Capped acyclic permutants of the circular protein kalata B1

The cyclotides are a family of head-to-tail cyclized peptides that display exceptionally high stability and a range of biological activities. Acyclic permutants that contain a break in the circular backbone have been reported to be devoid of the haemolytic activity of the prototypic cyclotide kalata B1, but the potential role of the charges at the introduced termini in this loss of membraneolytic activity has not been fully determined. In this study, acyclic permutants of kalata B1 with capped N- and C-termini were synthesized and found to adopt a native fold. These variants were observed to cause no measurable lysis of erythrocytes, strengthening the connection between backbone cyclization and haemolytic activity.     

Folding and stability of a primitive protein

We have previously attempted to simulate domain creation in early protein evolution by recombining polypeptide segments from non-homologous proteins, and we have described the structure of one such de novo protein, 1b11, a segment-swapped tetramer with novel architecture. Here, we have analyzed the thermodynamic stability and folding kinetics of the 1b11 tetramer and its monomeric and dimeric intermediates, and of 1b11 mutants with changes at the domain interface. Denatured 1b11 polypeptides fold into transient, folded monomers with marginal stability (DeltaG<1kcalmol(-1)) which convert rapidly ( approximately 6x10(4)M(-1)s(-1)) into dimers (DeltaG=9.8kcal/mol) and then more slowly ( approximately 3M(-1)s(-1)) into tetramers (DeltaG=28kcalmol(-1)). Segment swapping takes place during dimerization, as suggested by mass spectroscopic analysis of covalently linked peptides derived from proteolysis of a disulfide-linked dimer. Our results confirm that segment swapping and associated oligomerization are both powerful ways of stabilizing proteins, and we suggest that this may have been a feature of early protein evolution.     

Folding and stability of sweet protein single-chain monellin. An insight to protein engineering

Engineered single-chain monellin (SCM) proteins were constructed by recombinant technology without disrupting the topology and sweet activity of native protein. Data from 8-anilinonaphthalene-1-sulfonic acid fluorescence, size-exclusion chromatography, and heteronuclear NMR strongly suggest the presence of a folding intermediate at 1.5 m GdnHCl for SCM protein. The structural feature of the folding intermediate from NMR data reveals that the secondary structures became mostly unstable, and protein experiences a dynamic equilibrium between native and unfolded state. All backbone amide protons exchange within 10 min, which imply that no stable hydrogen bonds exist in the secondary structural regions in the folding intermediate. From equilibrium unfolding and mutagenesis studies, the unfolding transition midpoints of mutant proteins gradually shifted toward lower denaturant concentration, indicating stability reductions of mutant proteins. Our results suggest that stability and folding pathways of SCM proteins could be regulated by a combined study of spectroscopy and mutagenesis, and these studies will provide useful information for understanding the folding kinetics of novel engineered proteins.     

Drug users in contact with general practice.

A group of heroin users who are in contact with a general practice in north west Edinburgh are described. The study group was younger and included more women than previous studies. These people used a large variety of drugs and mainly purchased them locally. Frequent and often prolonged abstinent periods occurred with no prescribed opiate treatment. The group had experienced a high rate of drug related medical disorders. All these points raise the possibility that opiate users who are known to general practitioners may be a distinctly different population from those who attend drug dependency clinics. The frequency of remission and the prevalence of polydrug use have profound implications for planning and evaluating an effective medical response.     

Functional role of polyhydroxy compounds on protein structure and thermal stability studied by circular dichroism spectroscopy.

