Guidelines for membrane protein engineering derived from de novo designed model peptides

Notwithstanding great advances in the engineering and structural analysis of globular proteins, relatively limited success has been achieved with membrane proteins--due largely to their intrinsic high insolubility and the concomitant difficulty in obtaining crystals. Progress with de novo synthesis of model membrane-interactive peptides presents an opportunity to construct simpler peptides with definable structures, and permits one to approach an understanding of the properties of the membrane proteins themselves. In the present article, we review how our laboratory and others have used peptide approaches to assess the detailed interactions of peptides with membranes, and primary folding at membrane surfaces and in membranes. Structural studies of model peptides identified the existence of a "threshold hydrophobicity," which controls spontaneous peptide insertion into membranes. Related studies of the relative helicity of peptides in organic media such as n-butanol indicate that the helical propensity of individual residues--not simply their hydrophobicity--may dictate the conformations of peptides in membranes. The overall experimental results provide fundamental guidelines for membrane protein engineering.     

Functionalized de novo designed proteins: mechanism of proton coupling to oxidation/reduction in heme protein maquettes

Proton exchange with aqueous media coupled to heme oxidation/reduction is commonly seen but not understood in natural cytochromes. Our synthetic tetrahelix bundle heme protein maquettes successfully reproduce natural proton coupling to heme oxidation/reduction. Potentiometry reveals major pK shifts from 4.2 to 7.0 and from 9.4 to 10.3 in the maquette-associated acid/base group(s) upon heme reduction. Consequently, a 210 mV decrease in the heme redox potential is observed between the two extremes of pH. Potentiometry with resonance Raman and FTIR spectroscopy performed over a wide pH range strongly implicates glutamate side chains as the source of proton coupling below pH 8.0, whereas lysine side chains are suggested above pH 8.0. Remarkably, the pK values of several glutamates in the maquette are elevated from their solution value (4.4) to values as high as 7.0. It is suggested that these glutamates are recruited into the interior of the bundle as part of a structural rearrangement that occurs upon heme binding. Glutamate to glutamine variants of the prototype protein demonstrate that removal of the glutamate closest to the heme diminishes but does not abolish proton exchange. It is necessary to remove additional glutamates before pH-independent heme oxidation/reduction profiles are achieved. The mechanism of redox-linked proton coupling appears to be rooted in distributed partial charge compensation, the magnitude of which is governed by the dielectric distance between the ferric heme and acid/base side chains. A similar mechanism is likely to exist in native redox proteins which undergo charge change upon cofactor oxidation/reduction.     

Structural thermodynamics: prediction of protein stability and protein binding affinities

It has been demonstrated that the prognosis of ovarian cancer is influenced by the dose intensity of cytotoxic treatment. The impact of received dose intensity of platinum-based combination chemotherapy on disease outcome was analysed in 226 stage III-IV ovarian cancer patients entered into two prospective randomised trials. All patients received either cisplatin or carboplatin and cyclophosphamide with or without doxorubicin for six courses after primary surgery. The impact of the received dose intensity of each drug (RDI), the average received dose intensity of the treatment regimen (ARDI) and the relative total drug dose (RTD) on progression-free survival (PFS) and survival were analysed. In the 198 patients receiving the full six courses of treatment, RDI of cisplatin or carboplatin, ARDI and RTD were > 0.76 in 74.2, 61.1 and 65.1% of cases, respectively. Although the differences were not significant, pathological complete response was more frequently observed in the group of patients with ARDI < 0.75, whereas the partial response rate was higher in the ARDI > or = 0.76 group. Median survival and PFS were 19 and 13 months; 22 and 10 months; 23 and 13 months for the groups of patients receiving chemotherapy at a ARDI of < 0.75, > or = 0.76-0.99 and > 1.00, respectively (P = not significant). It appears that modest dose modifications and brief treatment delays during first-line platinum-based chemotherapy do not affect response rate, survival and PFS in advanced ovarian cancer patients.     

