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At least 73 records · Page 4Linked to original sources
PubMed1997

A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin.

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (i) the pKa values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (ii) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (iii) preventing rotation of the C13-C14 double bond by replacing the retinal with an all-trans locked analogue makes little difference to the Schiff base pKa. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.

Bacteriorhodopsins↗
PubMed1992

Role of protein conformation changes and transphosphorylations in the function of Na+/K(+)-transporting adenosine triphosphatase: an attempt at an integration into the Na+/K+ pump mechanism.

The particular aim of the review on some basic facets of the mechanism of Na+/K(+)-transporting ATPase (Na/K-ATPase) has been to integrate the experimental findings concerning the Na(+)- and K(+)-elicited protein conformation changes and transphosphorylations into the perspective of an allosterically regulated, phosphoryl energy transferring enzyme. This has led the authors to the following summarizing evaluations. 1. The currently dominating hypothesis on a link between protein conformation changes ('E1 in equilibrium with E2') and Na+/K+ transport (the 'Albers-Post scheme') has been constructed from a variety of partial reactions and elementary steps, which, however, do not all unequivocally support the hypothesis. 2. The Na(+)- and K(+)-elicited protein conformation changes are inducible by a variety of other ligands and modulatory factors and therefore cannot be accepted as evidence for their direct participation in effecting cation translocation. 3. There is no evidence that the 'E1 in equilibrium with E2' protein conformation changes are moving Na+ and K+ across the plasma membrane. 4. The allosterically caused ER in equilibrium with ET ('E1 in equilibrium with E2') conformer transitions and the associated cation 'occlusion' in equilibrium with 'de-occlusion' processes regulate the actual catalytic power of an enzyme ensemble. 5. A host of experimental variables determines the proportion of functionally competent ER enzyme conformers and incompetent ET conformers so that any enzyme population, even at the start of a reaction, consists of an unknown mixture of these conformers. These circumstances account for the occurrence of contradictory observations and apparent failures in their comparability. 6. The modelling of the mechanism of the Na/K-ATPase and Na+/K+ pump from the results of reductionistically designed experiments requires the careful consideration of the physiological boundary conditions. 7. Na+ and K+ ligandation of Na/K-ATPase controls the geometry and chemical reactivity of the catalytic centre in the cycle of E1 in equilibrium with E2 state conversions. This is possibly effected by hinge-bending, concerted motions of three adjacent, intracellularly exposed peptide sequences, which shape open and closed forms of the catalytic centre in lock-and-key responses. 8. The Na(+)-dependent enzyme phosphorylation with ATP and the K(+)-dependent hydrolysis of the phosphoenzyme formed are integral steps in the transport mechanism of Na/K-ATPase, but the translocations of Na+ and K+ do not occur via a phosphate-cation symport mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2005

Cantilever sensor for nanomechanical detection of specific protein conformations.

A fast, label-free, and multiplexed method based on piezoresistive cantilevers is reported for the detection of specific protein conformations at the nanoscale level. The ligand-binding domain of the human oestrogen receptor (ERalpha-LBD) is used as the experimental model system, and ERalpha-LBD with or without oestradiol (E2) is detected using the conformation-specific peptides alpha/betaI (Ser-Ser-Asn-His-Gln-Ser-Ser-Arg-Leu-Ile-Glu-Leu-Leu-Ser-Arg, which recognizes E2-bound ER) and alpha/betaII (Ser-Ala-Pro-Arg-Ala-Thr-Ile-Ser-His-Tyr-Leu-Met-Gly-Gly, which recognizes E2-free ER). Target-specific signals are obtained in situ at protein concentrations of 2.5-20 nM. The in-build electrical readout of the piezoresistive cantilevers provides a convenient alternative to the conventional optical detection, and the presented method offers the possibility of detecting protein conformational changes using miniaturized microarrays.

Biosensing Techniques↗
PubMed2006

Analytical model for studying how environmental factors influence protein conformational stability in solution.

