Frequency Dependent Non- Thermal Effects of Oscillating Electric Fields in the Microwave Region on the Properties of a Solvated Lysozyme System: A Molecular Dynamics Study.

The use of microwaves in every day's applications raises issues regarding the non thermal biological effects of microwaves. In this work we employ molecular dynamics simulations to advance further the dielectric studies of protein solutions in the case of lysozyme, taking into consideration possible frequency dependent changes in the structural and dynamic properties of the system upon application of electric field in the microwave region. The obtained dielectric spectra are identical with those derived in our previous work using the Fröhlich-Kirkwood approach in the framework of the linear response theory. Noticeable structural changes in the protein have been observed only at frequencies near its absorption maximum. Concerning Cα position fluctuations, different frequencies affected different regions of the protein sequence. Furthermore, the influence of the field on the kinetics of protein-water as well as on the water-water hydrogen bonds in the first hydration shell has been studied; an extension of the Luzar-Chandler kinetic model was deemed necessary for a better fit of the applied field results and for the estimation of more accurate hydrogen bond lifetime values.

One-shot LC-MS/MS analysis of post-translational modifications including oxidation and deamidation of rat lens α- and ß-crystallins induced by γ-irradiation.

The eye lens is a transparent organ that functions to focus light and images on the retina. The transparency and high refraction of the lens are maintained by the function of α-, ß-, and γ-crystallins. These long-lived proteins are subject to various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, which occur gradually during the aging process. Such modifications, which are generated by UV light and oxidative stress, decrease crystallin solubility and lens transparency, and ultimately lead to the development of age-related cataracts. Here, we irradiated young rat lenses with γ-rays (5-500 Gy) and extracted the water-soluble (WS) and water-insoluble (WI) protein fractions. The WS and WI lens proteins were digested with trypsin, and the resulting peptides were analyzed by one-shot LC-MS/MS to determine the specific sites of oxidation of methionine and tryptophan, deamidation sites of asparagine and glutamine, and isomerization of aspartyl in rat α- and ß-crystallins in the WS and WI fractions. Oxidation and deamidation occurred in several crystallins after irradiation at more than, respectively, 50 and 5 Gy; however, isomerization did not occur in any crystallin even after exposure to 500 Gy of irradiation. The number of oxidation and deamidation sites was much higher in the WI than in the WS fraction. Furthermore, the oxidation and deamidation sites in rat crystallins resemble those reported in crystallins from human age-related cataracts. Thus, this study on post-translational modifications of crystallins induced by ionizing irradiation may provide useful information relevant to the formation of human age-related cataracts.

Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus.

We have identified a single tryptophan (Trp) residue responsible for loss of binding and biological activity upon ultraviolet (UV) light irradiation in MEDI-493, a humanized monoclonal antibody (MAb) against respiratory syncytial virus (RSV). This finding provides a better understanding of structure-function relationship in a 150-kDa protein. Irradiation of MEDI-493 with UV light resulted in spectral changes typical of Trp photoproducts and in a progressive loss of MEDI-493 binding and biological activity as measured by ELISA, Biacore, and cell-based assays. Mass spectrometric characterization of the proteolytic peptides generated from the UV irradiated MEDI-493 confirmed that most methionine (Met) and a few Trp residues were oxidized to various extents upon exposure to UV light. Among Trp residues, only Trp-105, containing the most solvent-exposed indole moiety in MEDI-493 and residing in a complementary-determining region (CDR) of the heavy chain, was significantly oxidized. When bound to a synthetic antigenic peptide, MEDI-493 showed significant resistance toward binding activity loss during UV irradiation. A second MAb (MEDI-524) with Trp-105 replaced by phenylalanine (Phe) showed a similar pattern of Met oxidation, but no loss of binding and biological activity following irradiation. Treatment of both MAbs with Met- and Trp-specific oxidizing reagents showed that oxidation of Trp-105 correlated with the activity loss, whereas Met oxidation did not affect the activity. These results demonstrate that Trp-105 in MEDI-493 is responsible for the UV light-induced effects.

Structures of alpha-type ions formed in the 157 nm photodissociation of singly-charged peptide ions.

One hundred fifty-seven nm photodissociation of singly-charged peptide ions induces the cleavage of alpha-carbon to carbonyl-carbon bonds along the backbone. a(n) + 1 radical ions are observed as the primary photolysis products of peptides with N-terminal arginines in a linear ion trap mass spectrometer. The radical elimination pathways undertaken by the a(n) + 1 radical ions to form more stable even-electron species are studied in hydrogen-deuterium (H/D) exchange experiments. Two types of a(n) ions along with d-type ions are observed as secondary elimination products. The relative abundance of each depends on the C-terminal residue of the radical fragment ion.

