Untitled

Zolla, Lello; Timperio, Anna Maria; Testi, Maria Grazia; Bianchetti, Maria; Bassi, Roberto; Manera, Francesco; Corradini, Danilo

doi:10.1023/a:1006397419473

Cited by 20 publications

(8 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To verify if strong illumination induces degradation of the light harvesting proteins, as observed for core proteins, we have investigated the effect of light on both isolated and aggregated PSII antenna proteins from spinach and pea, using a number of techniques, including HPLC, SDS electrophoresis, mass spectrometry, and EPR. We found that separation of proteins by reversed-phase HPLC, previously developed in our laboratory ( , ), was particularly useful. Besides being more effective than SDS−PAGE electrophoresis, it also allows quantitative evaluation of the relative amount of each protein component.…”

Section: Resultsmentioning

confidence: 95%

Involvement of Active Oxygen Species in Degradation of Light-Harvesting Proteins under Light Stresses

Zolla

Rinalducci

2002

Biochemistry

Self Cite

View full text Add to dashboard Cite

This paper presents evidence for light-mediated degradation of isolated light-harvesting proteins (Lhc2) and involvement of oxygen free radicals in the process. The time course of light harvesting photodestruction is much slower than that of D1 protein (requiring hours for complete breakdown). By use of mass spectrometry and amino acid sequencing, it has been revealed that the primary cleavages take place in the hydrophilic portion of the NH(2) region where oxygen-containing radicals attack randomly and not at specific sites. Moreover, these chlorophyll binding proteins are completely fragmented. From the effectiveness of scavengers and the preliminary electron paramagnetic resonance measurements reported, it appears that singlet oxygen is involved as a short-lived species, and hydroxyl and alkoxyl radicals act at higher light intensity or over a longer time, whereas hydrogen peroxide and superoxide anions are not observed. Antenna proteins appear more resistance to photodestruction in their monomeric form than in trimeric form, while minor antenna are highly sensitive. However, the organization of both minor and major proteins in the photosystem II supracomplex affords some photoprotection. Interestingly, leaves exposed to strong light contained degraded major antenna, unlike those kept in the dark, which is consistent with studies on the illumination of isolated proteins, supporting the hypothesis that active oxygen species play a role in vivo in the short-term acclimative adaptation of plants.

show abstract

Section: Resultsmentioning

confidence: 95%

Involvement of Active Oxygen Species in Degradation of Light-Harvesting Proteins under Light Stresses

Zolla

Rinalducci

2002

Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Isolation (21). The light-harvesting complex was isolated from the PSII membranes as described previously (11) with the following modifications.…”

Section: Methodsmentioning

confidence: 99%

“…Interestingly, the other antenna proteins (Lhcb2, Lhcb4, and Lhcb5) were present only in single copies in all investigated species. The isomers of Lhcb1 were recognized previously because of the result that in most species, the major antenna system of PSII showed more than one major band or peak that could be resolved both by SDS-PAGE in a 15-cm gel (38) or by RP-HPLC (21). However, the difference in apparent mass of ϳ1,000 -2,000 mass units revealed by SDS-PAGE in combination with a specific antibody was too large to be correlated with the DNA sequences for genes of the same type (36).…”

Section: Revelation Of Proteomic Heterogeneity By Rp-hplc-esi-msmentioning

confidence: 99%

“…Traditionally, the protein components of the PS II major and minor antenna system are resolved by SDS-PAGE into several closely migrating protein bands (19,20). Additionally, reversed-phase HPLC (RP-HPLC) has been successfully applied to the fractionation of the various types of antenna proteins (21). Although single stage chromatographic separation systems offer high resolving power, the separation selectivity may not suffice to separate all protein isoforms having very subtle structural differences (22).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Isoforms of Photosystem II Antenna Proteins in Different Plant Species Revealed by Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Huber¹,

