. (6 July 2007), pp. 19534-19545.

Chara corallina class XI myosin is by far the fastest molecular motor. To investigate the molecular mechanism of this fast movement, we performed a kinetic analysis of a recombinant motor domain of Chara myosin. We estimated the time spent in the strongly bound state with actin by measuring rate constants of ADP dissociation from actinmiddle dotmotor domain complex and ATP-induced dissociation of the motor domain from actin. The rate constant of ADP dissociation from acto-motor domain was >2800 s-1, and the rate constant of ATP-induced dissociation of the motor domain from actin at physiological ATP concentration was 2200 s-1. From these data, the time spent in the strongly bound state with actin was estimated to be <0.82 ms. This value is the shortest among known values for various myosins and yields the duty ratio of <0.3 with a Vmax value of the actin-activated ATPase activity of 390 s-1. The addition of the long neck domain of myosin Va to the Chara motor domain largely increased the velocity of the motility without increasing the ATP hydrolysis cycle rate, consistent with the swinging lever model. In addition, this study reveals some striking kinetic features of Chara myosin that are suited for the fast movement: a dramatic acceleration of ADP release by actin (1000-fold) and extremely fast ATP binding rate. 10.1074/jbc.M611802200

Mechanochemical Coupling in the Myosin Motor Domain. II. Analysis of Critical Residues

Haibo Yu — 2008-02-27T07:14:16-00:00

PLoS Computational Biology, Vol. 3, No. 2. (1 February 2007), e23.

An important challenge in the analysis of mechanochemical coupling in molecular motors is to identify residues that dictate the tight coupling between the chemical site and distant structural rearrangements. In this work, a systematic attempt is made to tackle this issue for the conventional myosin. By judiciously combining a range of computational techniques with different approximations and strength, which include targeted molecular dynamics, normal mode analysis, and statistical coupling analysis, we are able to identify a set of important residues and propose their relevant function during the recovery stroke of myosin. These analyses also allowed us to make connections with previous experimental and computational studies in a critical manner. The behavior of the widely used reporter residue, Trp501, in the simulations confirms the concern that its fluorescence does not simply reflect the relay loop conformation or active-site open/close but depends subtly on its microenvironment. The findings in the targeted molecular dynamics and a previous minimum energy path analysis of the recovery stroke have been compared and analyzed, which emphasized the difference and complementarity of the two approaches. In conjunction with our previous studies, the current set of investigations suggest that the modulation of structural flexibility at both the local (e.g., active-site) and domain scales with strategically placed “hotspot” residues and phosphate chemistry is likely the general feature for mechanochemical coupling in many molecular motors. The fundamental strategies of examining both collective and local changes and combining physically motivated methods and informatics-driven techniques are expected to be valuable to the study of other molecular motors and allosteric systems in general.

Mechanochemical Coupling in the Myosin Motor Domain. I. Insights from Equilibrium Active-Site Simulations

Haibo Yu — 2008-02-27T07:02:49-00:00

PLoS Computational Biology, Vol. 3, No. 2. (1 February 2007), e21.

Although the major structural transitions in molecular motors are often argued to couple to the binding of Adenosine triphosphate (ATP), the recovery stroke in the conventional myosin has been shown to be dependent on the hydrolysis of ATP. To obtain a clearer mechanistic picture for such “mechanochemical coupling” in myosin, equilibrium active-site simulations with explicit solvent have been carried out to probe the behavior of the motor domain as functions of the nucleotide chemical state and conformation of the converter/relay helix. In conjunction with previous studies of ATP hydrolysis with different active-site conformations and normal mode analysis of structural flexibility, the results help establish an energetics-based framework for understanding the mechanochemical coupling. It is proposed that the activation of hydrolysis does not require the rotation of the lever arm per se, but the two processes are tightly coordinated because both strongly couple to the open/close transition of the active site. The underlying picture involves shifts in the dominant population of different structural motifs as a consequence of changes elsewhere in the motor domain. The contribution of this work and the accompanying paper [36] is to propose the actual mechanism behind these “population shifts” and residues that play important roles in the process. It is suggested that structural flexibilities at both the small and large scales inherent to the motor domain make it possible to implement tight couplings between different structural motifs while maintaining small free-energy drops for processes that occur in the detached states, which is likely a feature shared among many molecular motors. The significantly different flexibility of the active site in different X-ray structures with variable level arm orientations supports the notation that external force sensed by the lever arm may transmit into the active site and influence the chemical steps (nucleotide hydrolysis and/or binding).

Cooperative actions between myosin heads bring effective functions

Seiji Esaki — 2008-02-27T06:44:31-00:00

Biosystems, Vol. 88, No. 3. (April 2007), pp. 293-300.

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle.

Differential Regulation of Dynein and Kinesin Motor Proteins by Tau

Ram Dixit — 2008-01-20T05:11:55-00:00

Science (17 January 2008), 1152993.

Dynein and kinesin motor proteins transport cellular cargos toward opposite ends of microtubule tracks. In neurons, microtubules are abundantly decorated with microtubule-associated proteins (MAPs) such as tau. Motor proteins thus encounter MAPs frequently along their path. To determine the effects of tau on dynein and kinesin motility, we conducted single molecule studies of motor proteins moving along tau-decorated microtubules. Dynein tended to reverse direction whereas kinesin tended to detach at patches of bound tau. Kinesin was inhibited at ~10-fold lower tau concentration than dynein and the microtubule-binding domain of tau was sufficient to inhibit motor activity. The differential modulation of dynein and kinesin motility suggests that MAPs can spatially regulate the balance of microtubule-dependent axonal transport. 10.1126/science.1152993

Axle-Less F1-ATPase Rotates in the Correct Direction

Shou Furuike — 2008-02-22T10:24:05-00:00

Science, Vol. 319, No. 5865. (15 February 2008), pp. 955-958.

