maenas (intertidal zone) and Segonzacia mesatlantica - Station ...
Share Embed Flag
Download ePaper

maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

maenas (intertidal zone) and Segonzacia mesatlantica - Station ... maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

sb.roscoff.fr
from sb.roscoff.fr More from this publisher
24.06.2014 Views

The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 153 Light scattering is a phenomenon directly related to the mass of protein molecules in solution. As an electromagnetic wave passes through the particle, the oscillating electromagnetic field induces an oscillating dipole in the particle itself which in return reemits an electromagnetic wave: a particle excited by a laser beam will remit (scatter) light in all directions. Scattered intensity increases with increasing mass and concentration of the particles. Scattered intensity is also direction dependent. Small particles will scatter light equally in all directions while bigger particles will scatter light in an anisotropic way, depending on their size and shape. This information can be used either statically (static light scattering - SLS) by averaging the light intensity for estimating the mass of the scattering particle or dynamically (dynamic light scattering - DLS), by analyzing the fluctuation of the scattered light, for computing the hydrodynamic radius of the scattering particle (under Brownian motion hypothesis). The principle of Multi-Angle Laser Light Scattering (MALLS) is to illuminate a sample cell with a laser beam and to detect the intensities scattered by the particles at several angles around the cell. A relationship exists between scattered intensity at a given angle, molar mass, concentration, size and shape of the particle: when measuring the concentration by the mean of a refractive index (RI) detector, the other parameters can be deduced from the light-scattering pattern. The amount of scattered light is directly proportional to the product of the weight-average molar mass and the solute concentration: LS~Mw.c. This relationship is based on Zimm’s formalism of the Rayleigh Debye-Gans light scattering model for dilute polymer solutions [38-43]. While lowangle light scattering only provides mass determination, multi-angle detection also allows determination of size and shape. Since scattering intensity is strongly dependent on particle radius, a small amount of large materials in the sample would give a large response with the light scattering detector, although their concentration, as measured by UV or RI response, is very small. The MALLS detector is much less sensitive to small molecules [34]. To obtain accurate measurements, LS experiments must be carried out with a particle sorting system connected before the actual LS device. If no separation is performed before LS analysis, then average values of molar mass and root mean square (rms) radius are determined for all particles in the sample. The necessity for a stable scattering baseline when performing acquisition forbids use of solvent gradient as in affinity or reverse-phase chromatography. Commonly used systems are size-exclusion chromatography (SEC) or field flow fractionation (FFF) in which the eluting solvent has a constant composition. The quality of LS measurements depends on the separation performed before analysis: if two molecular species co-elute or overlap during elution, the molar mass values will be averaged and no precise determination can be made. Heterogeneity in a seemingly pure elution peak can be assessed by calculating the polydispersity over the peak. To obtain reliable values for masses and rms radius with MALLS the choice of the fitting method is very important. Three methods are usually employed (Zimm, Debye and Berry methods). They rely on plotting a function of scattered intensities versus sin 2 (/2) where is the scattering angle and on extrapolating the curves towards 0° angle value. Mass is calculated from the intercept and rms radius from the slope at 0°. If the molecules are small (

