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The side-chain dynamics of methyl groups in two structurally related proteins from the fibronectin type III (fnIII) superfamily, the third fnIII domain from human tenascin (TNfn3) and the tenth fnIII domain from human fibronectin (FNfn10), have been studied by NMR spectroscopy. Side-chain order parameters reveal that the hydrophobic cores of the two proteins have substantially different mobilities. The core of TNfn3 is very dynamic, with exceptionally low order parameters for the most deeply buried residues, while that of FNfn10 is more like those of other proteins which have been studied with this technique, having a relatively rigid core with uniformly distributed dynamics. The unusually dynamic core of TNfn3 appears to be related to its amino acid composition, which makes it more fluid-like. A further explanation for the mobility of the TNfn3 core may be found in the negative correlation between the order parameter and excess packing volume, which shows that the core of TNfn3 is less densely packed and consequently has lower methyl order parameters for its buried residues. Rotameric transitions, presumably facilitated by the lower packing density, appear to make an important contribution to lowering the order parameters, and have been probed by measuring three-bond scalar couplings. Overall, although backbone dynamics is generally similar for proteins with the same topology on a fast time scale (picoseconds to nanoseconds), this study shows that a single fold can accommodate a wide variation in the dynamic properties of its core.

Titin I27 shows a high resistance to unfolding when subject to external force. To investigate the molecular basis of this mechanical stability, protein engineering Phi-value analysis has been combined with atomic force microscopy to investigate the structure of the barrier to forced unfolding. The results indicate that the transition state for forced unfolding is significantly structured, since highly destabilising mutations in the core do not affect the force required to unfold the protein. As has been shown before, mechanical strength lies in the region of the A' and G-strands but, contrary to previous suggestions, the results indicate clearly that side-chain interactions play a significant role in maintaining mechanical stability. Since Phi-values calculated from molecular dynamics simulations are the same as those determined experimentally, we can, with confidence, use the molecular dynamics simulations to analyse the structure of the transition state in detail, and are able to show loss of interactions between the A' and G-strands with associated A-B and E-F loops in the transition state. The key event is not a simple case of loss of hydrogen bonding interactions between the A' and G-strands alone. Comparison with Phi-values from traditional folding studies shows differences between the force and "no-force" transition states but, nevertheless, the region important for kinetic stability is the same in both cases. This explains the correspondence between hierarchy of kinetic stability (measured in stopped-flow denaturant studies) and mechanical strength in these titin domains.

Theoretical studies of protein folding suggest that multiple folding pathways should exist, but there is little experimental evidence to support this. Here we demonstrate changes in the flux between different transition states on parallel folding pathways, resulting in unprecedented upward curvature in the denaturant-dependent unfolding kinetics of a beta-sandwich protein. As denaturant concentration increases, the highly compact transition state of one pathway becomes destabilized and the dominant flux of protein molecules shifts toward another pathway with a less structured transition state. Furthermore, point mutations alter the relative accessibility of the pathways, allowing the structure of two transition states on separate, direct folding pathways to be mapped by systematic Phi-value analysis. It has been suggested that pathways with diffuse rather than localized transition states are evolutionarily selected to prevent misfolding, and indeed we find that the transition state favored at high concentrations of denaturant is more polarized than the physiologically relevant one.

The advent of a new class of force microscopes designed specifically to “pull” biomolecules has allowed non-specialists to use force microscopy as a tool to study single-molecule protein unfolding. This powerful new technique has the potential to explore regions of the protein energy landscape that are not accessible in conventional bulk studies. It has the added advantage of allowing direct comparison with single-molecule simulation experiments. However, as with any new technique, there is currently no well described consensus for carrying out these experiments. Adoption of standard schemes of data selection and analysis will facilitate comparison of data from different laboratories and on different proteins. In this review, some guidelines and principles, which have been adopted by our laboratories, are suggested. The issues associated with collecting sufficient high quality data and the analysis of those data are discussed. In single-molecule studies, there is an added complication since an element of judgement has to be applied in selecting data to analyse; we propose criteria to make this process more objective. The principal sources of error are identified and standardised methods of selecting and analysing the data are proposed. The errors associated with the kinetic parameters obtained from such experiments are evaluated. The information that can be obtained from dynamic force experiments is compared, both quantitatively and qualitatively to that derived from conventional protein folding studies.

Atomic force microscopy is an exciting new single-molecule technique to add to the toolbox of protein (un)folding methods. However, detailed analysis of the unfolding of proteins on application of force has, to date, relied on protein molecular dynamics simulations or a qualitative interpretation of mutant data. Here we describe how protein engineering Φ value analysis can be adapted to characterize the transition states for mechanical unfolding of proteins. Single-molecule studies also have an advantage over bulk experiments, in that partial Φ values arising from partial structure in the transition state can be clearly distinguished from those averaged over alternate pathways. We show that unfolding rate constants derived in the standard way by using Monte Carlo simulations are not reliable because of the errors involved. However, it is possible to circumvent these problems, providing the unfolding mechanism is not changed by mutation, either by a modification of the Monte Carlo procedure or by comparing mutant and wild-type data directly. The applicability of the method is tested on simulated data sets and experimental data for mutants of titin I27.