F1adenosine triphosphatase (ATPase) is an ATP-driven rotary molecular motor in which the central gamma subunit rotates inside a cylinder made of three alpha and three subunits alternately arranged. The rotor shaft, an antiparallel alpha-helical coiled coil of the amino and carboxyl termini of the gamma subunit, deeply penetrates the central cavity of the stator cylinder. We truncated the shaft step by step until the remaining rotor head would be outside the cavity and simply sat on the concave entrance of the stator orifice. All truncation mutants rotated in the correct direction, implying torque generation, although the average rotary speeds were low and short mutants exhibited moments of irregular motion. Neither a fixed pivot nor a rigid axle was needed for rotation of F1-ATPase. 10.1126/science.1151343

Mechanisms of prion protein assembly into amyloid

Jan Stohr — 2008-02-20T02:01:23-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 7. (19 February 2008), pp. 2409-2414.

The conversion of the alpha-helical, cellular isoform of the prion protein (PrPC) to the insoluble, -sheet-rich, infectious, disease-causing isoform (PrPSc) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, alpha-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions. 10.1073/pnas.0712036105

Cooperative folding kinetics of BBL protein and peripheral subunit-binding domain homologues

Wookyung Yu — 2008-02-22T09:40:43-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 7. (19 February 2008), pp. 2397-2402.

Recent experiments claiming that Naf-BBL protein follows a global downhill folding raised an important controversy as to the folding mechanism of fast-folding proteins. Under the global downhill folding scenario, not only do proteins undergo a gradual folding, but folding events along the continuous folding pathway also could be mapped out from the equilibrium denaturation experiment. Based on the exact calculation using a free energy landscape, relaxation eigenmodes from a master equation, and Monte Carlo simulation of an extended MunozEaton model that incorporates multiscale-heterogeneous pairwise interactions between amino acids, here we show that the very nature of a two-state cooperative transition such as a bimodal distribution from an exact free energy landscape and biphasic relaxation kinetics manifest in the thermodynamics and foldingunfolding kinetics of BBL and peripheral subunit-binding domain homologues. Our results provide an unequivocal resolution to the fundamental controversy related to the global downhill folding scheme, whose applicability to other proteins should be critically reexamined. 10.1073/pnas.0708480105

Three-dimensional structure of vertebrate cardiac muscle myosin filaments

Maria Zoghbi — 2008-02-22T09:20:05-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 7. (19 February 2008), pp. 2386-2390.

Contraction of the heart results from interaction of the myosin and actin filaments. Cardiac myosin filaments consist of the molecular motor myosin II, the sarcomeric template protein, titin, and the cardiac modulatory protein, myosin binding protein C (MyBP-C). Inherited hypertrophic cardiomyopathy (HCM) is a disease caused mainly by mutations in these proteins. The structure of cardiac myosin filaments and the alterations caused by HCM mutations are unknown. We have used electron microscopy and image analysis to determine the three-dimensional structure of myosin filaments from wild-type mouse cardiac muscle and from a MyBP-C knockout model for HCM. Three-dimensional reconstruction of the wild-type filament reveals the conformation of the myosin heads and the organization of titin and MyBP-C at 4 nm resolution. Myosin heads appear to interact with each other intramolecularly, as in off-state smooth muscle myosin [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:43614366], suggesting that all relaxed muscle myosin IIs may adopt this conformation. Titin domains run in an elongated strand along the filament surface, where they appear to interact with part of MyBP-C and with the myosin backbone. In the knockout filament, some of the myosin head interactions are disrupted, suggesting that MyBP-C is important for normal relaxation of the filament. These observations provide key insights into the role of the myosin filament in cardiac contraction, assembly, and disease. The techniques we have developed should be useful in studying the structural basis of other myosin-related HCM diseases. 10.1073/pnas.0708912105

Transitive homology-guided structural studies lead to discovery of Cro proteins with 40% sequence identity but different folds

Christian Roessler — 2008-02-22T09:00:55-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 7. (19 February 2008), pp. 2343-2348.

Proteins that share common ancestry may differ in structure and function because of divergent evolution of their amino acid sequences. For a typical diverse protein superfamily, the properties of a few scattered members are known from experiment. A satisfying picture of functional and structural evolution in relation to sequence changes, however, may require characterization of a larger, well chosen subset. Here, we employ a "stepping-stone" method, based on transitive homology, to target sequences intermediate between two related proteins with known divergent properties. We apply the approach to the question of how new protein folds can evolve from preexisting folds and, in particular, to an evolutionary change in secondary structure and oligomeric state in the Cro family of bacteriophage transcription factors, initially identified by sequence-structure comparison of distant homologs from phages P22 and lambda. We report crystal structures of two Cro proteins, Xfaso 1 and Pfl 6, with sequences intermediate between those of P22 and lambda. The domains show 40% sequence identity but differ by switching of alpha-helix to -sheet in a C-terminal region spanning approx25 residues. Sedimentation analysis also suggests a correlation between helix-to-sheet conversion and strengthened dimerization. 10.1073/pnas.0711589105

The X-ray crystal structure of RNA polymerase from Archaea

Akira Hirata — 2008-01-31T11:58:31-00:00

Nature (30 January 2008)

Chaos in a long-term experiment with a plankton community

Elisa Beninca — 2008-02-13T20:43:08-00:00

Nature, Vol. 451, No. 7180. (14 February 2008), pp. 822-825.