154 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al. tion mass spectrometry (ESI-MS) is well suited for the detection of noncovalent protein complexes and opened a new era in protein characterization due to the high accuracy of molecular mass determination [50, 51]. Compared to MALDI, ESI is a gentle method of ionization-desorption of the sample at atmospheric pressure. In the last decades, it was successfully applied to biomolecules when it was made possible to transfer macromolecules into a gas phase. The inherently mild ionization has allowed the successful examination of noncovalent interactions of proteins with ligands, cofactors and prosthetic groups, including peptides, with other proteins and of the quaternary structure of proteins [5, 50-54]. Until ten years ago, observation of large noncovalent multi-protein complexes in aqueous solutions at neutral pH has been limited by the upper m/z range of about 4000 available on most commercial mass spectrometers. The development of orthogonal time-of-flight (ToF) mass analyzers [55, 56] and their commercial exploitation has dramatically extended the experimentally available m/z range to at least 25000. At the present time, the most appropriate mass spectrometers for studying proteins and protein complexes under native conditions have a time-offlight analyzer [56], with a theoretically unlimited m/z range in addition to parallel detection ensuring high sensitivity [55]. Hybrid instruments of the quadrupole-time-of-flight geometry are also well-suited for the analysis of native proteins and protein complexes [57]. In addition, nanospray development [58] has allowed the introduction of small amounts of sample and enabled the determination of the masses of very large protein complexes such as GroEL [59], the intact MS2 virus capsid [60] and ribosome [61]. Under standard denaturing conditions, the protein is dissolved in a mixture of water and organic solvent such as acetonitrile or methanol, with a trace of an added organic acid (e.g. formic acid). The resulting solution has a pH~3.0. Under these conditions noncovalent bonds are broken, the protein sample is protonated and generates an m/z spectrum that has a wide distribution of multiply charged ions from which the mass of the protein can be calculated according to the formula m /z = M + nH + , where M is the molecular mass of n the protein and n is the number of protons associated with it. The mass accuracy of the ESI-MS analysis under these denaturing conditions is typically 0.01% of the protein mass using a modern mass spectrometer. In addition, as ESI-MS analyses proteins from a liquid phase, the solvent conditions can be adapted so that the protein can be studied under more physiological conditions, for example in water alone or more typically in an aqueous, volatile buffer at pH 6-8. The use of a buffered solution at neutral pH is a typical method permitting preservation of the native state of the protein in solution, which is required for successful analysis of noncovalent macromolecular complexes. The solvent must not contain too much inorganic salts which would result in low signal/noise ratio, but must be of enough ionic strength to maintain the assembly. Besides, the use of a buffer can add a variety of metallic or other ions or small molecules to the analyte that may complicate the spectra due to formation of adduct with the protein. The choice of buffer and data interpretation are thus of paramount importance [62]. The two most commonly used buffers are ammonium acetate and ammonium hydrogen carbonate, due to their pH range and volatility, at concentrations of 1-100mM. In such cases, as the protein remains in its native, folded state, the generated m/z spectrum exhibits a narrower distribution of charge states. In general the protein is less charged because protonation is less efficient than in its denatured state. Noncovalent analysis requires adjusted parameters of the ESI source such as increased pressure in the nebulization chamber, decreased declustering potential and extended m/z detection range. For respiratory pigments, samples are usually dissolved in aqueous ammonium acetate 10 mM, pH 6.8 [62]. It has been shown that the experimental masses decreased slightly with increased declustering potential (60 to 160 V) and were generally 0.1 to 0.2 % higher than the calculated masses, due probably to complexation with cations and water molecules [63]. Such data not only allow the mass of the protein to be calculated, but also enable an assessment of the protein’s conformation to be made due to the preservation of its tertiary structure during the analysis [64]. Co-populated protein conformers can often be identified if each population gives rise to a unique charge state distribution [5, 65, 66]. The evaluation of quaternary structures such as noncovalently bound macromolecular protein complexes and the interaction of proteins with DNA, RNA and other ligands are also within reach. The first examples of non-covalently bound protein complexes monitored by ESI-MS were reported in 1991 [5, 52, 67], just two years after ESI-MS development. Since then, there has been significant progress in this field: many instrumental developments have been made and a wealth of examples of quaternary structures increasing in size and complexity has accumulated. The high precision of the method also allows determination of post-translational modifications such as glycosylation [68, 69]. By using reducing conditions, occurrence of intraand inter-chain disulfide bridges can be investigated and monomers from covalent complexes identified. Carbamidomethylation of free cysteine (Cys) can provide the number of free Cys residues. Characteristic differences in successive species masses can be linked with modifications such as methylation, phosphorylation and others. As the intensity of the detected signal depends on the ionization behavior of the molecule, quantification from deconvoluted spectrum should be approached with extreme care. No absolute quantification can be made but abundance of identical species in different samples can be compared. The technique is of great interest when studying macromolecular complexes as it gives insight into the subunit composition and their structural relation (disulfide bridges) [70]. From the subunit masses models can be constructed provided the macromolecular complex mass is known. Noncovalent ESI-MS was recently successfully applied to invertebrate hemoglobins [63, 71, 72] and to crustacean hemocyanins [62, 73, 74]. An important issue in noncovalent ESI-MS is whether the detected species are relevant to the species present in biological conditions. Since different buffer conditions (e.g. pH, ionic strength) are sufficient to induce dissociation or aggregation in solution, the question of the effect of a trans- 75