The role of conserved proline residues in fibronectin type III (fnIII) domains is investigated. Surprisingly, none of the standard set of explanations for residue conservation applies. The proline residues are not apparently conserved for function, or stability, or to nucleate folding, or to promote stabilising interactions across domain boundaries. However, when the most highly conserved proline residues are mutated to alanine there is an increase in the rate of aggregation of a fnIII double-module construct. The results suggest that proline residues may be conserved at domain-domain boundaries in fnIII domains to prevent aggregation in multi-modular proteins.

Titin is a giant, multidomain muscle protein forming a major component of the sarcomere in vertebrate striated muscle. As for many other multidomain proteins, the properties of titin are often studied by characterisation of the constituent domains in isolation. This raises the question of to what extent the properties of the isolated domains are representative of the domains in the wild-type protein. We address this question for the I-band region of titin, which is of particular biological interest due to its role in muscle elasticity, by determining the properties of five immunoglobulin domains from the I-band in three different contexts; firstly as isolated domains with the boundaries defined conservatively, secondly, with a two amino acid extension at both the N and C terminus and thirdly as part of multidomain constructs. We show that adjacent domains in the titin I-band have very different kinetic properties which, in general, undergo only a small change in the presence of neighbouring domains and conclude that, provided that care is taken in the choice of domain boundaries, the properties of the titin I-band are essentially "the sum of its parts". From this and other work we propose that variation in kinetic properties between adjacent domains may be a general property of the I-band thereby preventing misfolding events on muscle relaxation.

Anfinsen (1961) demonstrated that the information for a protein to fold is entirely contained within its sequence. More recently, the importance of unfolded, but functional, proteins has been recognised. These so-called intrinsically disordered proteins (IDPs) lack a three-dimensional structure, but may fold upon interacting with a partner protein. Where is the folding information for these coupled folding and binding reactions contained? Is the amino acid sequence of the IDP the sole determinant of the folding reaction? Or are they dictated by the partner protein? We examine these questions by comparing and contrasting multiple interactions between IDPs and their structured partners, utilising the Bcl-2 family as a model system.

Introduction Evolution and design of protein complexes are frequently viewed through the lens of amino acid mutations at protein interfaces, but we showed previously that residues distant from interfaces are also commonly involved in the evolution of alternative quaternary structures. We hypothesized that in these protein families, the difference in oligomeric state is due to a change in intersubunit geometry. The indirect mutations would act by changing protein conformation and dynamics, similar to the way in which allosteric small molecules introduce functional conformational change. We refer to these substitutions as “allosteric mutations.” Rationale In this work, we investigate the mechanism of action of allosteric mutations on oligomeric state in the PyrR family of pyrimidine operon attenuators. In this family, an entirely sequence-conserved helix that forms a tetrameric interface in the thermophilic ortholog (BcPyrR) switches to being solvent-exposed in the mesophilic ortholog (BsPyrR). This results in a homodimeric structure in which the two subunits are clearly rotated relative to their orientation in the tetramer. What is the origin of this rotation and the change in quaternary structure? To dissect the role of the 49 substitutions between BsPyrR and BcPyrR, we used ancestral sequence reconstruction in combination with structural and biophysical methods to identify a set of allosteric mutations that are responsible for this shift in conformation. We compared the conformational changes introduced by the mutations to the protein motion during allosteric regulation by guanosine monophosphate (GMP). Results We identified 11 key mutations controlling oligomeric state, all distant from the interfaces and outside ligand-binding pockets. We confirmed the role of these allosteric mutations by engineering a shift in oligomeric state in an inferred ancestral PyrR protein (intermediate in sequence between the extant orthologs). We further used the inferred ancestral states and their mutants to show that the allosteric mutations are part of a downhill adaptation of the PyrR proteins to lower temperatures. We compared the x-ray crystal structures of ancestral and engineered PyrR proteins to the free and GMP-bound structure of the mesophilic BsPyrR, which shifts its equilibrium from dimer to tetramer upon ligand binding. Binding of the allosteric molecule introduces a change in intersubunit geometry that is equivalent to the evolutionary difference in intersubunit geometry between the dimeric and tetrameric homologs. We further find that the difference in oligomeric state is coupled to the difference in intrinsic dynamics of the dimers. Finally, we used the residue-residue contact network approach to show that the residues corresponding to the allosteric mutations undergo large contact rewiring when the intersubunit geometry and, in turn, oligomeric state change, either by GMP binding or by the introduction of allosteric mutations. Conclusion We show that evolution employs the intrinsic dynamics of this protein to toggle a conformational switch in a manner similar to that of small molecules. Shifting the relative populations of different states by subtle modifications is a process central to protein function and, as shown here, also to protein evolution. This suggests that we can learn from evolution and design proteins with multiple conformational states.

