Convex Constraint Analysis Research Articles

Combining X-ray scattering and spectroscopy as structure resolving tool

Modern applications and basic research in medicine, biotechnology and materials are often concerned with hybrid (organic-inorganic) and synergetic systems. In other words, systems that brings enhanced properties and performances. The challenge is now in the understanding of the complex interactions leading to their assembly and operation. Due to their inherent chemical and structural complexities a combination of several techniques is necessary to determine unambiguously the molecular mechanisms of assembly and operation. [1, 2] To address this need, we have implemented a simultaneous measurement platform called SURF [3] that consists of SAXS, UV-Vis, Raman and fluorescence techniques. The SURF platform provides simultaneous measurements on the same sample volume and a multivariate framework to associate the spectroscopic and X-ray scattering information. Convex constraint analysis (CCA) and two dimensional correlation analyses (2DCOS and 2DHCOS) had been introduced to enhance the interpretation and integration of the data from the different techniques producing self-consistent models and resolving complex behaviour details (structure and chemistry). Additional benefits of the SURF are sample quality control and "on line" diagnostics. In this contribution, we illustrate the benefits of the SURF approach on selected examples. Acknowledgements: The SURF set-up has been mainly financial supported by MAX IV laboratory. S. Haas has a postdoctoral grant of MAX IV lab. S Canton and Q. Zhu are acknowledged for fruitful discussions.

The kinetics of thermal stress induced denaturation of Aquaporin 0

Aquaporin 0 (AQP0) is an integral membrane protein that facilitates water transport and cellular adhesion in the lens. Its dysfunction has been associated with cataractogenesis. Our earlier studies showed AQP0 undergoes aggregation when subjected to thermal stress and this aggregation seems to have been facilitated by mechanical agitation brought about by gentle stirring. The purpose of this study is to determine the secondary structural changes that precede aggregation and the role that α-crystallin plays in inhibiting those structural changes. Detergent solubilized calf lens AQP0 was subjected to thermal stress at 50°C for varying times. Secondary structural changes were measured by Circular Dichroism (CD) spectropolarimetry. Convex constraint analysis was used to deconvolute the CD spectra into pure component curves representing the secondary structural elements. Our results showed that under thermal stress, the α-helix content of AQP0 decreased from 50% to 7% with a concomitant increase from 0% to 52% in β-sheet content. The time-dependent loss of α-helical structure and gain of β-sheet structure appear to follow first-order kinetics with very similar values (∼30min) suggesting a single transition. In the presence of α-crystallin, this conversion to β-sheet is minimized, suggesting that the protein structure that binds to the molecular chaperone is mostly the α-helical structure of AQP0.

Combined SAXS/UV–vis/Raman as a Diagnostic and Structure Resolving Tool in Materials and Life Sciences Applications

In order to diagnose and fully correlate structural, chemical, and functional features of macromolecules and particles in solution, we propose the integration of spectroscopy and scattering on the same measuring volume and at the same time in a dedicated sample environment with multiple probes. Combined SAXS/UV-vis and SAXS/Raman information are employed to study the radiation damage effect in proteins in solution and the scattering from single wall carbon nanotubes (SWNTs) in SDS dispersion, respectively. In the first case, a clear correlation is observed between the time dependence of the radius of gyration (Rg) of the protein determined by SAXS and the turbidity of the protein solution extracted from simultaneous UV-vis measurements. In the second case, the ratio of bundled/isolated carbon nanotubes is obtained unambiguously through proper modeling of the scattering data and cross-validated with the Raman information. The uses of convex constraint analysis (CCA) and two-dimensional correlation analyses (2DCOS and 2DHCOS) are introduced to fully explore the combination of data sets from different techniques and to extract unique insights from the sample.

Heat-Induced Conformational Unfolding of Fatty Acid-Free and Fatted Camel Serum Albumin: A Comparative Study

Albumin is a multifunctional non-glycosylated, negatively charged plasma protein, with extraordinary ligand-binding and transport properties, antioxidant functions, and enzymatic activities. Physiologically, albumin transports free fatty acids in plasma and contributes in maintaining colloid osmotic pressure. Recent progresses in using albumin as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of peptide or protein-based drugs, increased the attempts for improving albumin stability. Studying the thermal stability of camel albumin may provide us not only new clues for designing recombinant albumins, but also molecular insights on camel physiology. This study aims to determine the thermal stability of camel albumin. Fatted camel serum albumin (FCSA) was purified from blood via combination of Cohn's method and anion-exchange chromatography. Activated charcoal treatment was used to obtain defatted camel serum albumin (CSA). Fluorescence spectroscopy and differential scanning calorimetry (DSC) were used to study thermal denaturation of this protein. The set of fluorescence spectra were deconvoluted using the convex constraint analysis method (CCA). The results from deconvolution of fluorescence spectroscopy and DSC showed three and two components for CSA and FCSA, respectively. The bimodal DSC transition can be attributed to a crevice between domains I and II and formation of two independent thermodynamic domains. The crevice formation can be prevented by fatty acid binding between domains I and II. The calculated values of ∆H v/∆H cal, approximately 0.4 for CSA and near 1 for FCSA, confirmed the presence of at least one intermediate in thermal unfolding of CSA and the absence of the intermediate for FCSA. The obtained midpoint transition temperature (T m) of FCSA was about 20°C higher than that of CSA. Such enormous stabilizing effect may be attributed to the fact that fatty acid serves as glue which preserves different domains beside each other and prevents formation of the mentioned intermediate.