We introduce an analytical modeling strategy for probing the conformational stability of globular proteins in aqueous solution. In this approach, the intrinsic (i.e., infinite dilution) thermodynamic stability and coarse structural properties of the proteins, as well as the effective protein-protein interactions, derive from a heteropolymer collapse theory that incorporates predicted temperature- and pressure-dependent hydrophobic interactions. Protein concentration effects are estimated by integrating this information into a molecular thermodynamic model, which is an ad hoc generalization of the exact equilibrium theory of a one-dimensional binary mixture of square-well particles that interconvert through an isomerization (i.e., folding) reaction. The end result is an analytical multiscale modeling approach which, although still schematic, can predict that folded proteins exhibit a closed-loop region of stability in the pressure-temperature plane and that protein concentration has a nonmonotonic effect on protein stability, results consistent with qualitative trends observed in both experiments of protein solutions and simulations of coarse-grained protein models.

Computer Simulation↗
PubMed2006

Molecular mechanism based on self-replicating protein conformation for the inheritance of acquired information in humans.

Recent evidence challenges the paradigmatic view of nucleic acids as the sole mediators of hereditary information. Here I present a molecular mechanism that can explain how acquired information in humans in a DNA independent mode becomes innate and heritable. The model is based on self-replicating protein conformations, a concept derived from prion and amyloid biology. Information is stored in specific beta-sheet protein conformations that can act as cytoplasmic molecular memories. The conformational information can be transmitted to next generations in a non-nucleic acid based inheritance system utilizing the self-perpetuating potential of such beta-rich protein aggregates. Chaperones play a crucial role in the model by regulating and balancing the process of folding and misfolding; they also assist in preventing the development of aggregation-based disease. The protein conformation-mediated information system could represent an evolutionary conserved primordial mechanism: while the main strategy has been to ensure rapid folding of polypeptides into the native, functional conformation, the disfolded, beta-rich amyloidogenic state has provided advantage by providing a cytoplasmic, protease-resistant self-perpetuating DNA-independent adaptive inheritance system. The model offers an explanation for the problematic question of the evolution of complex behavioural traits and has even impact in the context of mammalian cloning: the protein conformation-based information localized in the somatic cytoplasm is lost when transferring nuclei only into enucleated oocytes. The protein conformation-based model presented herein postulates that proteins may contain much more information than determined by the nucleotide-triplet controlled peptide sequence and that there exists cross-talk and information exchange between proteins.

Amyloid↗
PubMed1992

Detection of native-like models for amino acid sequences of unknown three-dimensional structure in a data base of known protein conformations.

We present an approach which can be used to identify native-like folds in a data base of protein conformations in the absence of any sequence homology to proteins in the data base. The method is based on a knowledge-based force field derived from a set of known protein conformations. A given sequence is mounted on all conformations in the data base and the associated energies are calculated. Using several conformations and sequences from the globin family we show that the native conformation is identified correctly. In fact the resolution of the force field is high enough to discriminate between a native fold and several closely related conformations. We then apply the procedure to several globins of known sequence but unknown three dimensional structure. The homology of these sequences to globins of known structures in the data base ranges from 49 to 17%. With one exception we find that for all globin sequences one of the known globin folds is identified as the most favorable conformation. These results are obtained using a force field derived from a data base devoid of globins of known structure. We briefly discuss useful applications in protein structural research and future development of our approach.

Algorithms↗
PubMed2006

[Study on the membrane protein conformational changes and mechanisms of myocardial cell irradiated by pulse microwave].