Amino acid residues responsible for the meta-III decay rates in rod and cone visual pigments.

Vertebrate retinas have two types of photoreceptor cells, rods and cones, which contain visual pigments with different molecular properties. These pigments diverged from a common ancestor, and their difference in molecular properties originates from the difference in their amino acid residues. We previously reported that the difference in decay times of G protein-activating meta-II intermediates between the chicken rhodopsin and green-sensitive cone (chicken green) pigments is about 50 times. This difference only originates from the differences of two residues at positions 122 and 189 (Kuwayama, S., Imai, H., Hirano, T., Terakita, A., and Shichida, Y. (2002) Biochemistry 41, 15245-15252). Here we show that the meta-III intermediates exhibit about 700 times difference in decay times between the two pigments, and the faster decay in chicken green can be converted to the slower decay in rhodopsin by replacing the residues in chicken green with the corresponding rhodopsin residues. However, the inverse directional conversion did not occur when the two residues in rhodopsin were replaced by those of chicken green. Analysis using chimerical mutants derived from these pigments has demonstrated that amino acid residues responsible for the slow rhodopsin meta-III decay are situated at several positions throughout the C-terminal half of rhodopsin. Considering that rhodopsins evolved from cone pigments, it has been suggested that the molecular properties of rhodopsin have been optimized by mutations at several positions, and the chicken green mutants at two positions could be rhodopsin-like pigments transiently produced in the course of molecular evolution.

Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis.

The characterization of protein modifications is essential for the study of protein function using functional genomic and proteomic approaches. However, current techniques are not efficient in determining protein modifications. We report an approach for sequencing proteins and determining modifications with high speed, sensitivity and specificity. We discovered that a protein could be readily acid-hydrolyzed within 1 min by exposure to microwave irradiation to form, predominantly, two series of polypeptide ladders containing either the N- or C-terminal amino acid of the protein, respectively. Mass spectrometric analysis of the hydrolysate produced a simple mass spectrum consisting of peaks exclusively from these polypeptide ladders, allowing direct reading of amino acid sequence and modifications of the protein. As examples, we applied this technique to determine protein phosphorylation sites as well as the sequences and several previously unknown modifications of 28 small proteins isolated from Escherichia coli K12 cells. This technique can potentially be automated for large-scale protein annotation.

The expression and sequences of T cell antigen receptor beta-chain genes in the thymus at an early stage after sublethal irradiation.

The sequential appearance of the thymocyte subpopulations and TCR gene messages occurred in the thymus of AKR mice (H-2k, Mlsa) from 7 to 14 days after sublethal irradiation. The thymocytes on day 7 after irradiation were composed of a large number of CD4+CD8+ blast-like cells and a relatively high proportion of CD4-CD8- cells (15 to 25%) but few CD3highCD4+CD8-/CD4-CD8+ cells. Approximately 22% of the CD4-CD8- cells were CD3high and -27% of the CD3highCD4-CD8- cells (-6% of whole CD4-CD8- cells) were F23.1+. The thymocytes on day 7 expressed a large amount of gamma- and delta-chain gene transcripts but reduced levels of alpha- and beta-chain gene transcripts. The V gene repertoire of 18 functional beta-chain cDNA derived from the thymocytes on day 7 was compared with those of 20 functional beta-chain cDNA derived from the thymocytes on day 14 which were composed of a large number of CD3lowCD4+CD8+ small-sized cells and a small number of CD3highCD4+CD8- cells. It is noteworthy that the distribution of V beta genes expressed in the thymocytes on day 7 was much the same as that in the thymocytes on day 14 but significantly different from that in normal BALB/c thymocytes as previously described. Interestingly, neither V beta 8.1 nor V beta 6 genes, which are important for recognition of the product of the Mlsa locus, was detected in these two cDNA libraries. These results suggest that clonal selection of TCR V beta repertoire, irrespective of positive or negative selection, appears to occur at the early stage of T cell differentiation, i.e., on the blast-like CD4+CD8+ thymocytes.

[Changes in the collagen amino acid composition of calf skin after gamma-irradiation in an aqueous solution]. / Izmenenie aminokislotnogo sostava kollagena kozhi telenka pri gamma-obluchenii v vodnom rastvore.

A study was made of the amino acid composition of calf skin collagen after gamma-irradiation (60Co) of 2.5 X 10(-6) M aerated aqueous protein solution within the dose range from 30 to 2000 Gy. The radiosensitivity of amino acid residues was compared.