The inherent flexibility of peptides and protein fragments quantitized by CD in conjunction with CCA+

ECD spectroscopy is traditionally used for rapid, non-atomic level structure analysis of natural products such as peptides and proteins. Unlike globular proteins, peptides less frequently adopt a single 3D-fold in a time average manner. Moreover, they exhibit an ensemble of conformers composed of a multitude of substantially different structures. In principle, both ECD- and vibrational circular dichroism (VCD)-spectroscopy are sensitive enough to pick up structural information on these dynamic ensembles. However, the interpretation of the raw spectral data of these highly dynamic molecular systems can be cumbersome. The herein presented Convex Constraint Analysis Plus method, or CCA+ for short (http://www.chem.elte.hu/departments/protnmr/cca/), provides a unique opportunity for spectral ensemble analysis of peptides, glycopeptides, peptidomimetics, and other foldamers. The precision and accuracy of the approach is presented here through different peptide model systems. An interesting temperature and pH dependent folding and unfolding of a miniprotein (e.g. Tc5b variant) is also described. Analysis of CD spectra sets strongly affected by solvent and ion type is also introduced to account for severe environmental-induced structure influencing effect(s). The deconvolution makes always possible the quantitative data analysis even when the interpretation of the deconvolution resulted in pure CD curves is complex.

Spherical polyelectrolyte brushes as carrier particles for proteins: An investigation of the structure of adsorbed and desorbed bovine serum albumin

The change in the secondary and tertiary structure of bovine serum albumin (BSA) induced by the interaction with spherical polyelectrolyte brushes (SPB) has been investigated using fluorescence and circular dichroism (CD) spectroscopy. The SPB consist of poly(acrylic acid) chains grafted to a poly(styrene) core. The colloidal SPB represent a new substrate for protein immobilization because their protein binding capacity can be controlled by the ionic strength of the solution: SPB bind large amounts of BSA at low ionic strength (pH=6.1), but they are largely protein resistant at moderate salt concentrations of 500 mM. The conformation of BSA which was labeled with the environmentally sensitive dansyl fluorophore was studied before adsorption to the SPB, in the adsorbed state, and after desorption from the SPB. In the adsorbed state the obtained fluorescence spectrum is red‐shifted which indicates a hydration of the dansyl fluorophores due to a distortion of the tertiary structure of BSA. Fluorescence and CD spectroscopic analysis of BSA that was desorbed from the SPB shows that the adsorption‐induced conformational changes are largely reversible. Convex constraint analysis of the observed CD spectra of BSA yield α‐helix fractions of 68% and 57% before adsorption to and after desorption from the SPB, respectively. In a general view, the results of this study demonstrate that spherical polyelectrolyte brushes are suitable for a controlled immobilization and release of proteins without major conformational changes.

Analysis of the structural organization and thermal stability of two spermadhesins. Calorimetric, circular dichroic and Fourier-transform infrared spectroscopic studies.

The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel beta-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgroup of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5 degrees C and 78.6 degrees C, respectively. The calorimetric enthalpy changes (delta Hcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that beta-structure and turns are the major structural element of both PSP-I/PSP-II (53% of beta-sheet, 21% of turns) and aSFP (44% of beta-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, alpha-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-beta-structure proteins, and would support a beta-barrel topology like that predicted for the CUB domain. Other beta-structure folds, such as the Greek-key pattern characteristic of many carbohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms.

The measurement of transmembrane helices by the deconvolution of CD spectra of membrane proteins: A review

AbstractThe interpretation of the CD spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as x‐ray or nmr data. Therefore, these methods are inappropriate for a CD data base whose secondary structures are unknown, as in the case of the membrane proteins. The Convex Constraint Analysis algorithm [A. Perczel, M. Hollósi, G. Tusnády, and G. D. Fasman (1991) Protein Engineering, Vol. 4, 669–679], on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived “pure” CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five‐component spectra so obtained from the deconvolution consisted of two different types of α‐helices (the α‐helix in the soluble domain and the αT‐helix, for the transmembrane α‐helix), a β‐pleated sheet, a class C‐like spectrum related to β‐turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the αT‐helix was characterized by a positive red‐shifted band in the range 195–200 nm (+95,000 deg cm2 dmol−l), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222 nm band (−50,000 and −60,000 deg cm2 dmol−1, respectively) in comparison with the regular α‐helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of + 70,000, −30,000, and −30,000 deg cm2 dmol−1, respectively. © 1994 John Wiley & Sons, Inc.