Micro-Fourier transform infrared spectroscopy (FTIR) technique was applied to study the membrane protein conformational and functional changes of myocardial cell irradiated by pulse microwave. The results show that pulse microwave could influence the membrane protein structure markedly. The stretching vibration of lipid--CH2--, lecithoid C=O, amide I and II region was decreased or displaced. The secondary structures of membrane protein were also changed by irradiation. The percentage of alpha-helix and beta-pleated sheet structure decreased remarkably, and the disordering of secondary membrane proteins increased. All the above changes are correlated with the irradiation dosage. The results indicated that the integrality of myocardial cell membrane was injured by pulse microwave, and the membrane fluidity and stability decreased. Multi-biochemically active structures were damaged. Then all the changes could make a biochemical foundation of pathologic effects, which included membrane function decline, cell morphological change, configuration injuring and apoptosis etc. This paper is from a new view of protein conformation to explore the molecular pathologic mechanism of the damage caused by pulse microwave irradiation.

Animals↗
PubMed2005

Detection of Bax protein conformational change using a surface plasmon resonance imaging-based antibody chip.

We describe an antibody chip technology that uses a surface plasmon resonance (SPR) imaging system to examine the conformational change of a protein. In this study, we used Bax protein, a pro-apoptotic member of the Bcl-2 family of proteins, as a model protein to investigate the conformational alteration triggered by a TNF-related apoptosis-inducing ligand (TRAIL), a potent inducer of apoptosis. To develop the antibody chip for detecting the Bax conformational change, we immobilized Bax monoclonal antibody 6A7, which recognizes only a conformationally changed Bax protein on a gold surface. The resultant immobilized Bax antibodies provided specific and accurate measurements of the active conformation-specific epitope in the apoptotic cancer cells treated with the TRAIL; these measurements corresponded to the data obtained by immunoprecipitation analysis using an active conformation-specific Bax antibody (6A7). The results of our study indicated that TRAIL-induced Bax structural change could be monitored quickly and simply using an SPR imaging system, thus demonstrating the potential for using such a system for the analysis of conformational properties of target proteins.

Antibodies↗
PubMed2005

Protein conformation significantly influences immune responses to prion protein.

In prion diseases, such as variant Creutzfeldt-Jakob disease normal cellular prion protein (PrPC), a largely alpha-helical structure is converted to an abnormal conformational isoform (PrPSc) that shows an increase in beta-sheet content. Similarly, the recombinant form of PrPC (ralpha-PrP) can be converted to a conformation dominated by beta-sheet (rbeta-PrP) by reduction and mild acidification in vitro, a process that may mimic in vivo conversion following PrPC internalization during recycling. Despite PrPSc accumulation and prion propagation in the lymphoreticular system before detectable neuroinvasion, no Ab response to PrP has been detected, probably due to immune tolerance. To investigate how the immune system may respond to alpha- and beta-PrP, we immunized Prnp(0/0) mice that are not tolerant of PrP with ralpha-PrP and rbeta-PrP. In this study, we show that although T cells stimulated by these differently folded conformers PrP recognize similar immunodominant epitopes (residues 111-130 and 191-210) the cytokine profile in response to ralpha- and rbeta-PrP was different. Challenge with ralpha-PrP elicited a strong response of IL-5 and IL-10, whereas rbeta-PrP led to an early increased production of IFN-gamma. In addition, immunization with ralpha-PrP led to production of predominantly IgG1 isotype Ab in the sera, whereas after immunization with rbeta-PrP, IgG2b was significantly produced. Thus, both humoral and cellular responses to these differently folded isoforms of the same protein are different, indicating a possible involvement of Th1 and Th2 pathway activation. These differences may be exploitable diagnostically and therapeutically for prion diseases, such as variant Creutzfeldt-Jakob disease.

Animals↗
PubMedDate not supplied

Ultrastructural and biophysical studies on protein conformations of epithelium and stroma in benign prostatic hyperplasia before and after transurethral resection of the prostate.

