Determination of the size distribution of blood microparticles directly in plasma using atomic force microscopy and microfluidics.

Microparticles, also known as microvesicles, found in blood plasma, urine, and most other body fluids, may serve as valuable biomarkers of diseases such as cardiovascular diseases, systemic inflammatory disease, thrombosis, and cancer. Unfortunately, the detection and quantification of microparticles are hampered by the microscopic size of these particles and their relatively low abundance in blood plasma. The use of a combination of microfluidics and atomic force microscopy to detect microparticles in blood plasma circumvents both problems. In this study, capture of a specific subset of microparticles directly from blood plasma on antibody-coated mica surface is demonstrated. The described method excludes isolation and washing steps to prepare microparticles, improves the detection sensitivity, and yields the size distribution of the captured particles. The majority of the captured particles have a size ranging from 30 to 90 nm, which is in good agreement with prior results obtained with microparticles immediately isolated from fresh plasma. Furthermore, the qualitative shape of the size distribution of microparticles is shown not to be affected by high-speed centrifugation or the use of the microfluidic circuit, demonstrating the relative stable nature of microparticles ex vivo.

Study of the binding of single-stranded DNA-binding protein to DNA and poly(rA) using electric field induced birefringence and circular dichroism spectroscopy.

Binding of the single-stranded DNA-binding protein (SSB) of Escherichia coli to single-stranded (ss) polynucleotides produces characteristic changes in the absorbance (OD) and circular dichroism (CD) spectra of the polynucleotides. By use of these techniques, complexes of SSB protein and poly(rA) were shown to display two of the binding modes reported by Lohman and Overman [Lohman, T.M., & Overman, L. (1985) J. Biol. Chem. 260, 3594-3603]. The circular dichroism spectra of the "low salt" (10 mM NaCl) and "high salt" (greater than 50 mM NaCl) binding mode are similar in shape, but not in intensity. SSB binding to poly(rA) yields a complexed CD spectrum that shares several characteristics with the spectra obtained for the binding of AdDBP, GP32, and gene V protein to poly(rA). We therefore propose that the local structure of the SSB-poly(rA) complex is comparable to the structures proposed for the complexes of these three-stranded DNA-binding proteins with DNA (and RNA) and independent of the SSB-binding mode. Electric field induced birefringence experiments were used to show that the projected base-base distance of the complex is about 0.23 nm, in agreement with electron microscopy results. Nevertheless, the local distance between the successive bases in the complex will be quite large, due to the coiling of the DNA around the SSB tetramer, thus partly explaining the observed CD changes induced upon complexation with single-stranded DNA and RNA.

Structure calculations for single-stranded DNA complexed with the single-stranded DNA binding protein GP32 of bacteriophage T4: a remarkable DNA structure.

In this study it is established by calculation which regular conformations single-stranded DNA and RNA can adopt in the complex with the single-stranded DNA binding protein GP32 of bacteriophage T4. In order to do so, information from previous experiments about base orientations and the length and diameter of the complexes is used together with knowledge about bond lengths and valence angles between chemical bonds. It turns out that there is only a limited set of similar conformations which are in agreement with experimental data. The arrangement of neighboring bases is such that there is ample space for aromatic residues of the protein to partly intercalate between the bases, which is in agreement with a previously proposed model for the binding domain of the protein [Prigodich, R. V., Shamoo, Y., Williams, K. R., Chase, J. W., Konigsberg, W. H., & Coleman, J. E. (1986) Biochemistry 25, 3666-3671]. Both C2'endo and C3'endo sugar conformations lead to calculated DNA conformations that are consistent with experimental data. The orientation of the O2' atoms of the sugars in RNA can explain why the binding affinity of GP32 for polyribonucleotides is lower than for polydeoxyribonucleotides.

A refined calculation of the solution dimensions of the complex between gene 32 protein and single stranded DNA based on estimates of the bending persistence length.

The rotation diffusion coefficient of a complex of GP32, the single stranded DNA binding protein of the bacteriophage T4, with a single stranded DNA fragment with about 270 bases was determined to obtain further information on the flexibility of this particle. The rotation diffusion of these molecules is used as a sensitive measure of the flexibility of different DNA protein complexes. Using the theory of Hagerman and Zimm (Biopolymers 20, 1481 (1981)) and assuming a bending persistence length of about 35 nanometer it can be shown that the axial increment for GP32 complexes with single stranded DNA is close to 0.5 nm per base. The value for the bending persistence length is in agreement with values found for much larger DNA protein complexes using light scattering experiments. This value for the persistence length also implies that the complex is thin. The radius is estimated to be around 1.7 nm, which shows a moderate degree of hydration. With this set of parameters we can describe all the hydrodynamic experiments on GP32 complexes from 76 to more than 7000 bases obtained using electric birefringence, quasi-elastic light scattering and sedimentation experiments performed in our group over the last few years.