BACKGROUND: Robotic surgery is the latest innovation in the field of minimally invasive surgery. In the case of robotic surgery, instead of directly moving the instruments the surgeon uses a robotic system to control the instruments for surgical procedures. Robotic surgical systems have been used in various gynaecological surgeries for benign disease, such as hysterectomy (removal of the uterus), myomectomy (removal of uterine leiomyomas) and tubal reanastomosis (the reuniting of a divided tube). The mounting evidence demonstrates the feasibility and safety of robotic surgery in benign gynaecological disease. Robotic surgery is advertised as having promising advantages including more precise vision and procedures, improved ergonomics and shorter length of hospital stay. However, the main disadvantages of the robotic surgical system should not be overlooked, including the high cost of disposable instruments and retraining for both surgeons and nurses. OBJECTIVES: To assess the effectiveness and safety of robot‐assisted surgery in the treatment of benign gynaecological disease. SEARCH METHODS: We searched the Cochrane Menstrual Disorders and Subfertility Group's Trial Register, the Cochrane Central Register of Controlled Trials (CENTRAL) (Issue 2, 2011), MEDLINE and EMBASE up to November 2011 and citation lists of relevant publications. SELECTION CRITERIA: All randomised controlled trials (RCTs) comparing robotic surgery for benign gynaecological disease to laparoscopic or open surgical procedures. RCTs comparing different types of robotic assistants were also included. We contacted study authors for unpublished information, but failed in obtaining a response. DATA COLLECTION AND ANALYSIS: Two review authors independently screened studies for inclusion. The domains assessed for risk of bias were allocation concealment, blinding, incomplete outcome data and selective outcome reporting. Odds ratios (OR) were used for reporting dichotomous data with 95% confidence intervals (CI), whilst mean differences (MD) were determined for continuous data. Statistical heterogeneity was assessed using the I(2) statistic. We contacted the primary authors for missing data but failed in obtaining a response. MAIN RESULTS: Two trials involving 158 participants were included. Since one included trial was published in conference proceedings, limited usable data were available for further analysis. The only analysis in this trial showed comparable rates of conversions to open surgery between the robotic group and the laparoscopic group (OR 1.41, 95% CI 0.22 to 9.01; P = 0.72). One RCT showed longer operation time (MD 66.00, 95% CI 40.93 to 91.07; P < 0.00001), higher cost (MD 1936.00, 95% CI 445.69 to 3426.31; P = 0.01) in the robotic group compared with the laparoscopic group. Also, both studies reported that robotic and laparoscopic surgery seemed comparable regarding intraoperative outcome, complications, length of hospital stay and quality of life. AUTHORS' CONCLUSIONS: Currently, the limited evidence showed that robotic surgery did not benefit women with benign gynaecological disease in effectiveness or in safety. Further well‐designed RCTs with complete reported data are required to confirm or refute this conclusion.

Protein–protein interactions are at the heart of regulatory and signaling processes in the cell. In many interactions, one or both proteins are disordered before association. However, this disorder in the unbound state does not prevent many of these proteins folding to a well-defined, ordered structure in the bound state. Here we examine a typical system, where a small disordered protein (PUMA, p53 upregulated modulator of apoptosis) folds to an α-helix when bound to a groove on the surface of a folded protein (MCL-1, induced myeloid leukemia cell differentiation protein). We follow the association of these proteins using rapid-mixing stopped flow, and examine how the kinetic behavior is perturbed by denaturant and carefully chosen mutations. We demonstrate the utility of methods developed for the study of monomeric protein folding, including β-Tanford values, Leffler α, Φ-value analysis, and coarse-grained simulations, and propose a self-consistent mechanism for binding. Folding of the disordered protein before binding does not appear to be required and few, if any, specific interactions are required to commit to association. The majority of PUMA folding occurs after the transition state, in the presence of MCL-1. We also examine the role of the side chains of folded MCL-1 that make up the binding groove and find that many favor equilibrium binding but, surprisingly, inhibit the association process.

Blends of a highly epoxidized natural rubber (ENR50) with unmodified natural rubber (NR) and ethylene propylene elastomers (EPDM) were produced to evaluate the mixing and curing characteristics. Dodecyl succinic anhydride was used to cross-link the ENR50 component and the reactivity was assessed by monitoring the evolution of the torque in an oscillatory co-axial cylinder rheometer, as well as by DSC thermal analysis. A physical model was used to obtain a single parameter for the reactivity of the system, which corresponds to the rate constant for first order curing reactions. Although the blends were thermodynamically immiscible, displaying no significant change in Tg, the components were well dispersed at microscopic level. Better mechanical properties were obtained for blends with EPDM.

The kinase-inducible domain interacting (KIX) domain of CREB binding protein binds to multiple intrinsically disordered transcription factors in vivo at two distinct sites on its surface. Several reports have been made of allosteric communication between these two sites in this well-characterized model system. In this work, we have performed fluorescence stopped-flow measurements to investigate the kinetics of binding of five KIX binding proteins. We find that they all have similar association and dissociation rate constants for complex formation, despite their wide range of intrinsic helical propensities. Furthermore, by careful arrangement of pseudofirst-order conditions, we have been able to show that both association and dissociation rate constants are decreased when a partner is bound at the alternative site. These decreases suggest that positive allosteric effects are not mediated by structural changes in binding sites but rather, through a more general mechanism, largely mediated through dissociation, which we propose is largely related to changes in the flexibility of the KIX domain itself.

It is thankfully rare for extenuating circumstances to fully test the processes and procedures enshrined in national and world antidoping authorities’ rules and laws. It is also thankfully very rare that a failed drugs test can have some positive implications. Antidoping laws are undoubtedly focused on ensuring fair competition, however, there are occasions when honest athletes discover medical diagnoses through failed antidoping tests. The purpose of this paper is to broadly discuss antidoping considerations encountered, based on the four principles of medical ethics and to propose simple solutions to these problems. Unfortunately, extreme medical circumstances will often test the limits of antidoping and medical processes and with open channels for feedback, these systems can improve. Performance enhancement seems an illogical concept if an athlete’s medical treatment and disease are more inherently performance harming than unintended potential doping, but needs to be carefully managed to maintain fair sport.