Conformational change of chaperone Hsc70 upon binding to a decapeptide: a circular dichroism study.

The conformation of bovine Hsc70, a 70-kDa heat shock cognate protein, and its conformational change upon binding to decapeptides, was studied by CD spectroscopy and secondary structure prediction (Chou, P.Y. & Fasman, G.D., 1974, Biochemistry 13, 222-245). The CD spectra were analyzed by the LINCOMB method, as well as by the convex constraint analysis (CCA) method (Perczel, A., Park, K., & Fasman, G.D., 1992, Anal. Biochem. 203, 83-93). The result of the CD analysis of Hsc70 (15% alpha-helix, 24% beta-sheet, 24% beta-turn, and 38% remainder) was very similar to the predicted secondary structure for the beta-sheet (24%) and the beta-turn (29%). However, there is disagreement between the alpha-helical content by CD analysis (15%) and the predicted structure (30%). In spite of the fact that the decapeptides contained a considerable amount of beta-sheet (22%), the interaction of the heat shock protein with the peptide resulted in an overall decrease in the content of beta-sheet conformation (-15%) of the complex. This may be due to induction of a molten globule state. The result of the CCA analysis indicated that the Hsc70 undergoes a conformational change upon binding the decapeptides.

Analysis of the circular dichroism spectrum of proteins using the convex constraint algorithm: A practical guide

Due to the time scale of circular dichroism (CD) measurements, it is theoretically possible to deconvolute such a spectrum if the pure CD spectra differ significantly from one another. In the last decade several methods have been published aiming at obtaining the conformational weights, or percentages (which are the coefficients for a linear combination) of the so-called typical secondary structural elements making up the three-dimensional structure of proteins. Two methods that can be used to determine the secondary structures of proteins are described here. The first method, called LINCOMB, is a simple algorithm based on a leastsquares fit with a set of reference spectra representing the known secondary structures and yielding an estimation of weights attributed to α-helix, β-pleated sheet (mainly antiparallel), β-turns, unordered form, and aromatic/disulfide (or nonpeptide) contributions of the protein being analyzed. This method requires a “template” or reference curve set, which was obtained from the second method. The second method, “convex constraint analysis,” is a general deconvolution method for a CD spectra set of any variety of conformational type. The algorithm, based on a set of three constraints, is able to deconvolute a set of CD curves to its common “pure”-component curves and conformational weights. To analyze a single CD spectrum with this method, the spectrum is appended to the data set used as a reference data set. As a way to determine the reliability of the algorithm and provide a guideline to its usage, some applications are presented.

Deconvolution of the circular dichroism spectra of proteins: the circular dichroism spectra of the antiparallel beta-sheet in proteins.

A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure beta-pleated sheet in globular proteins. On the basis of X-ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:669-679, 1991), was improved by the addition of proteins with high beta-pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (alpha-helix, antiparallel beta-pleated sheet, beta-turns, and unordered conformation), as well as a curve attributed to the "aromatic contribution" in the wavelength range of 195-240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X-ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X-ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the alpha-helical structure, but upon analysis yielded a considerable amount of beta-sheet in agreement with the X-ray structure.

Convex constraint analysis: a natural deconvolution of circular dichroism curves of proteins

A new algorithm, called convex constraint analysis, has been developed to deduce the chiral contribution of the common secondary structures directly from experimental CD curves of a large number of proteins. The analysis is based on CD data reported by Yang, J.T., Wu, C.-S.C. and Martinez, H.M. [Methods Enzymol., 130, 208-269 (1986)]. Application of the decomposition algorithm for simulated protein data sets resulted in component spectra [B (lambda, i)] identical to the originals and weights [C (i, k)] with excellent Pearson correlation coefficients (R) [Chang, C.T., Wu, C.-S.C. and Yang, J.T. (1978) Anal. Biochem., 91, 12-31]. Test runs were performed on sets of simulated protein spectra created by the Monte Carlo technique using poly-L-lysine-based pure component spectra. The significant correlational coefficients (R greater than 0.9) demonstrated the high power of the algorithm. The algorithm, applied to globular protein data, independent of X-ray data, revealed that the CD spectrum of a given protein is composed of at least four independent sources of chirality. Three of the computed component curves show remarkable resemblance to the CD spectra of known protein secondary structures. This approach yields a significant improvement in secondary structural evaluations when compared with previous methods, as compared with X-ray data, and yields a realistic set of pure component spectra. The new method is a useful tool not only in analyzing CD spectra of globular proteins but also has the potential for the analysis of integral membrane proteins.