The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein <strong>and</strong> Peptide Science, 2008, Vol. 9, No. 2 153<br />

Light scattering is a phenomenon directly related to the<br />

mass of protein molecules in solution. As an electromagnetic<br />

wave passes through the particle, the oscillating electromagnetic<br />

field induces an oscillating dipole in the particle itself<br />

which in return reemits an electromagnetic wave: a particle<br />

excited by a laser beam will remit (scatter) light in all directions.<br />

Scattered intensity increases with increasing mass <strong>and</strong><br />

concentration of the particles. Scattered intensity is also direction<br />

dependent. Small particles will scatter light equally<br />

in all directions while bigger particles will scatter light in an<br />

anisotropic way, depending on their size <strong>and</strong> shape. This<br />

information can be used either statically (static light scattering<br />

- SLS) by averaging the light intensity for estimating the<br />

mass of the scattering particle or dynamically (dynamic light<br />

scattering - DLS), by analyzing the fluctuation of the scattered<br />

light, for computing the hydrodynamic radius of the<br />

scattering particle (under Brownian motion hypothesis).<br />

The principle of Multi-Angle Laser Light Scattering<br />

(MALLS) is to illuminate a sample cell with a laser beam<br />

<strong>and</strong> to detect the intensities scattered by the particles at several<br />

angles around the cell. A relationship exists between<br />

scattered intensity at a given angle, molar mass, concentration,<br />

size <strong>and</strong> shape of the particle: when measuring the concentration<br />

by the mean of a refractive index (RI) detector, the<br />

other parameters can be deduced from the light-scattering<br />

pattern. The amount of scattered light is directly proportional<br />

to the product of the weight-average molar mass <strong>and</strong> the solute<br />

concentration: LS~Mw.c. This relationship is based on<br />

Zimm’s formalism of the Rayleigh Debye-Gans light scattering<br />

model for dilute polymer solutions [38-43]. While lowangle<br />

light scattering only provides mass determination,<br />

multi-angle detection also allows determination of size <strong>and</strong><br />

shape. Since scattering intensity is strongly dependent on<br />

particle radius, a small amount of large materials in the sample<br />

would give a large response with the light scattering detector,<br />

although their concentration, as measured by UV or<br />

RI response, is very small. The MALLS detector is much<br />

less sensitive to small molecules [34].<br />

To obtain accurate measurements, LS experiments must<br />

be carried out with a particle sorting system connected before<br />

the actual LS device. If no separation is performed before<br />

LS analysis, then average values of molar mass <strong>and</strong> root<br />

mean square (rms) radius are determined for all particles in<br />

the sample. The necessity for a stable scattering baseline<br />

when performing acquisition forbids use of solvent gradient<br />

as in affinity or reverse-phase chromatography. Commonly<br />

used systems are size-exclusion chromatography (SEC) or<br />

field flow fractionation (FFF) in which the eluting solvent<br />

has a constant composition. The quality of LS measurements<br />

depends on the separation performed before analysis: if two<br />

molecular species co-elute or overlap during elution, the<br />

molar mass values will be averaged <strong>and</strong> no precise determination<br />

can be made. Heterogeneity in a seemingly pure elution<br />

peak can be assessed by calculating the polydispersity<br />

over the peak.<br />

To obtain reliable values for masses <strong>and</strong> rms radius with<br />

MALLS the choice of the fitting method is very important.<br />

Three methods are usually employed (Zimm, Debye <strong>and</strong><br />

Berry methods). They rely on plotting a function of scattered<br />

intensities versus sin 2 (/2) where is the scattering angle<br />

<strong>and</strong> on extrapolating the curves towards 0° angle value. Mass<br />

is calculated from the intercept <strong>and</strong> rms radius from the slope<br />

at 0°. If the molecules are small (

logo

Produits

Ressources

Entreprises

Terms of service
Privacy policy
Cookie policy
Cookie settings
Imprint
Change language
Made with love in Switzerland
© 2024 Yumpu.com all rights reserved

Hooray! Your file is uploaded and ready to be published.

Saved successfully!

Ooh no, something went wrong!