Many cellular proteins are ‘disordered’ in isolation. A subset of these intrinsically disordered proteins (IDPs) can, upon binding another molecule, fold to a well-defined three-dimensional structure. In the structurally heterogeneous, unbound ensemble of these IDPs, conformations are likely to exist that, in part, resemble the final bound form. It has been suggested that these conformations, displaying ‘residual structure’, could be important for the mechanism of such coupled folding and binding reactions. PUMA, of the BCL-2 family, is an IDP in isolation but will form a single, contiguous α-helix upon binding the folded protein MCL-1. Using the helix-breaking residue proline, we systematically target each potential turn of helix of unbound PUMA and assess the binding to MCL-1 using time-resolved stopped-flow techniques. All proline-containing mutants bound, and although binding was weaker than the wild-type protein, association rate constants were largely unaffected. We conclude that population of particular residual structure, containing a specific helical turn, is neither required for the binding nor for fast association of PUMA and MCL-1.

IDPs are overrepresented in processes such as signalling and transcription, where proteins often interact with a range of partners. One much-studied key hub protein is the coactivator CBP/p300, whose folded KIX domain binds to a number of different intrinsically disordered transcription factors. The interaction of KIX with several of its ligands has been well studied by equilibrium methods, and structural information is available for many of the complexes. By careful control and consideration of experimental conditions such as temperature and ionic strength we have been able to perform kinetic studies that reveal the mechanism of the association reaction of KIX with cMyb; the fastest protein-protein interaction yet reported. Furthermore, through comparative studies with several binding partners we shed light on an important outstanding question in the IDP field: what is the advantage of disorder to a protein?View Large Image | View Hi-Res Image | Download PowerPoint Slide

Oligomerisation is essential for the function of some proteins. The PyrR family of proteins are involved in pyrimidine operon attenuation. They are regulated by the presence of certain nucleotides such as guanosine monophosphate (GMP), which stabilises the tetrameric state. Notably, some members of this family can adopt a tetrameric oligomerisation state regardless of the presence of the GMP. Perica et al. (2012) previously found that this family, among several others, have differences in sequence outside the oligomeric interfaces and these are sufficient to explain the changes to their subunit geometry and oligomerisation states. Here, we compared the differences in structural flexibility linked to the changes in oligomeric state caused by a) specific mutations and, b) by the presence of bound GMP. We calculated the coarse-grained normal modes of dimeric units of the PyrR dimers and tetramers using an Elastic Network Model implemented in the Molecular Modelling ToolKit. We conducted several analyses including comparing the normal modes of these proteins with each other using the Bhattacharyya Coefficient similarity measure, correlations matrices, and the conformational overlap analysis, by which the contribution of these modes to the transition from one state to another can be quantified. Firstly, we found that while the dynamics were very similar between all the structures, there was a noticeable difference between the dimeric and tetrameric units. We also show that both sets of proteins transition from tetrameric to dimeric states similarly, indicating some overlap between the effects of allostery and evolution on oligomerisation in PyrR.

Force spectroscopy has emerged as a new tool to study protein folding, in which force replaces the chemical denaturant used in traditional folding experiments. This new technique complements older methods and allows a range of new questions to be investigated. What sort of protein is able to resist mechanical unfolding, and to what extent is mechanical stability dictated by fold or function? What is the effect of force on the unfolding energy surface? Do proteins unfold by the same pathway in mechanical and chemical denaturation experiments? Answers to these are starting to emerge based on a combination of experimental and computational approaches. We present some of the forced unfolding experiments to date and simple methods for characterizing the unfolding potential from the results. Several studies have also begun a more fine-grained description of mechanical unfolding, for example by invoking intermediates to explain features seen in unfolding traces and by using mutagenesis to try to localize the origin of mechanical stability. We propose further experimental approaches to this goal using the protein engineering method to characterize transition states, similar to those used in conventional folding experiments. However, it is likely that a high-resolution picture of mechanical unfolding will only emerge through a combined interpretation of careful experimental work and computer simulation.

The potential advantages of introducing anisotropic properties into a rubber compound by replacing some particulate filler with short fibre have been investigated. The control compound (containing no fibre) was based on NR and contained 60 pphr (N330) carbon black. In the fibre filled compound, 15 pphr of the carbon black was replaced with an equivalent volume of Santoweb fibre (10 pphr). Specimens with strong fibre orientation were produced and tensile, tear, compression, and shear properties measured at different angles to the orientation. Stretching parallel to fibre orientation resulted in a stiffness at low elongations × 3·5 higher than in the control compound. However, the tensile and tear strengths were lower than the control compound, probably owing to the high stresses at the fibre surfaces leading to fibre pullout. Maximum tensile and tear strengths were obtained when fibre orientation was at angles of 20° and 45°, respectively, to the stretching direction. Stiffness at a small (5%) comp...