Pharmacological distribution diagrams: a tool for de novo drug design

BACKGROUND: There is no empirical data available on attitudes concerning AIDS and habits towards HIV infected patients of physicians in general or private practice. In this study results of a self-evaluation are presented. METHODS: 178 physicians working with out-patients in different medical fields were randomly selected for a cross sectional study and interviewed using a standardised questionnaire. RESULTS: 89% think that they are sufficiently informed about AIDS (in the USA 20%). They regarded the risk of infection to be lower than the Anglo-American physicians. They believed there is a lack of interchange of information between colleagues regarding the degree of infectiousness of referred patients. A third of the physicians fear that other patients will go elsewhere if they find out that their physician is treating AIDS patients. 54% would hold special clinic sessions for HIV-patients outside the normal schedule for practice times. 89% believed that HIV patients were partly to blame for their illness. CONCLUSIONS: Although the physicians recognise the problem of HIV-infection, they partly deny the real necessities and facts. A reason for this could be the emotions underlying the general attitude to everything pertaining to HIV-disease. Attitudes to HIV-disease and the dealing with it in daily practice must be considered on the basis of individual emotional motives.     

Structural and functional aspects of basic helix-loop-helix protein folding by heat-shock protein 90

The assembly and folding of nascent polypeptides are mediated in vivo by molecular chaperones, many of which are members of the heat-shock family of proteins. We have shown previously that heat-shock protein 90 (HSP90) folds an inactive fraction of a recombinant basic helix-loop-helix (bHLH) protein generated in Escherichia coli (MyoD) into its active conformation. We show here that HSP90 also folds another bHLH protein (E12) and heterodimers of E12/MyoD into their active conformations. By purifying inactive heterodimers of E12/MyoD and subsequently rendering them active in binding DNA by treatment with HSP90, we show that one folding step mediated by HSP90 occurs after oligomerization of the bHLH protein monomers. A series of deletion mutants is used to identify the 48-amino acid region of HSP90 that confers bHLH folding activity, which lies near the COOH terminus. This region is required for activation of DNA binding of MyoD and E12 homodimers and E12/MyoD heterodimers.     

Structural dynamism in the concept of self: A flexible model for a malleable concept.

The self-concept is theorized to play an important role in many psychological processes. Numerous theories rest on assumptions concerning the differential accessibility of pieces of self-knowledge, but relatively little attention during the past decade has centered on examining the underlying structure of the self-concept that mediates such differences. In the present article, we suggest a new model of the self-concept that incorporates recent advances in knowledge regarding conceptual structure. We envision the self as a representation in working memory with inherently flexible content and organization. Initial empirical evidence supporting this view is reviewed and followed by a discussion of the implications of this model for many phenomena involving the self-concept. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Correlation between protein stability and crystal properties of designed ROP variants

Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-alpha-helical bundles, have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolution X-ray diffraction studies. For all mutants crystal size, sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry, in a manner which is not yet understood in detail.     

A fusion protein designed for noncovalent immobilization: stability, enzymatic activity, and use in an enzyme reactor

We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion. Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation. Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling. The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form. Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts.     

Contact interactions method: a new algorithm for protein folding simulations

Computer simulations of simple exact lattice models are an aid in the study of protein folding process; they have sometimes resulted in predictions experimentally proved. The contact interactions (CI) method is here proposed as a new algorithm for the conformational search in the low-energy regions of protein chains modeled as copolymers of hydrophobic and polar monomers configured as self-avoiding walks on square or cubic lattices. It may be regarded as an extension of the standard Monte Carlo method improved by the concept of cooperativity deriving from nonlocal contact interactions. A major difference with respect to other algorithms is that criteria for the acceptance of new conformations generated during the simulations are not based on the energy of the entire molecule, but cooling factors associated with each residue define regions of the model protein with higher or lower mobility. Nine sequences of length ranging from 20 to 64 residues were used on the square lattice and 15 sequences of length ranging from 46 to 136 residues were used on the cubic lattice. The CI algorithm proved very efficient both in two and three dimensions, and allowed us to localize energy minima not localized by other searching algorithms described in the literature. Use of this algorithm is not limited to the conformational search, because it allows the exploration of thermodynamic and kinetic behavior of model protein chains.     

Non-native local interactions in protein folding and stability: introducing a helical tendency in the all beta-sheet alpha-spectrin SH3 domain

The relative importance of secondary structure interactions versus tertiary interactions for stabilising and guiding the folding process is a matter for discussion. Phenomenological models of protein folding assign an important role to local contacts in protein folding and stability. On the other hand, simplistic lattice simulations find that secondary structure is mainly the product of protein compaction and that optimisation of folding speed seems to require small contributions of local contacts to the stability of the folded state. To examine the extent to which secondary structure propensities influence protein folding and stability, we have designed mutations that introduce a strong non-native helical propensity in the first 19 residues of the alpha-spectrin SH3 domain. The mutant proteins have the same three-dimensional structure as the wild-type, but they are less stable and have less co-operative folding transitions. There seems to be a relationship between the non-native helical propensity and the compaction of the denatured state. This suggests that in the denatured ensemble under native conditions there is a significant proportion of compact structures with non-native secondary structures. Our results demonstrate that non-local interactions can overcome strong non-native secondary structure propensities and, more important, that optimisation of folding speed and co-operativity requires the latter to be relatively small.     

Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics

By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence-specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non-native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non-native conformations of hydrophobic-polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non-native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non-native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.     

Prenatal diagnosis of a stable de novo centric fission: a case report

Adenosine has become the treatment of choice for paroxysmal supraventricular tachycardia because of its safety and efficacy. There have been no reports of malignant arrhythmias occurring after adenosine administration. This case report presents the occurrence of a malignant wide complex tachycardia after intravenous adenosine administration in a 10-year-old boy 2 days after a Fontan procedure. Thus the administration of adenosine in a critically ill postoperative patient can have morbidity or potential mortality and must be monitored closely.     

Cystic fibrosis: a disease of altered protein folding

Patch clamp technique was employed to record single Na channel currents in isolated guinea-pig ventricular myocytes. Burst mode could be elicited by step depolarization and terminates immediately after repolarization. The unitary current of burst mode was not only dependent on Na concentration in the pipettes but also on the test voltage. The open time constant increased as testing voltage becomes more positive. The results from stepwise-depolarization and ramp depolarization experiments showed that the more steps or the faster the upstroke velocity of depolarization used, the more the burst mode would occure.     

Structural resolution of the folding pathway of a protein by correlation of phi-values with inter-residue contacts

The phenotypic identification of the classical propionibacteria is essentially still problematic and alternative techniques for the identification of the various species are required. A rapid and sensitive technique for the routine identification of the classical propionibacteria, based on the amplification of 16S rRNA genes using the polymerase chain reaction and the subsequent restriction endonuclease digestion of the PCR products, was previously described. Although this technique enabled differentiation between the various classical species examined it was only evaluated on a limited number of type and reference strains. During this study, the taxonomic relationship between 135 Propionibacterium strains from diverse ecological niches, representing four classical species was investigated using this PCR/RFLP technique. Visual differentiation between the classical Propionibacterium was possible after restriction endonuclease digestion of the PCR products obtained using primers 16sP1-16sP4 and 16sP3-16sP4 with the restriction endonucleases HaeIII, AluI and HpaIII, respectively. With the exception of strains independently identified as "P. rubrum" and "P. sanguineum", the results of this study confirm the consolidation of the "old" species into the various classical species as they currently exist. It was therefore concluded that the PCR/RFLP protocol is suitable for the rapid and routine identification of the classical propionibacteria.     

Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high alpha-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum-derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in alpha-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.     

Structure and stability of monomeric lambda repressor: NMR evidence for two-state folding

The absence of equilibrium intermediates in protein folding reactions (i.e., two-state folding) simplifies thermodynamic and kinetic analyses but is difficult to prove rigorously. We demonstrate a sensitive method for detecting partially folded species based on using proton chemical shifts as local probes of structure. The coincidence of denaturation curves for probes throughout the molecule is a particularly stringent test for two-state folding. In this study we investigate a new form of the N-terminal domain of bacteriophage lambda repressor consisting of residues 6-85 (lambda 6-85) using nuclear magnetic resonance (NMR) and circular dichroism (CD). This truncated version lacks the residues required for dimerization and is monomeric under the conditions used for NMR. Heteronuclear NMR was used to assign the 1H, 15N, and backbone 13C resonances. The secondary and tertiary structure of lambda 6-85 is very similar to that reported for the crystal structure of the DNA-bound 1-92 fragment [Beamer, L. J., and Pabo, C. O. (1992) J. Mol. Biol. 227, 177-196], as judged by analysis of chemical shifts, amide hydrogen exchange, amide-alpha coupling constants, and nuclear Overhauser enhancements. Thermal and urea denaturation studies were conducted using the chemical shifts of the four aromatic side chains as local probes and the CD signal at 222 nm as a global probe. Plots of the fraction denatured versus denaturant concentration obtained from these studies are identical for all probes under all conditions studied. This observation provides strong evidence for two-state folding, indicating that there are no populated intermediates in the folding of lambda 6-85.