Polyhydroxy compounds such as cyclitols, acyclic polyols and sugars are produced by a wide variety of organisms under stressful conditions in order to protect macromolecular structure. Plants undergoing abiotic stresses like heat and dehydration accumulate enormous amounts of polyhydroxy compounds (up to 400 mM) in their cellular tissues. Not only do they serve as osmoprotectants ("compatible solutes"), they also protect membrane structure and preserve enzymatic activity. To gain further insight into the mechanism of protein protection by polyhydroxy compounds, we examined the structural and thermal stability of six model proteins (bovine serum albumin, glutamine synthetase of Escherichia coli, malate dehydrogenase of pig heart, SH2 domain of phospholipaseCgamma1, SH2_Myc and GST_MycMax fusion proteins) upon the addition of various polyhydroxy compounds by circular dichroism spectroscopy. Our results show that D-pinitol (1D-3-O-methyl-chiro-inositol), L-quebrachitol (1L-2-O-methyl-chiro-inositol), myo-inositol, D-chiro-inositol, mannitol, glucose and trehalose promoted improved structural and thermal stability for each protein, whereas conduritol (1,4/2,3-cyclohexanetetrol) and glycerol were not effective. An increase in the midpoint denaturation temperature (T(m)) of 3.3 degrees C to 4.7 degrees C was observed for each protein upon the addition of 400 mM myo-inositol. Although the apparent T(m) of each protein was shifted by the addition of polyhydroxy compounds, the influence seems to be dependent on attributes like the protein surface topology, the hydration shell and on the nature of the protective solute, as well as on its concentration. The O-methylated cyclitols D-pinitol and L-quebrachitol were more effective preservatives than the less hydrophobic non-methylated myo-inositol and D-chiro-inositol. Amongst various polyhydroxy compounds, hydrophobic cyclitols were the most effective stabilizers.     

Folding and stability of the extracellular domain of the human amyloid precursor protein

The beta-amyloid peptide (A beta), the major component of the senile plaques found in the brains of Alzheimer's disease patients, is derived from proteolytic processing of a transmembrane glycoprotein known as the amyloid precursor protein (APP). Human APP exists in various isoforms, of which the major ones contain 695, 751, and 770 amino acids. Proteolytic cleavage of APP by alpha- or beta-secretases releases the extracellular soluble fragments sAPP alpha or sAPP beta, respectively. Despite the fact that sAPP alpha plays important roles in both physiological and pathological processes in the brain, very little is known about its structure and stability. We have recently presented a structural model of sAPP alpha 695 obtained from small-angle x-ray scattering measurements (Gralle, M., Botelho, M. M., Oliveira, C. L. P., Torriani, I., and Ferreira, S. T. (2002) Biophys. J. 83, 3513-3524). We now report studies on the folding and stabilities of sAPP alpha 695 and sAPP alpha 770. The combined use of intrinsic fluorescence, 4-4'-Dianilino-1,1'binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, circular dichroism, differential ultraviolet absorption, and small-angle x-ray scattering measurements of the equilibrium unfolding of sAPP alpha 695 and sAPP alpha 770 by GdnHCl and urea revealed multistep folding pathways for both sAPP alpha isoforms. Such stepwise folding processes may be related to the identification of distinct structural domains in the three-dimensional model of sAPP alpha. Furthermore, the relatively low stability of the native state of sAPP alpha suggests that conformational plasticity may play a role in allowing APP to interact with a number of distinct physiological ligands.     

The thermodynamics of protein stability. Cold destabilization as a general phenomenon

A theoretical analysis of the temperature/stability profiles of proteins shows that, where a two-state model represents the denaturation, and where the free energy of denaturation delta G(T) shows a strong temperature dependence, then the protein becomes subject to both high- and low-temperature destabilization. In the simplest case delta G(T) is parabolic, therefore the high temperature TH, where delta (G(TH) = 0, is complemented by a low temperature TL, where delta G(TL) = 0. It is generally stated that the partial molal heat capacity change delta C accompanying the heat denaturation is positive and independent of the temperature. This implies that heating the protein through TL results in a negative delta C which seems physically unsatisfactory. The constant delta C model is explored and a physically more realistic model is advanced which allows for a temperature-dependent delta C which changes sign at some temperature within the range of stability of the native protein; delta G(T) then has the form of a skewed parabola. Experimental heat capacity data for native lysozyme and for a flexible polymer lend support to this model. The molecular basis of cold inactivation of proteins is discussed in the light of the thermodynamic analysis.     

Induction of growth arrest by a temperature-sensitive p53 mutant is correlated with increased nuclear localization and decreased stability of the protein.

A temperature-sensitive mutant of p53, p53Val-135, was found to be able to arrest cell proliferation when overexpressed at 32.5 degrees C. While much of the protein was cytoplasmic in cells proliferating at 37.5 degrees C, it became predominantly nuclear at 32.5 degrees C. Concomitantly, p53Val-135 became destabilized, although not to the extent seen in primary fibroblasts.     