Background: Do proteins that have the same structure fold by the same pathway even when they are unrelated in sequence? To address this question, we are comparing the folding of a number of different immunoglobulin-like proteins. Here, we present a detailed protein engineering φ value analysis of the folding pathway of TI I27, an immunoglobulin domain from human cardiac titin. Results: TI I27 folds rapidly via a kinetic intermediate that is destabilized by most mutations. The transition state for folding is remarkably native-like in terms of solvent accessibility. We use φ value analysis to map this transition state and show that it is highly structured; only a few residues close to the N-terminal region of the protein remain completely unfolded. Interestingly, most mutations cause the transition state to become less native-like. This anti-Hammond behavior can be used as a novel means of obtaining additional structural information about the transition state. Conclusions: The residues that are involved in nucleating the folding of TI I27 are structurally equivalent to the residues that form the folding nucleus in an evolutionary unrelated fibronectin type III protein. These residues form part of the common structural core of Ig-like domains. The data support the hypothesis that interactions essential for defining the structure of these β sandwich proteins are also important in nucleation of folding.

Much published literature on the way in which phase morphology and filler distribution affect blend properties is contradictory or confusing. Experiments were carried out to elucidate the relationships and to determine whether the use of compatibilizers or special mixing techniques might have a beneficial effect on natural rubber:butadiene rubber (NR:BR) blend properties. NR:BR blends were prepared using both a masterbatch method and a single-stage mixing method. A cure system which gave an even distribution of crosslinks between the phases was used. The morphology, tensile strength and tear strength properties of the blends were measured. Results indicated a high degree of compatibility with fine textured blends (domain sizes < 1µm) being quickly and easily produced, even from masterbatches of very different viscosities. Strength properties of these fine textured masterbatch blends could be predicted by applying the simple rule of mixtures to properties of individual compounds mixed under the same conditions. Although in particular situations a coarse morphology could result in high tear resistance values, for most applications a fine textured morphology gives the most satisfactory overall tensile and tear strength properties. For blends mixed in a single-stage process, development of a fine textured morphology was much quicker than that of filler dispersion. For NR:BR blends containing an optimum cure system it was concluded that the mixing cycle should be chosen to optimize filler dispersion and that use of a compatibilizer will not significantly shorten the mixing cycle or improve the properties of the blend.

To identify the contacts that stabilise the rate-limiting transition state for folding of FNfn10 (the tenth fnIII domain of human fibronectin), 42 mutants have been analysed at 29 positions across this domain. An anomalous response to mutation means that structure formation in the A, B and G strands cannot be evaluated by this method. In all the residues analysed, phi-values are fractional and no completely structured region is observed. The analysis reveals that hydrophobic residues from the central strands of the beta-sandwich form a large core of interactions in the transition state. Brønsted analysis shows that the stabilisation energy from the amino acid side-chains in the transition state is approximately 40 % of that in the native state. The protein folds by a nucleation-condensation mechanism, and tertiary interactions within the core make up the folding nucleus. Local interactions, in turns and loops, are apparently much less significant. Comparison with an homologous domain from human tenascin (TNfn3), shows that FNfn10 has a more extended, structured transition state spanning three different "layers" of the beta-sandwich. The results support the hypothesis that interactions in the common structural core guide the folding of these domains.

It is proposed that a non-polar filler can reduce interfacial energies between polar and non-polar polymers. Experiments have been carried out to test this hypothesis using carbon black as the filler in blends of natural rubber (NR) and a nitrile rubber (NBR) with an acrylonitrile content of 45%. Blends of NR–NBR (70/30) were prepared in an internal mixer with varying amounts of carbon black. The dramatic decrease in domain size on addition of carbon black was nonetheless lower than that predicted. Further experiments showed that the amount of carbon black available at the interface for compatibilisation was influenced by preferential incorporation into the lower viscosity elastomer (NBR). Thus, elastomers of similar viscosity should be added to the mixer prior to the carbon black in order to maximise the amount of ‘free’ unwetted carbon black present when the elastomers are blended together. Blending experiments carried out under these conditions resulted in a morphology close to the prediction b...

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering.

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended beta-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.

What are the selective pressures on protein sequences during evolution? Amino acid residues may be highly conserved for functional or structural (stability) reasons. Theoretical studies have proposed that residues involved in the folding nucleus may also be highly conserved. To test this we are using an experimental "fold approach" to the study of protein folding. This compares the folding and stability of a number of proteins that share the same fold, but have no common amino acid sequence or biological activity. The fold selected for this study is the immunoglobulin-like beta-sandwich fold, which is a fold that has no specifically conserved function. Four model proteins are used from two distinct superfamilies that share the immunoglobulin-like fold, the fibronectin type III and immunoglobulin superfamilies. Here, the fold approach and protein engineering are used to question the role of a highly conserved tyrosine in the "tyrosine corner" motif that is found ubiquitously and exclusively in Greek key proteins. In the four model beta-sandwich proteins characterised here, the tyrosine is the only residue that is absolutely conserved at equivalent sites. By mutating this position to phenylalanine, we show that the tyrosine hydroxyl is not required to nucleate folding in the immunoglobulin superfamily, whereas it is involved to some extent in early structure formation in the fibronectin type III superfamily. The tyrosine corner is important for stability, mutation to phenylalanine costs between 1.5 and 3 kcal mol(-1). We propose that the high level of conservation of the tyrosine is related to the structural restraints of the loop connecting the beta-sheets, representing an evolutionary "cul-de-sac".

