Biophysical Reviews and Letters

The most important function of proteins may well be to bind to other biomolecules. It has long been felt that kinetic rates of bond formation and dissociation between soluble receptors and ligands might account for most features of the binding process. Only theoretical considerations allowed to predict the behaviour of surface-attached receptors from the properties of soluble forms. During the last decade, experimental progress essentially based on flow chambers, atomic force microscopes or biomembrane force probes allowed direct analysis of biomolecule interaction at the single bond level and gave new insight into previously ignored features such as bond mechanical properties or energy landscapes. The aim of this review is (i) to describe the main advances brought by laminar flow chambers, including information on bond response to forces, multiplicity of binding states, kinetics of bond formation between attached structures, effect of molecular environment on receptor efficiency and behaviour of multivalent attachment, (ii) to compare results obtain by this and other techniques on a few well defined molecular systems, and (iii) to discuss the limitations of the flow chamber method. It is concluded that a new framework may be needed to account for the effective behaviour of biomolecule association.

Antifreeze Proteins (AFPs) modify the growth rates and orientations of ice crystal facets through attainment of the best structural match. The insect-type AFPs have ice binding surfaces (IBS) with regularly spaced binding intervals in two directions that engage the primary ice surfaces and reduce their growth rates preferentially. The primary ice surfaces are kinetically stable and have fixed orientations, since they are strongly bonded in two directions. The fish-type AFPs have one-dimensional helical and irregular globular IBSs that are either linearly extended with regular ice binding intervals, or have ice binding sites lacking spacing regularity. They adjust the orientations of the secondary ice surfaces, that have indeterminate face indices since they are strongly bonded in only one direction. The fish-type AFPs stabilize secondary ice surfaces and adjust their orientations by mimicking strong bonding directions, that are not present in the ice structure. The theory agrees with experimental observation and explains hitherto unexplained phenomena. The observed broad variation in prismatic and, more importantly, pyramidal ice crystallites produced in the presence of fish-type AFPs is explained, since these faces are secondary. The observed crystals triggered by most insect-type AFPs are disk shaped, because they consist of primary ice surfaces. The allegedly exceptional ice pyramids triggered by the insect-type TmAFP are the primary ice pyramid with fixed indices, and entirely different from the pyramids of the fish-type AFPs.

The effect of Antifreeze Protein Type I (AFP I, one type of fish antifreeze protein) on ice crystallization was examined quantitatively based on a "micro-sized ice nucleation" technique. It is found that Antifreeze Proteins can inhibit the ice nucleation process by adsorbing onto both the surface of ice nuclei and that of foreign dusts. This leads to an increase of the ice nucleation barrier and the desolvation kink kinetics barrier. Based on the latest nucleation model, the increases in the ice nucleation barrier and the kink kinetics barrier were measured. This enables us to quantitatively examine the antifreeze mechanism of AFP I.

Recently, the stretched intermediate model was proposed for the B–Z deoxyribonucleic acid (DNA) transition based on simulation results carried out using the Stochastic Difference Equation (SDE) that showed unwinding and elongation of the oligomer during the transition. This model has proven to be successful in resolving the steric dilemma in short oligomers. However, extending the simulation method to larger DNA strands may prove to be computationally challenging. Such difficulty has led us to adopt a mean field approach using phenomenological interaction potentials to simulate the transition. Like the atomistic approach, the SDE simulations based on the mean field approach, also suggest the presence of a stretched intermediate during the transition.

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) is an appropriate method for measuring diffusion constants and the number of fluorescent molecules very close to the coverglass surface. Recently, we have reported the application of TIR-FCS to cell biology, measuring membrane-binding farnesylated green fluorescent proteins (EGFP-F) in living cells. In this research, we measured the signal transduction molecule, protein kinase C (PKC), fused with EGFP in living HeLa cells by using TIR-FCS. We observed two different diffusional mobilities of PKCβII-EGFP, three-dimensional faster diffusion near the plasma membrane and slower lateral diffusion on the plasma membrane after adinosine tri phosphate (ATP) activation. These results indicate that it is possible to use TIR-FCS in the study of molecular dynamics and interactions of signal transduction proteins on the plasma membrane of the living cell.

This organic thin-film systems that are based on silicon-carbon covalent bonds have been shown to lead to densely packed alkyl monolayers that have potential bio-passivation or bio-sensing applications. Presented are electrical impedance spectroscopy (EIS) characterisations of a series of alkyl monolayers [CH₃(CH₂)_mCH=CH₂; m = 7, 9, 11, 13, 15] that were covalently linked to Si(111) wafers. The characterizations reveal capacitance, conductance and geometrical properties of the monolayers. The capacitance properties yield estimates of thicknesses for the monolayers that increase proportionally with each additional CH₂ unit and are consistent with the known physical properties of these films such as dielectric constants and chain canting angles. This study illustrates that EIS charcterizations are able to probe immobilized surfaces on silicon with sub-atomic resolution which is so important in the development of practical bio-passivation or bio-sensing applications.

The lifetime of the asymmetric fundamental stretching 2218 cm^-1 vibration of the anesthetic gas nitrous oxide (N₂O) dissolved in octanol and olive oil is reported. These solvents are model systems commonly used to assess anesthetic potency. Picosecond time-scale molecular dynamics simulations have suggested that protein dynamics or membrane dynamics play a role in the molecular mechanism of anesthetic action. Ultrafast infrared spectroscopy with 100 fs time resolution is an ideal tool to probe dynamics of anesthetic molecules on such timescales. Pump-probe studies centered at the peak of the vibrational band yield a lifetime of 55 ± 1 ps in olive oil and 52 ± 1 ps in octanol. The similarity of lifetimes suggests that energy relaxation of the anesthetic is determined primarily by the hydrophobic nature of the environment, consistent with models of anesthetic action. The results show that nitrous oxide is a good model system for probing anesthetic-solvent interactions using nonlinear infrared spectroscopy.

Macromolecules crowd defined spaces, thereby excluding other like-sized molecules from the volume they occupy. These excluded-volume effect(s) (EVE) are well characterized for intracellular and partially for extracellular compartments such as blood plasma. We showed that EVE in fibroblast culture leads to faster enzymatic procollagen conversion and matrix deposition. Apparently, EVE can be applied to emulate in vivo conditions in an in vitro setting. Thus, we attempted to quantitatively capture the crowding potential of various macromolecules using dynamic light scattering under physiological conditions. We found that charged macromolecules like dextran sulfate (negative, 500 kDa) have a hydrodynamic radii of 46.4 ± 0.3 nm i.e. ~4 fold larger than that of neutral macromolecules like Ficoll (neutral, 400 kDa) and thus show greater EVE potential. At biologically effective concentrations viscosity was not increased. Unexpectedly, we observed a dramatic drop of hydrodynamic radii of all macromolecules tested above a threshold concentration. This suggested a hyper-crowding state in which the crowders compacted themselves mutually. We will use this hyper-crowding threshold to determine retrogradely rules that allow to predict the conditions for optimum crowding effects (such as the half-hyper-crowding concentration) in biological systems. We propose Dynamic Light Scattering (DLS) as a potential tool to estimate EVE in biotechnical applications.