Complex formation between the adenovirus DNA-binding protein and single-stranded poly(rA). Cooperativity and salt dependence.

The complex formed between adenovirus DNA-binding protein (AdDBP) and poly(rA) was investigated with circular dichroism spectroscopy. The binding process was studied at a variety of salt concentrations, and the titration curves were analyzed according to the contiguous cooperative binding model given by McGhee and von Hippel [McGhee, J.D., & von Hippel, P.H. (1974) J. Mol. Biol. 86, 469-489]. The cooperativity factor omega of the binding process is low (omega approximately 20-30) and independent of the salt concentration. This in contrast to the binding constant K for which a moderately strong salt dependence is observed: delta log (K omega)/delta log [NaCl] = -3.1. The size of the binding site was consistently calculated to be about 13. We also studied the C-terminal 39-kDa fragment which is sufficient for DNA replication in vitro. Complex formation between this fragment of AdDBP and poly(rA) appeared to be characterized by spectroscopic and binding properties similar to those of the intact protein. Only, the binding constant in 50 mM NaCl is a factor of 5 lower.

A model for the complex between the helix destabilizing protein GP32 of bacteriophage T4 and single-stranded DNA.

A model for the structure of the complex between the helix-destabilizing protein of bacteriophage T4, GP32, and single-stranded DNA is proposed. In this model the bases are arranged in a helix, that is characterized by a relatively large distance between successive bases, a substantial base tilt, in combination with a small rotation per base. This helix is further organized into a tertiary structure, possibly a superhelix, of which the corresponding protein shell corresponds to the relatively rigid and rod-like structure that is observed in hydrodynamic experiments. It is proposed that similar structural features apply to other single-stranded DNA binding proteins in complex with polynucleotides.

The internal dynamics of gene 32 protein-DNA complexes studied by quasi-elastic light scattering.

The hydrodynamic properties of large homodisperse single stranded DNAs complexed with the helix destabilizing protein of phage T4, the product of gene 32 (GP32), have been measured. The results suggest a size of the binding site between 8 and 10 nucleotides/GP32 molecule, in reasonable agreement with earlier work on a complex between GP32 and single stranded 145 base DNA. From static light scattering experiments it is concluded that the persistence length of these complexes is about 30 nm, distinctly smaller than the generally accepted value for double stranded DNA. The quasi-elastic light scattering properties of the DNA-GP32 complexes were determined. The variation of the apparent translation diffusion coefficient Dapp with the scattering vector q was analyzed using the discrete ISMF and Rouse-Zimm models [S.C. Lin et al., Biopolymers 17 (1978) 425]. The model parameters that followed from the fit of Dapp versus q2 and from an extensive global analysis of the actually measured autocorrelation functions agreed with the notion that these DNA-protein complexes are indeed rather flexible. The continuous Soda model [K. Soda, Macromolecules 17 (1984) 2365] could successfully explain the variation of Dapp versus q2, assuming a persistence length of 30 nm and a base-base distance in the complex of 0.44 nm.

Linear dichroism of the complex between the gene 32 protein of bacteriophage T4 and poly(1,N6-ethenoadenylic acid).

We performed linear dichroism measurements in compressed polyacrylamide gels on the complex between the helix-destabilizing protein of bacteriophage T4, GP32 and poly(1,N6-ethenoadenylic acid), which is used as a model system for single-stranded DNA. A strong hyperchromism for poly(1,N6-ethenoadenylic acid) in the complex indicates a strongly altered conformation. The positive linear dichroism in the wavelength region where the bases absorb must be explained by a strong tilting of the bases in the complex. This finding is in accordance with results from earlier studies, using electric birefringence and circular dichroism measurements. Our measurements show that the angle between the bases and the local helix axis is 42(+/- 6)degrees. In addition, a pronounced contribution from the tryptophan residues of GP32 can be recognized, indicating that several of these residues have a specific orientation in the complex. The sign of the dichroism due to the tryptophan residues is the same as that due to the DNA bases. However, it is not sufficient to assume that all the observed dichroism is due to one or more intercalated tryptophan residues and there must be one or more additional tryptophan residues that make an angle of less than 40 degrees with the local helix axis. Some possible structures of the DNA-protein complex are discussed.

Hydrodynamic studies of a DNA-protein complex. Dimensions of the complex of single-stranded 145 base DNA with gene 32 protein of phage T4 deduced from quasi-elastic light scattering.

The translational diffusion coefficient of the saturated complex of single-stranded 145 base DNA and the helix-destabilizing protein of phage T4, GP32, can be measured at equilibrium by means of quasi-elastic light scattering. If the complex is considered as a rigid rod one can estimate its dimensions by combining the translational diffusion coefficient with earlier data on rotational diffusion. It was found that the average base-base distance of the 145 base DNA in the complex is between 4.3 and 4.7 A, while the diameter of the complex is between 44 and 68 A. This suggests that the conformation of the complex must be such that a large amount of water is trapped.