With the ultimate aim of improving mixer geometry by using a mathematical model for rubber mixing, this paper concentrates on the dispersive mixing process of filler disagglomeration. Specially designed elongational flow experiments were used to achieve dispersive mixing under conditions of known stress and strain rate history. It was found that the disagglomeration process is satisfactorally described by a first order differential equation with the rate constant proportional to the power density experienced by the compound during mixing. The kinetic model was implemented in original finite element software to obtain a 2-dimensional simulation of mixing in a twin rotor internal mixer. It was concluded that the 2-dimensional simulation was not only a necessary precursor to 3-dimensional modeling, but would in itself be useful for relating cross-sectional rotor geometry to efficiency of filler disagglomeration, and hence, dispersive mixing.

There has been a large body of work on the deposition of colloidal particles onto many different kinds and shapes of surfaces. Nearly all work in the past has involved flowing systems in which a well-defined hydrodynamic layer exists at the collector surface with certain advantages and disadvantages. We have chosen a still system in which transport of particles to the collector surface occurs solely by diffusion. Both particles and surfaces were negatively charged, so that deposition was driven by dispersion forces. A photomicroscopic technique has been used to determine absolute values of initial rates of deposition. Three monodisperse polystyrene colloids of various particle sizes and surface charge densities were synthesized. Their zeta potentials were determined by microelectrophoresis in Ba(NO 3 ) 2 solutions. Collector surfaces were glass and glass coated with polystyrene. Their zeta potentials were determined from their streaming potentials in various concentrations of electrolyte. Direct determination of diffusion coefficients indicate that the particles behave normally. Initial deposition rates exhibit strong dependence on ionic strength, indicating large electrical double layer effects. Like the work of others, rates of deposition fell off with time, perhaps due to the presence of `hot spots', i.e. highly localized areas of greater reactivity. The most regular behavior occurred in deposition on polystyrene-coated glass, presumably because roughness and highly reactive sites on the glass surface were largely eliminated. Absolute rates were several times theoretical for fastest deposition. It is speculated that the hydrophobic effect plays a significant role in accelerating particles towards the collector. Plots of log (initial rate constant) vs. log (ionic strength) were linear and generally in the order anticipated by Reerink–Overbeek theory.

The third fibronectin type III domain from human tenascin adopts a compact beta-sandwich fold. Its boundaries were originally selected to encode a 90-residue domain (TNfn31-90). We conclude that the dynamic properties of TNfn3 are more accurately represented when the C terminus is extended by the two naturally succeeding residues. Longitudinal (R1) and transverse (R2) 15N relaxation rates, and ¿1H-15N¿ NOE enhancements at pH 4.9 and 300 K are presented for TNfn31-90 and TNfn31-92, the extended form, at two field strengths (11.74 and 14.10 T). Nearly identical results confirm their similar motional properties over a broad range of timescales. However, a number of residues near the C terminus in TNfn31-90 exhibit elevated transverse relaxation rates and broadened signals in 1H-15N HSQC spectra. Explicit rates of chemical exchange for five residues in TNfn31-90 were determined by measuring transverse relaxation rates in a series of CPMG experiments with spin-echo refocusing delays increasing from 311 to 1436 micros. Calculated exchange rates average 1000(+/-311) s-1, with individual uncertainties near 20%. Homonuclear TOCSY experiments collected between pH 4 and 7 reveal the coincident titration of two acidic clusters in TNfn31-90 at pH 5. 64(+/-0.47). The repulsive electrostatic interaction of the C-terminal carboxylate with one of these clusters may promote chemical exchange in the shorter domain. Additionally, NOE and chemical shift data suggest hydrogen bond formation between the added residues and adjacent loops. The data affirm the importance of judiciously selecting domain boundaries prior to the characterization of molecular properties.

The structural and dynamic properties of an immunoglobulin superfamily domain (IgSF), Ig 18′, have been characterised by NMR at 285 K, in the presence of 4.2 M and 6.0 M urea, respectively. Analysis of chemical shift deviations, 3 J HNHα coupling constants, sequential NOE pattern, and 15 N relaxation data reveals that although the two urea-denatured states are highly disordered, some local turn-like residual structures do exist. Moreover, some distinct differences between the properties of the two denatured states are observed. In 4.2 M urea-denatured Ig 18′, regions 80–83 and 86–92 adopt turn-like conformations, furthermore, region 84–93 is involved in slow exchange processes that occur on a micro- to millisecond time-scale. In the 6.0 M urea-denatured state, these turn-like conformations are less occupied, and chemical exchange processes in region 84–93 are largely reduced. In contrast, region 32–36 has persistent turn-like structures in both urea-denatured states. Some correlation between the spectral density function at 0 frequency, J eff (0), for the urea-denatured states and the secondary structure elements of the folded state have been observed. Except for the terminal regions, residues corresponding to β-strands have higher J eff (0) values compared to residues corresponding to loops. The characterisation and comparison of the two urea-denatured states highlight residues that possess properties that may be crucial for the initiation of folding of this domain.