Folding of staphylococcal nuclease A studied by equilibrium and kinetic circular dichroism spectra

The urea-induced unfolding of staphylococcal nuclease A has been studied by circular dichroism both at equilibrium and by the kinetics of unfolding and refolding (pH 7.0 and 4.5 degrees C), as a function of Ca2+ and thymidine 3',5'-diphosphate (pdTp) concentration. The results are as follows. (1) The unfolding transition is shifted to higher concentrations of urea by Ca2+ and pdTp, and the presence of both ligands further stabilizes the protein. (2) In the first stage of kinetic refolding, the peptide ellipticity changes rapidly within the dead time of stopped-flow measurement (15 ms), indicating accumulation of a transient intermediate. This intermediate is remarkably less stable than those of other globular proteins previously studied. (3) Dependence of the folding and unfolding rate constants on urea concentration indicates that the critical activated state of folding ("transition state") has considerable structural organization. The transition state does not, however, have the capacity to bind Ca2+ and pdTp, as indicated by the effects of these ligands on the unfolding rate constant. (4) There are at least four different phases in the refolding kinetics in native conditions below 1 M urea. In the absence of pdTp, there are two phases in unfolding, while in the presence of pdTp the unfolding kinetics show a single phase. Some characteristics of the transient intermediate and of the transition state for folding are discussed.     

Mutational analysis of acylphosphatase suggests the importance of topology and contact order in protein folding.

Muscle acylphosphatase (AcP) is a small protein that folds very slowly with two-state behavior. The conformational stability and the rates of folding and unfolding have been determined for a number of mutants of AcP in order to characterize the structure of the folding transition state. The results show that the transition state is an expanded version of the native protein, where most of the native interactions are partially established. The transition state of AcP turns out to be remarkably similar in structure to that of the activation domain of procarboxypeptidase A2 (ADA2h), a protein having the same overall topology but sharing only 13% sequence identity with AcP. This suggests that transition states are conserved between proteins with the same native fold. Comparison of the rates of folding of AcP and four other proteins with the same topology, including ADA2h, supports the concept that the average distance in sequence between interacting residues (that is, the contact order) is an important determinant of the rate of protein folding.     

Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate.

The replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline-limited reaction that is unusually slow in the refolding of the wild-type protein. The distribution of fast- and slow-folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp-59 is in close contact to Pro-39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with the trans----cis reisomerization of the Tyr-38-Pro-39 bond at the stage of a native-like folding intermediate. The steric hindrance is abolished either by changing Trp-59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild-type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal that trans----cis isomerization of Pro-39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate rate-limiting steps by trapping partially folded structures.     

Folding of DsbB in mixed micelles: a kinetic analysis of the stability of a bacterial membrane protein

Measuring the stability of integrated membrane proteins under equilibrium conditions is hampered by the nature of the proteins' amphiphilic environment. While intrinsic fluorescence is a useful probe for structural changes in water-soluble proteins, the fluorescence of membrane proteins is sensitive to changes in lipid and detergent composition. As an attempt to overcome this problem, I present a kinetic analysis of the folding of a membrane protein, disulfide bond reducing protein B (DsbB), in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate (SDS) and dodecyl maltoside (DM). This analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. Refolding and unfolding occur on the second to millisecond timescale and involve only one relaxation phase, when monitored by conventional stopped-flow. The kinetic data indicate that denaturation occurs around 0.3 mole fraction of SDS, in agreement with CD analysis and acrylamide quenching data. The rate constants have been fit to a three-state folding scheme involving the SDS-denatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of SDS. The stability of DsbB is around 4.4 kcal/mol in DM, and this is halved upon reduction of the two periplasmic disulfide bonds, and is sensitive to mutagenesis. With the caveat that kinetic data are always open to alternative interpretations, time-resolved studies in mixed micelles provide a useful approach to measure membrane protein stability over a wide range of concentrations of SDS and DM, as well as a framework for the future characterization of the DsbB folding mechanism.     

Folding energetics of a multidomain protein, flagellin.