The alterations in protein conformation of epithelium and stroma in human benign prostatic hyperplasia (BPH) undergoing prostatic needle biopsy (PNB) and transurethral resection of the prostate (TURP) were isolated in vitro and investigated using attenuated total reflectance (ATR)/Fourier transform infrared (FT-IR) microspectroscopy. Second-derivative spectral analysis and a curve-fitting program were used to verify and obtain the protein secondary conformation and composition of the samples. TURP uses an electric cutting heat but PNB is a nonheating process. The results indicate a predominantly higher proportion of beta-sheet conformation of the epithelium or stroma in BPH tissue at 1633 or 1630 cm(-1) in the amide I band, but after treatment with TURP, both maximum peaks shifted to 1644 or 1646 cm(-1) due to random coil structure, respectively. This conversion suggests that the heat generated by the cutting loop could increase the temperature in the tissue surrounding the prostate, thereby modifying the protein conformational structure of epithelium and stroma in BPH tissue.

Aged↗
PubMed1996

Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: glass transition and protein conformation.

The effects of glass transition of, and protein conformation in, the dried solid on the storage stability of freeze-dried recombinant human interleukin-1 receptor antagonist (rhIL-1ra) were examined. Glass transition is a temperature-dependent phenomenon. Amorphous materials become hard and brittle at temperatures below their characteristic glass transition temperatures (Tg) such that diffusion of molecules along the matrix is not sufficient to cause large-scale structural changes. To ascertain the importance of the glass transition in protein storage stability, we compared 10 different lyophilized rhIL-1ra formulations, with Tgs ranging from 20 to 56 degrees C, during several weeks of storage at temperatures above and below the samples' Tgs. Protein degradation, both deamidation and aggregation, was greatly accelerated at temperatures above Tg, but for some formulations also arose below Tg. Thus, storage of dried proteins below the Tg is necessary but not sufficient to ensure long-term stability. To examine the effects of protein structure in the dried solid, we prepared formulations with various sucrose concentrations, all of which had a Tg = 66 +/- 2.5 degrees C. With infrared spectroscopy, we determined that the protein lyophilized with </=1% sucrose was unfolded in the initial dried solid. In contrast, in those formulations with >/=5% sucrose, conformational change was inhibited during lyophilization. When stored at 50 degrees C, degradation of the freeze-dried protein varied inversely with sucrose concentration. These results indicate that structural changes arising during the lyophilization process led to damage during subsequent storage, even if the storage temperature was less than the Tg. Together the results of these studies document that to obtain optimum stability of dried rhIL-1ra it was necessary to inhibit conformational change during lyophilization and to store at temperatures below the Tg of the dried formulation.

Amides↗
PubMed2000

Crystallographic analysis of protein conformational changes in the bacteriorhodopsin photocycle.

A variety of neutron, X-ray and electron diffraction experiments have established that the transmembrane regions of bacteriorhodopsin undergo significant light-induced changes in conformation during the course of the photocycle. A recent comprehensive electron crystallographic analysis of light-driven structural changes in wild-type bacteriorhodopsin and a number of mutants has established that a single, large protein conformational change occurs within 1 ms after illumination, roughly coincident with the time scale of formation of the M(2) intermediate in the photocycle of wild-type bacteriorhodopsin. Minor differences in structural changes that are observed in mutants that display long-lived M(2), N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. These observations support a model for the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M(1)) are well approximated by one protein conformation in which the Schiff base has extracellular accessibility, while the structures of the later intermediates (M(2), N and O) are well approximated by the other protein conformation in which the Schiff base has cytoplasmic accessibility.

Bacteriorhodopsins↗
PubMed2005

A fluorescent probe designed for studying protein conformational change.