There is an unanswered question from previous studies of 1H/2H-exchange of amide protons of barnase. Under certain conditions, there is a relatively abrupt change from EX2 towards EX1 kinetics as the temperature is slightly increased. The change in kinetics for different mutants is not directly related to their changes in stability. We have measured the stability of the folding intermediate of barnase (I) in 2H2O under a variety of conditions and calculated its population at different temperatures. The change in kinetics correlates with the change in the population of the folding intermediate. At higher temperatures and pH, the free energy of I becomes higher than that of the denatured state, D, and the kinetics becomes EX1. The data fit a simple kinetic scheme. Such changes in kinetics may be used to detect the presence of intermediates in the folding reaction at equilibrium in native conditions, but cannot distinguish whether they are on or off-pathway.

Amide hydrogen-deuterium exchange is a sensitive probe of the structure, stability and dynamics of proteins. The significant increase in the number of small, model proteins that have been studied has allowed a better understanding of the structural fluctuations that lead to hydrogen exchange. Recent technical advances enable the methodology to be applied to the study of protein—protein interactions in much larger, more complex systems.

Residual structure in the denatured state of a protein may contain clues about the early events in folding. We have simulated by molecular dynamics the denatured state of barnase, which has been studied by NMR spectroscopy. An ensemble of 10 4 structures was generated after 2 ns of unfolding and following for a further 2 ns. The ensemble was heterogeneous, but there was nonrandom, residual structure with persistent interactions. Helical structure in the C-terminal portion of helix α1 (residues 13–17) and in helix α2 as well as a turn and nonnative hydrophobic clustering between β3 and β4 were observed, consistent with NMR data. In addition, there were tertiary contacts between residues in α1 and the C-terminal portion of the β-sheet. The simulated structures allow the rudimentary NMR data to be fleshed out. The consistency between simulation and experiment inspires confidence in the methods. A description of the folding pathway of barnase from the denatured to the native state can be constructed by combining the simulation with experimental data from φ value analysis and NMR.

A bell-mouthed die geometry was designed to cause convergent flow at a constant, uniform, elongational strain rate. An equation was derived, which showed that steady-state elongational viscosity could be calculated from a plot of pressure drop due to elongation against a simple function of die length. To obtain values of pressure drop due to elongation, it was necessary to correct the total pressure drop measured across the bell-mouthed dies for the contribution from shear occurring near the die wall. For this purpose, a simplified shape for the bell-mouthed dies was assumed, comprising several parallel sided segments. Applying a formula to pressure drop data measured across straight dies corresponding to these segments gave an estimate of the pressure drop due to shear across the bell-mouthed dies. Pressure drops due to elongation were determined by subtracting the pressure drop due to shear from the total pressure drop measured across the bell-mouthed dies. Measurements were also carried out with lubrication to validate the shear correction method. The results indicate that for the compound used in this study, a combination of bell-mouthed and straightsided dies can be used in a conventional capillary rheometer to determine steady-state elongational viscosity. An elongational viscosity of 190 kPa s at 90°C and at a strain rate of 10 s−1 was determined for a simple styrene-butadiene rubber compound. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 66: 1139–1150, 1997

This article explores the problematic of negotiating academic genre in the context of higher education (HE) and the implications for students’ writing. The growth of multi‐disciplinary, modular programmes and a more heterogeneous student population requires that we consider the challenges faced by undergraduates, both within and between disciplines, and suggest ways in which navigational difficulties can be reduced. Our research is informed by our own multi‐disciplinarity which draws on the substantive areas of education, applied linguistics and sociology. Using case studies of students’ writing we have analysed the ways in which undergraduates negotiate two disciplines concurrently. Our findings suggest that students’ knowledge of one discipline influences another, rather in the same way that a first language affects second language acquisition. The resulting work may fail to hit the target discourse or may recontextualize material in creative but discipline‐challenging ways. Our explorations be...

Hydrogen exchange is an attractive method for observing small populations of partly unfolded states of proteins at equilibrium. It has been suggested that these represent folding intermediates so that hydrogen exchange can offer a short cut for studying protein-folding pathways. This cannot work in theory because it is not possible to tell whether they are intermediates or side reactions. Experimental studies of barnase and chymotrypsin inhibitor 2 show that there is no obvious relationship between hydrogen exchange at equilibrium and their folding pathways.

Interdomain interactions of spectrin are critical for maintenance of the erythrocyte cytoskeleton. In particular, “head-to-head” dimerization occurs when the intrinsically disordered C-terminal tail of β-spectrin binds the N-terminal tail of α-spectrin, folding to form the “spectrin tetramer domain”. This non-covalent three-helix bundle domain is homologous in structure and sequence to previously studied spectrin domains. We find that this tetramer domain is surprisingly kinetically stable. Using a protein engineering Φ-value analysis to probe the mechanism of formation of this tetramer domain, we infer that the domain folds by the docking of the intrinsically disordered β-spectrin tail onto the more structured α-spectrin tail.

Coupled folding and binding of intrinsically disordered proteins (IDPs) is prevalent in biology. As the first step toward understanding the mechanism of binding, it is important to know if a reaction is 'diffusion-limited' as, if this speed limit is reached, the association must proceed through an induced fit mechanism. Here, we use a model system where the 'BH3 region' of PUMA, an IDP, forms a single, contiguous α-helix upon binding the folded protein Mcl-1. Using stopped-flow techniques, we systematically compare the rate constant for association (k(+)) under a number of solvent conditions and temperatures. We show that our system is not 'diffusion-limited', despite having a k(+) in the often-quoted 'diffusion-limited' regime (10(5)-10(6) M(-1) s(-1) at high ionic strength) and displaying an inverse dependence on solvent viscosity. These standard tests, developed for folded protein-protein interactions, are not appropriate for reactions where one protein is disordered.