Thermodynamic investigations of flagellin from Salmonella typhimurium and its proteolytic fragments were conducted by differential scanning calorimetry (DSC) and circular dichroism (CD) melting measurements. A new method of analysis for a multi-state transition based on our original theoretical treatment of thermodynamic equations has been developed to analyze those data. The analysis of DSC curves confirmed the three thermodynamic domains of flagellin. The thermodynamic parameters of each domain were revised from those previously reported and the new values of the parameters have a good correlation to the apparent molecular masses of the morphological domains. CD melting measurements at far and near-UV wavelengths showed sequential unfolding of the domains. Therefore, we could reasonably assign the thermodynamically identified domains to the morphological domains. Further analysis of both DSC and CD data provided insights into the folding energetics of the multidomain structure of flagellin. An inner domain (Df1) of flagellin in the filament unfolds through a relatively broad transition, while the two outer domains unfold cooperatively and show sharp transitions. This indicates that the interdomain interactions between Df1 and D2 has different characteristics from the apparently more intimate interactions between D2 and D3. These characteristics suggest that flagellin is organized with relatively flexible domains and rigid domains, which appears to be responsible for the well-regulated assembly mechanism of the bacterial flagellar filament.     

Feasibility of contact surveys in general practice.

Surveys to evaluate risk factors for disease in the general population are popular with health authorities for assessing the effectiveness of their preventive measures. A contact survey of the lifestyles of 2000 randomly selected patients aged 25-64 was conducted in five general practices over 18 months; the medical records of the patients selected were tagged, and when the patients first visited the surgery they were given a questionnaire by the receptionists, which they completed in the waiting room. Over the 18 months at least 1400 of these patients visited the practices, of whom 1106 (55%) completed a questionnaire and 20 refused to do so; 896 (81%) completed it within one year. Information on the patients who were not surveyed was obtained by sending the questionnaire by post and by audit of medical records. The population surveyed on contact with the surgeries contained a higher proportion of young women, and possibly a higher proportion of patients from social classes IIIM-V, than the other patients. No important or consistent bias towards unhealthy patients at high risk was identified in the contact survey. A one year contact survey of a random, tagged sample is feasible in estimating the risk factors in a population and may be the method of choice in general practice because of its low cost and adaptability.     

Accuracy of circular contact area measurements with thin-film pressure sensors

Contact area is often used to characterize the biomechanical properties of joints, especially in testing of injury and joint replacement. Several methods have been developed to measure contact area, including piezo-resistive thin-film arrays. The purpose of this study was to determine the accuracy with which one of these systems (Tekscan, Inc., South Boston, MA) could measure the contact area of flat-ended circular indenters of varying known sizes. Static loads ranging from 1000 to 7000 N were applied to four flat, circular indenters (1140, 2027, 3167, and 4560 mm(2)) and the contact areas were recorded with Tekscan 5076 sensor. Similar testing was carried out on a 4000 sensor. I-scan software (Tekscan Inc., South Boston, MA) was used to analyze the Tekscan-recorded area measurements. The Tekscan data were also post-processed to filter out sensel signal intensity values that were at least two standard deviations from the average sensel signal intensity values of the sensor matrix. Unprocessed Tekscan measurements with the 5076 sensor had area percent errors ranging from 5% to 27%. The filtering algorithm reduced most errors to less than 1%. Similar trends of improved accuracy with post-filtering were found with the 4000 sensor. While this method of thresholding out the sensels with the lowest signal intensity values may not work for all surfaces and indenter shapes, it provides a new approach to improve the accuracy of contact area measurements collected with the Tekscan system.     

Folding of a beta-sheet protein monitored by real-time NMR spectroscopy

At low ionic strength, apoplastocyanin forms an unfolded state under non-denaturing conditions. The refolding of this state is sufficiently slow to allow real-time NMR experiments to be performed. Folding of apoplastocyanin, initiated by the addition of salt and followed by real-time 2D 1H-15N heteronuclear single quantum coherence (HSQC) spectroscopy, is highly cooperative. A concomitant increase in the intensity of both sequential and long-range nuclear Overhauser effects (NOEs) between backbone amide protons in successive acquisitions of 1H-15N HSQC-NOESY-HSQC spectra provides the first direct observation of the development of structure-specific NOEs as a protein folds. Our results show that the local and long-range interactions in the native apoplastocyanin are formed simultaneously, consistent with highly cooperative formation of the native structure.