The usefulness of fluorescence in studying protein motions derives from its sensitivity, kinetic resolution, and compatibility with both live cells and physiological assays. Recent advances in microscopy and membrane protein purification have permitted the observation of fluorescence changes that accompany the functional transitions of complex eukaryotic membrane proteins. These techniques rely on probes that can clearly report the environmental changes of specific residues, but most commonly available side-chain-reactive probes are not well suited for this purpose. Here, we introduce a red Cys-reactive probe, aminophenoxazone maleimide (APM), designed with improved chemical and spectral properties for reporting protein conformational change. APM is compact, uncharged, and has a short linker between probe and protein, all of which ensure that it can closely track the motions of the side chain to which it is attached. It undergoes large polarity-dependent changes in Stokes shift, as well as large bathochromic shifts in both excitation maximum (from 521 nm in toluene to 598 nm in water) and emission maximum (580 nm to 633 nm). These polarity-dependent spectral changes offer a potentially simple means of relating fluorescence to local structure and motion, although they are partially offset by some complicating factors in APM fluorescence. We find that, like a rhodamine maleimide, APM senses the conformational changes underlying voltage sensing in the Shaker potassium channel, and it is superior at a site that shows limited reactivity to the rhodamine. The spectral characteristics of APM can also report subtle differences between aqueous positions in purified preparations of the beta2 adrenergic receptor.

Fluorescent Dyes↗
PubMed1987

Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: II. Intrinsic Protein Fluorescence.

The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32 degrees C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30 degrees C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32 degrees C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14 degrees C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32 degrees C which could account for the complex temperature dependence of the barley root plasma membrane ATPase.

Journal Article↗
PubMed2001

The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo.

Intermolecular and intramolecular FRET between two spectrally overlapping green fluorescent protein variants fused to two different host proteins or at two different sites within the same protein offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes. By using fluorescence digital imaging microscopy, one can visualize the location of green fluorescent proteins within a living cell and follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules.

Calcium Signaling↗
PubMed2006

Modeling protein conformational changes by iterative fitting of distance constraints using reoriented normal modes.

Recently we have developed a normal-modes-based algorithm that predicts the direction of protein conformational changes given the initial state crystal structure together with a small number of pairwise distance constraints for the end state. Here we significantly extend this method to accurately model both the direction and amplitude of protein conformational changes. The new protocol implements a multisteps search in the conformational space that is driven by iteratively minimizing the error of fitting the given distance constraints and simultaneously enforcing the restraint of low elastic energy. At each step, an incremental structural displacement is computed as a linear combination of the lowest 10 normal modes derived from an elastic network model, whose eigenvectors are reorientated to correct for the distortions caused by the structural displacements in the previous steps. We test this method on a list of 16 pairs of protein structures for which relatively large conformational changes are observed (root mean square deviation >3 angstroms), using up to 10 pairwise distance constraints selected by a fluctuation analysis of the initial state structures. This method has achieved a near-optimal performance in almost all cases, and in many cases the final structural models lie within root mean square deviation of 1 approximately 2 angstroms from the native end state structures.

Computer Simulation↗
PubMed1993

A multi-level description scheme of protein conformation.

We propose a novel description scheme of protein backbone conformation that can model the important factors of protein structure formation, such as global interaction and geometric constraints. This description scheme represents a protein conformation with several symbolic sequences of multiple levels of abstraction. Each symbol in the sequence denotes the class of abstracted topology of subconformation with the size specific to the level. Low level sequences of this description represent fine structures of high resolution, and high level sequences represent the abstracted topologies of large scale. The classification of protein backbone subconformations of various sizes is the most important base for this description scheme. This has never been tried so far due to the complexity in dealing with the number of degrees of freedom in subconformations. However, the proposed technique solved this problem by abstracting the topology of middle and large scale subconformations. This linear expansion technique extracts a fixed number of parameters as the expansion coefficients from the co-ordinate representation of subconformations. In this case, the simple reverse-transformation from the expansion coefficients reconstructs the three-dimensional topology of a subconformation. The analysis of the relation between primary structure of a region and the subconformation of that region at each level in this description helps to model both local and global interactions of protein structure formation. Further, the statistic analysis of overlapping patterns of two subconformations models the geometric constraints important for a structure prediction system in generating a conformation which is geometrically sound.

Amino Acid Sequence↗