Acceleration of catalysis in dihydrofolate reductase by transient, site-specific photothermal excitation.

We have studied the role of protein dynamics in chemical catalysis in the enzyme dihydrofolate reductase (DHFR), using a pump-probe method that employs pulsed-laser photothermal heating of a gold nanoparticle (AuNP) to directly excite a local region of the protein structure and transient absorbance to probe the effect on enzyme activity. Enzyme activity is accelerated by pulsed-laser excitation when the AuNP is attached close to a network of coupled motions in DHFR (on the FG loop, containing residues 116-132, or on a nearby alpha helix). No rate acceleration is observed when the AuNP is attached away from the network (distal mutant and His-tagged mutant) with pulsed excitation, or for any attachment site with continuous wave excitation. We interpret these results within an energy landscape model in which transient, site-specific addition of energy to the enzyme speeds up the search for reactive conformations by activating motions that facilitate this search.

Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates.

Functional enzyme-nanoparticle bioconjugates are increasingly important in biomedical and biotechnology applications such as drug delivery and biosensing. Optimization of the function of such bioconjugates requires careful control and characterization of their structures and activity, but current methods are inadequate for this purpose. A key shortcoming of existing approaches is the lack of an accurate method for quantitating protein content of bioconjugates for low (monolayer) surface coverages. In this study, an integrated characterization methodology for protein-gold nanoparticle (AuNP) bioconjugates is developed, with a focus on site-specific attachment and surface coverage of protein on AuNPs. Single-cysteine-containing mutants of dihydrofolate reductase are covalently attached to AuNPs with diameters of 5, 15, and 30 nm, providing a range of surface curvature. Site-specific attachment to different regions of the protein surface is investigated, including attachment to a flexible loop versus a rigid α helix. Characterization methods include SDS-PAGE, UV-vis spectrophotometry, dynamic light scattering, and a novel fluorescence-based method for accurate determination of low protein concentration on AuNPs. An accurate determination of both protein and AuNP concentration in conjugate samples allows for the calculation of the surface coverage. We find that surface coverage is related to the surface curvature of the AuNP, with a higher surface coverage observed for higher surface curvature. The combination of these characterization methods is important for understanding the functionality of protein-AuNP bioconjugates, particularly enzyme activity.

A rapid ambient ionization-mass spectrometry approach to monitoring the relative abundance of isomeric glycerophospholipids.

Glycerophospholipids with two, non-equivalent fatty acyl chains can adopt one of two isomeric forms depending on the relative position of substitutions on the glycerol backbone. These so-called sn-positional isomers can have distinct biophysical and biochemical behaviors making it desirable to uniquely assign their regiochemistries. Unambiguous assignment of such similar molecular structures in complex biological extracts is a significant challenge to current analytical technologies. We have recently reported a novel mass spectrometric method that combines collision- and ozone-induced dissociation in series (CID/OzID) to yield product ions characteristic of acyl chain substitution patterns in glycerophospholipids. Here phosphatidylcholines are examined using the CID/OzID protocol combined with desorption electrospray ionization (DESI) to facilitate the rapid exploration of sample arrays comprised of a wide variety of synthetic and biological sources. Comparison of the spectra acquired from different extracts reveals that the sn-positional isomers PC 16:0/18:1 and PC 18:1/16:0 (where the 18:1 chain is present at the sn-2 and sn-1 position of the glycerol backbone, respectively) are most often found together in lipids of either natural or synthetic origin. Moreover, the proportions of the two isomers vary significantly between extracts from different organisms or even between adjacent tissues from the same organism.

Combining liquid chromatography with ozone-induced dissociation for the separation and identification of phosphatidylcholine double bond isomers.

Revealing the inherent molecular diversity of lipid biology requires advanced analytical technologies. Distinguishing phospholipids that differ in the position(s) of carbon-carbon double bonds within their acyl chains presents a particular challenge because of their similar chromatographic and mass spectral behaviours. Here-for the first time-we combine reversed-phase liquid chromatography for separation of isomeric phospholipids with on-line mass spectral analysis by ozone-induced dissociation (OzID) for unambiguous double bond position assignment. The customised tandem linear ion-trap mass spectrometer used in our study is capable of acquiring OzID scans on a chromatographic timescale. Resolving the contributions of isomeric lipids that are indistinguishable based on conventional mass spectral analysis is achieved using the combination of liquid chromatography and OzID. Application of this method to the analysis of simple (egg yolk) and more complex (sheep brain) extracts reveals significant populations of the phosphatidylcholine PC 16:0_18:1(n-7) alongside the expected PC 16:0_18:1(n-9) isomer.

Emerging mass spectrometry-based technologies for analyses of chromatin changes: analysis of histones and histone modifications.

Mass spectrometry (MS) is rapidly becoming an indispensable tool for the analysis of posttranslational modifications (PTMs) of proteins, and particularly histone PTMs that regulate physiological processes. The more traditional bottom-up approach of searching for modifications on peptides rather than intact proteins (top-down) has proven useful for finding phosphorylation, acetylation, and ubiquitination sites. With the use of modern instrumentation and various MS-based techniques, peptides and their PTMs can be characterized in a high-throughput manner while still maintaining high sensitivity and specificity. In complement to bottom-up MS, recent advances in MS technology, such as high-field Fourier transform ion cyclotron resonance (FTICR)-mass spectrometry, have permitted the study of intact proteins and their modifications. On-line and off-line protein separation instruments coupled to FTICR-MS allow the characterization of PTMs previously undetectable with bottom-up approaches. The use of unique fragmentation techniques in FTICR-MS provides a viable option for the study of labile modifications. In this chapter, we provide a detailed description of the analytical tools - mass spectrometry in particular - that are used to characterize modifications on peptides and proteins. We also examine the applicability of these mass spectrometric techniques to the study of PTMs on histones via both the bottom-up and top-down proteomics approaches.