Timperio²,

Zolla³

2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The high selectivity offered by reversed-phase highperformance liquid chromatography on-line coupled to electrospray ionization mass spectrometry has been utilized to characterize the major and minor light-harvesting proteins of photosystem II (Lhcb). Isomeric forms of the proteins, revealed either on the basis of different hydrophobicity enabling their chromatographic separation or on the basis of different molecular masses identified within one single chromatographic peak, were readily identified in a number of monocot and dicot species. The presence of several Lhcb1 isoforms (preferably in dicots) can explain the tendency of dicot Lhcb1 to form trimeric aggregates. The Lhcb1 molecular masses ranged from 24,680 to 25,014 among different species, whereas within the same species, the isoforms differed by 14 -280 mass units. All Lhcb1 proteins appear to be highly conserved among different species such that they belong to a single gene group that has several different gene family members. In all species examined, the number of isoforms corresponded more or less to the genes cloned previously. Two isoforms of Lhcb3 were found in petunia and tomato. For Lhcb6, the most divergent of all light-harvesting proteins, the greatest number of isoforms was found in petunia, tobacco, tomato, and rice. Lhcb2, Lhcb4, and Lhcb5 were present in only one form. The isoforms are assumed to play an important role in the adaptation of plants to environmental changes. Photosynthesis in higher plants begins with the absorption of light by the light-harvesting complex of photosystem II (LH-CII)1 through two types of light-harvesting proteins: the major antenna, comprising the proteins Lhcb1, Lhcb2, and Lhcb3, and the minor antenna, comprising the proteins Lhcb4, Lhcb5, and Lhcb6. Although the LHCII has been extensively studied since its discovery more than 35 years ago, there are still several questions about the number of polypeptides it contains and its supramolecular organization. It is known that LHCII apoproteins are encoded by families of nuclear genes (1-3) and over 40 nucleotide sequences encoding LHCII proteins from more than 15 different plant species have been cloned and sequenced (4). Moreover, a large number of protein bands can be resolved by gel electrophoresis from LHCII preparations (3). The precise relationship between the multiple functional roles played by LHCII, the diversity of polypeptides found in the membrane, and the complexity of the gene family that encodes the LHCII apoproteins has not yet been fully established. The observed protein heterogeneity has, in fact, a number of possible origins, thus the different protein isoforms could be: (i) products of distinct genes (5), (ii) post-translational modifications of one or a few primary translation products (6 -8), (iii) cleavage products of a common precursor at successive maturation stages (9), or (iv) artifacts introduced during sample workup and analysis. The antenna system is organized into a mobile complex containing homotrimers of Lhcb1 and heterotrimers co...

show abstract

“…Moreover, these matrices also have excellent chemical stability within the working range of biological separations. In (Table 1) the relative advantages and disadvantages of separations performed by HPLC systems are summarized together with alternative separation systems such as sodium dodecyl sulfate-polyacrylamide gel photosynthetic apparatus [30,35]. Clearly, using organic solvents and acids would cause the biological activity of the protein to be lost.…”

Section: Thylakoid Proteins Separationmentioning

confidence: 99%

Liquid Extraction-Ultracentrifugation-Liquid Chromatography-Mass Spectrometry: A Potent Tool for Separation and Identification of Thylakoid Membrane Proteins

Zolla

2006

CAC

Self Cite

View full text Add to dashboard Cite

This review of current HPLC techniques concludes that they represent a valuable tool for the characterization of virtually any hydrophobic protein, given the wide versatility, relative ease of use, and high resolution of the reversed phase column. Moreover, since the procedure does not destroy the sample, it allows for protein identification by coupling the column outlet on line with a mass spectrometer interfaced with an electrospray source. Thus, using the thylakoid membrane of the photosynthetic apparatus as a model, we have demonstrated that by taking intact mass measurements (IMM), each protein may be identified on the basis of the close correspondence between the molecular masses measured by RP-HPLC-ESI-MS with those expected from the DNA sequence, in those cases where post-translational modifications may be supposed absent. This was corroborated by the evidence that proteins assigned by IMM are also confirmed by 'in solution' trypsin digestion and peptide fragment fingerprinting (PFF) of each protein isolated by RP-HPLC using a preparative scale column. On the other hand, even when denaturated these highly hydrophobic proteins are barely digested, resulting the small number of peptides not sufficient for unequivocal protein identification by mass peptide fingerprinting. Furthermore, because IMM reflects the full protein sequence, it is possible to elucidate in a short time whether the protein has undergone any post-translational modifications. In the presence of single or multiple phosphorylations, for example, it is possible to estimate approximately the amount of phosphorylated protein present as a percentage of the total protein by comparing the intensity of deconvolution of each protein.

show abstract

Untitled

Cited by 20 publications

References 37 publications

Involvement of Active Oxygen Species in Degradation of Light-Harvesting Proteins under Light Stresses

Involvement of Active Oxygen Species in Degradation of Light-Harvesting Proteins under Light Stresses

Isoforms of Photosystem II Antenna Proteins in Different Plant Species Revealed by Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Liquid Extraction-Ultracentrifugation-Liquid Chromatography-Mass Spectrometry: A Potent Tool for Separation and Identification of Thylakoid Membrane Proteins

Contact Info

Product

Resources

About