Backgound: Methods have been developed recently for probing local fluctuations of protein structure using H/ 2 H-exchange of amide protons at equilibrium. It has been suggested that equilibrium exchange methods can identify the order of events in folding pathways and detect folding cores. We have applied the procedure of measuring the effects of denaturant on the H/ 2 H-exchange of amide protons of barnase, the folding pathway of which is well established. Results The addition of relatively low concentrations of denaturant causes the mechanism of exchange of amide protons of barnase to change from EX2 to EX1 for the residues that require global unfolding for exchange to occur. This change of mechanism, which would have been missed by some of the standard tests, causes artefacts that could be easily misinterpreted. We also present the thermodynamic argument that measurements at equilibrium cannot give the order of events in folding. Conclusion Measurement of H/ 2 H-exchange of amide protons at equilibrium, when applied correctly, is an excellent method for analyzing the equilibrium distribution of unfolded and partly folded states. It cannot, in theory and in practice, be used for determining protein folding pathways by itself.

In this series of papers, we examine the effects of introducing disulfide bond on the properties, structure and thermodynamics of a small globular protein, barnase. Three mutants have been made, in each of which a single crosslink confers different properties. Two of the disulfide bonds, between residues 43 and 80 (43–80) and between residues 85 and 102 (85–102), stabilise the protein, relative to both wild-type and the corresponding (reduced) dithiol forms: 85–102 is more stable than predicted from the entropic destabilisation of the unfolded state; 43–80 is less stable than predicted. The third disulfide bond, between residues 70 and 92 (70–92) destabilises the protein relative to both wild-type and the corresponding dithiol form, implying significant disruption of the folded protein on formation of the disulfide bond. Crystal structures of three mutant proteins have been solved. All three proteins have essentially the same fold as wild-type, but with left-handed disulfide bonds, which have dihedral geometries that have not been observed in naturally occurring disulfides. In the very stable mutant 85–102, there is no significant difference between the mutant and wild-type structures: these data do not explain the large stability of this protein. The disulfide bond at 43–80 induces small structural rearrangements close to the site of the disulfide bond, associated with some local disorder: the crosslink appears to decrease the stability of the native form of the protein. The destabilizing disulfide bond at 70–92 induces considerable structural change, with displacement of a loop and consequent disruption of a stabilizing salt-bridge. Our studies do not support the view that the conformation of the disulfide bond is crucial in determining the stability of the mutant proteins.

Disorder in protein ranges from regions of the chain that are highly dynamic through to entirely unstructured regions or entire domains of larger proteins. This disorder can play a functional role, promoting cooperativity and imparting both structural integrity and allosteric coupling. I will describe some of our current studies of very different protein folding systems.

Highlights ► We review the recent advances made from the study of related proteins. ► We relate pathway malleability to the balance between foldons and helical propensity. ► We speculate why different topologies respond differently to mutation. ► We discuss the role of kinetic intermediates in folding pathways. ► We explain why it is important to study several members from each protein fold., The ‘Fold Approach’ involves a detailed analysis of the folding of several topologically, structurally and/or evolutionarily related proteins. Such studies can reveal determinants of the folding mechanism beyond the gross topology, and can dissect the residues required for folding from those required for stability or function. While this approach has not yet matured to the point where we can predict the native conformation of any polypeptide chain in silico, it has been able to highlight, amongst others, the specific residues that are responsible for nucleation, pathway malleability, kinetic intermediates, chain knotting, internal friction and Paracelsus switches. Some of the most interesting discoveries have resulted from the attempt to explain differences between homologues.

Staphylococcus aureus and Staphylococcus epidermidis form communities (called biofilms) on inserted medical devices, leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. As biofilms are resistant to antibiotics, device removal is often required to resolve the infection. Thus, there is a need for new therapeutic strategies and molecular data that might assist their development. Surface proteins S. aureus surface protein G (SasG) and accumulation-associated protein ( S. epidermidis ) promote biofilm formation through their “B” regions. B regions contain tandemly arrayed G5 domains interspersed with approximately 50 residue sequences (herein called E) and have been proposed to mediate intercellular accumulation through Zn 2+ -mediated homodimerization. Although E regions are predicted to be unstructured, SasG and accumulation-associated protein form extended fibrils on the bacterial surface. Here we report structures of E–G5 and G5–E–G5 from SasG and biophysical characteristics of single and multidomain fragments. E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. E and G5 domains lack a compact hydrophobic core, and yet G5 domain and multidomain constructs have thermodynamic stabilities only slightly lower than globular proteins of similar size. Zn 2+ does not cause SasG domains to form dimers. The work reveals a paradigm for formation of fibrils on the 100-nm scale and suggests that biofilm accumulation occurs through a mechanism distinct from the “zinc zipper.” Finally, formation of two domains by each repeat (as in SasG) might reduce misfolding in proteins when the tandem arrangement of highly similar sequences is advantageous.