Mapping the amide I absorption in single bacteria and mammalian cells with resonant infrared nanospectroscopy.

Infrared (IR) nanospectroscopy performed in conjunction with atomic force microscopy (AFM) is a novel, label-free spectroscopic technique that meets the increasing request for nano-imaging tools with chemical specificity in the field of life sciences. In the novel resonant version of AFM-IR, a mid-IR wavelength-tunable quantum cascade laser illuminates the sample below an AFM tip working in contact mode, and the repetition rate of the mid-IR pulses matches the cantilever mechanical resonance frequency. The AFM-IR signal is the amplitude of the cantilever oscillations driven by the thermal expansion of the sample after absorption of mid-IR radiation. Using purposely nanofabricated polymer samples, here we demonstrate that the AFM-IR signal increases linearly with the sample thickness t for t > 50 nm, as expected from the thermal expansion model of the sample volume below the AFM tip. We then show the capability of the apparatus to derive information on the protein distribution in single cells through mapping of the AFM-IR signal related to the amide-I mid-IR absorption band at 1660 cm(-1). In Escherichia Coli bacteria we see how the topography changes, observed when the cell hosts a protein over-expression plasmid, are correlated with the amide I signal intensity. In human HeLa cells we obtain evidence that the protein distribution in the cytoplasm and in the nucleus is uneven, with a lateral resolution better than 100 nm.

Electrocatalytic interconversion of NADH and NAD(+) by Escherichia coli flavohemoglobin.

E. coli flavohemoglobin, oriented at electrodes via amphiphilic polymyxin B, electrocatalytically interconverts NADH and NAD(+) at its heme potentials operating as an electron transfer relay between the electrode and the protein FAD, where NADH/NAD(+) is transformed. The results are crucial for the development of NAD(+)-dependent bioelectrodes for biosynthesis, biosensors and biofuel cells.

Folding propensity and biological activity of peptides: New insights from conformational properties of a novel peptide derived from Vitreoscilla haemoglobin.

The synthetic peptide Vitr-p-13 (YPIVGQELLGAIK-NH(2)), derived from the bacterial dimeric Vitreoscilla haemoglobin (VHb) in the position 95-107, is characterized by a pre-eminent "statistical coil" conformation in water as demonstrated by CD experiments and long time-scale MD simulations. In particular, Vitr-p-13 does not spontaneously adopt an alpha-helix folding in water, but it is rather preferentially found in beta-hairpin-like conformations. Long time-scale MD simulations have also shown that Vitr-p-13 displays a "topological-trigger" which initiates alpha-helix folding within residues 7-10, exactly like seen in the temporins, a group of linear, membrane-active antimicrobial peptides of similar length. At variance with temporins, in Vitr-p-13 such a process is energetically very demanding (+10 kJ/mol) in water at 300 K, and the peptide was found to be unable to bind model membranes in vitro and was devoid of antimicrobial activity. The present results, compared with previous studies on similar systems, strengthen the hypothesis of the requirement of a partial folding when still in aqueous environment to allow a peptide to interact with cell-membranes and eventually exert membrane perturbation-related antibiotic effects on target microbial cells.

The distal heme pocket of Escherichia coli flavohemoglobin probed by infrared spectroscopy.

The infrared absorption spectra of ferric cyanide and ferrous carbonmonoxy Escherichia coli flavohemoglobin have been measured in order to probe the fine structural properties of the distal heme pocket, characterized by the presence of a tyrosine in position B10 and a glutamine in position E7. The stretching frequency of iron bound cyanide occurs at 2136 cm(-1), an unusually high value if compared to other heme proteins. The infrared spectrum of the CO bound derivative displays two peaks centered at 1960 cm(-1) and at 1909 cm(-1) respectively. H(2)O effects have been studied in both the ferric cyanide and ferrous CO derivatives in order to establish the presence of a distal hydrogen bonding to the iron bound ligand. The observed isotope shifts indicate that in the ferric cyanide derivative a hydrogen bond is donated from a residue in the distal pocket to the biatomic ligand whereas in the ferrous carbon monoxy derivative only the 1909 cm(-1) component is most likely hydrogen bonded to the phenolic group of TyrB10.