Calorimetric, spectroscopic, and model studies provide insight into the transport of Ti(IV) by human serum transferrin

Evidence suggests that transferrin can bind Ti(IV) in an unhydrolyzed form (without bound hydroxide or oxide) or in a hydrolyzed form. Ti(IV) coordination by N,N'-di(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) at different pH values models the two forms of Ti(IV)-loaded transferrin spectrally and structurally. 13C NMR and stopped-flow kinetic experiments reveal that when the metal is delivered to the protein using an unhydrolyzed source, Ti(IV) can coordinate in the typical distorted octahedral environment with a bound synergistic anion. The crystal structure of TiHBED obtained at low pH models this type of coordination. The solution structure of the complex compares favorably with the solid state from pH 3.0 to 4.0, and the complex can be reduced with E1/2 = -641 mV vs NHE. Kinetic and thermodynamic competition studies at pH 3.0 reveal that Ti(citrate)3 reacts with HBED via a dissociative mechanism and that the stability of TiHBED (log beta = 34.024) is weaker than that of the Fe(III) complex. pH stability studies show that Ti(IV) hydrolyzes ligand waters at higher pH but still remains bound to HBED until pH 9.5. Similarly, at a pH greater than 8.0 the synergistic anion that binds Ti(IV) in transferrin is readily displaced by irreversible metal hydrolysis although the metal remains bound to the protein until pH 9.5. Thermal denaturation studies conducted optically and by differential scanning calorimetry reveal that Ti(IV)-bound transferrin experiences only minimal enhanced thermal stability unlike when Fe(III) is bound. The C- and N-lobe transition Tm values shift to a few degrees higher. The stability, competition, and redox studies performed provide insight into the possible mechanism of Ti2-Tf transport in cells.     

High-order structure and dissociation of gaseous peptide aggregates that are hidden in mass spectra

Injected-ion mobility and high-pressure ion mobility techniques have been used to examine the conformations of bradykinin, insulin chain A, and several other peptide ions in the gas phase. Under the experimental conditions employed, evidence for multimer formation in the mass spectra of peptides is minimal or absent altogether. However, ion mobility distributions show that aggregates of peptides (containing a single charge per monomer unit) are observed at the same mass-to-charge ratios as the singly charged parent ions. Collision cross sections for these clusters show that they have tightly packed roughly spherical conformations. We have bracketed the average density as 0. 87 ρ < ρ < 1. 00 g cm^?3. In some cases, specific stable aggregate forms within a cluster size can be distinguished indicating that some high order structures are favored in the gas phase. Multimer formation between different sizes of polyalanine peptides shows no evidence for size specificity in aggregate formation. Collisional and thermal excitation studies have been used to examine structural transitions and dissociation of the multimers. Aggregates appear to dissociate via loss of singly charged monomers. The observation that peptide multimers can be concealed in mass spectral data requires that fragmentation patterns and reactivity studies of singly charged monomers be undertaken with care.     

Overtone mobility spectrometry: Part 2. Theoretical considerations of resolving power

The transport of ions through multiple drift regions is modeled to develop an equation that is useful for an understanding of the resolving power of an overtone mobility spectrometry (OMS) technique. It is found that resolving power is influenced by a number of experimental variables, including those that define ion mobility spectrometry (IMS) resolving power: drift field (E), drift region length (L), and buffer gas temperature (T). However, unlike IMS, the resolving power of OMS is also influenced by the number of drift regions (n), harmonic frequency value (m), and the phase number (Φ) of the applied drift field. The OMS resolving power dependence upon the new OMS variables (n, m, and Φ) scales differently than the square root dependence of the E, L, and T variables in IMS. The results provide insight about optimal instrumental design and operation.     

Overtone mobility spectrometry: Part 1. Experimental observations

A new method that allows a linear drift tube to be operated as a continuous ion mobility filter is described. Unlike conventional ion mobility instruments that use an electrostatic gate to introduce a packet of ions into a drift region, the present approach uses multiple segmented drift regions with modulated drift fields to produce conditions that allow only ions with appropriate mobilities to pass through the instrument. In this way, the instrument acts as a mobility filter for continuous ion sources. By changing the frequency of the applied drift fields it is possible to tune this instrument to transmit ions having different mobilities. A scan over a wide range of drift field frequencies for a single ion species shows a peak corresponding to the expected resonance time of the ions in one drift region segment and a series of peaks at higher frequencies that are overtones of the resonant frequency. The measured resolving power increases for higher overtones, making it possible to resolve structures that were unresolved in the region of the fundamental frequency. We demonstrate the approach by examining oligosaccharide isomers, raffinose and melezitose as well as a mixture of peptides obtained from enzymatic digestion of myoglobin.     

Magnetic susceptibility-induced alignment of proteins in reverse micelles

Proteins encapsulated within the aqueous core of reverse micelles are found to partially align in a magnetic field. The degree of alignment is sufficient to result in sizable residual 15N-1H dipolar couplings that can be easily measured. It is found that the magnetic susceptibility of the reverse micelle particle is not dominated by the encapsulated protein. The residual dipolar couplings are found to be structurally meaningful.     

Nativelike structure in designed four alpha-helix bundles driven by buried polar interactions

We show that a single internal polar interaction per helix is sufficient to engender structural specificity in that helix in helical bundle proteins. Furthermore, we use histidine-binding cofactors of different shapes which bind directly into the core, demonstrating that this structural specificity is not the result of a prescribed complimentary, "knobs in holes" core packing. We show that we can switch structural specificity of individual helices on and off by ligating cofactors, singly and in pairs, which bind either one or two histidine ligands. To our knowledge, this is the first demonstration of such extensive manipulation of protein structure by ligand binding, an important result of general interest to those working with self-assembled molecular systems. Finally, as these proteins were designed without the use of computational modeling, we not only demonstrate that designing a uniquely structured cofactor binding protein is not as difficult as is generally believed, we have determined why this is so: hydrophobic core complementarity, which is very difficult to design, is not necessary. Instead, a much simpler design process entails the creation of core polar interactions which themselves can drive conformational specificity.     

Thermal lensing, thermal management and transverse mode control in microchip VECSELs

Finite-element analysis is used to explore the practicalities and power-scaling potential of quasi-monolithic microchip vertical-external-cavity surface-emitting lasers: thermal lensing and its implications for transverse mode control are emphasised. A comparison is made between the use of sapphire and diamond heat spreaders. The experimental characterisation of an InGaAs/sapphire microchip VECSEL is presented as an exemplar system and the factors affecting slope efficiency, threshold and output power roll-over are examined. By comparing experimental measurements with the finite-element model, the key role of thermal lensing in transverse mode control is demonstrated. PACS 42.55.Xi; 42.55.Rz; 42.55.Px