Lee Makowski

Gap junction structures: V. Structural chemistry inferred from X-ray diffraction measurements on sucrose accessibility and trypsin susceptibility

X-ray diffraction patterns have been recorded from partially oriented specimens of gap junctions isolated from mouse liver and suspended in sucrose solutions of different concentration and thus of different electron density. Analysis of these diffraction patterns has shown that sucrose is excluded from the 6-fold rotation axis of the junction lattice for a length of about 100 A. This indicates that the aqueous channel of the junctions is in the closed, high resistance state in these preparations. Mapping of the sucrose-accessible space in the junction indicates that the cross-sectional area of the channel entrance on the cytoplasmic side of the membrane could be up to five times larger than the area of the transmembrane channel. Sucrose does not penetrate more than 20 A into the membrane along the channel. Apparently the aqueous channel, 8 to 10 A in radius for most of its length, is narrowed or blocked by a small feature about 50 A from the center of the gap. Very close interactions exist between the gap junction protein and the lipid polar head groups on the cytoplasmic surface of the membrane. In this region, the protein intercalates between the polar head groups. These results suggest that the gap junction protein may have a functional two-domain structure. One domain, with a molecular weight of about 15,000, spans one bilayer and half of the gap and is contained largely within a radius of 25 A from the 6-fold axis. The second domain is smaller and occupies the cytoplasmic surface of the gap junction membrane. Trypsin digestion removes about 4000 Mr from the cytoplasmic surface domain of the junction protein. Most of the material susceptible to trypsin digestion is located more than 28 A from the 6-fold axis.

Biological sensors based on Brownian relaxation of magnetic nanoparticles

We experimentally demonstrate a biomagnetic sensor scheme based on Brownian relaxation of magnetic nanoparticles suspended in liquids. The characteristic time scale of the Brownian relaxation can be determined directly by ac susceptibility measurements as a function of frequency. The peak in the imaginary part of the ac susceptibility shifts to lower frequencies upon binding the target molecules to the magnetic nanoparticles. The frequency shift is consistent with an increase of the hydrodynamic radius corresponding to the size of the target molecule.

Multidomain assembled states of Hck tyrosine kinase in solution

An approach combining small-angle X-ray solution scattering (SAXS) data with coarse-grained (CG) simulations is developed to characterize the assembly states of Hck, a member of the Src-family kinases, under various conditions in solution. First, a basis set comprising a small number of assembly states is generated from extensive CG simulations. Second, a theoretical SAXS profile for each state in the basis set is computed by using the Fast-SAXS method. Finally, the relative population of the different assembly states is determined via a Bayesian-based Monte Carlo procedure seeking to optimize the theoretical scattering profiles against experimental SAXS data. The study establishes the concept of basis-set supported SAXS (BSS-SAXS) reconstruction combining computational and experimental techniques. Here, BSS-SAXS reconstruction is used to reveal the structural organization of Hck in solution and the different shifts in the equilibrium population of assembly states upon the binding of different signaling peptides.

Quantitative Assessment of Peptide Sequence Diversity in M13 Combinatorial Peptide Phage Display Libraries

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0+/-1.6% of the random dodecapeptides and 7.9+/-2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a beta-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

A Rapid Coarse Residue-Based Computational Method for X-Ray Solution Scattering Characterization of Protein Folds and Multiple Conformational States of Large Protein Complexes

We present a coarse residue-based computational method to rapidly compute the solution scattering profile from a protein with dynamical fluctuations. The method is built upon a coarse-grained (CG) representation of the protein. This CG representation takes advantage of the intrinsic low-resolution and CG nature of solution scattering data. It allows rapid scattering determination from a large number of conformations that can be extracted from CG simulations to obtain scattering characterization of protein conformations. The method includes several important elements, effective residue structure factors derived from the Protein Data Bank, explicit treatment of water molecules in the hydration layer at the surface of the protein, and an ensemble average of scattering from a variety of appropriate conformations to account for macromolecular flexibility. This simplified method is calibrated and illustrated to accurately reproduce the experimental scattering curve of Hen egg white lysozyme. We then illustrated the applications of this CG method by computing the solution scattering patterns of several representative protein folds and multiple conformational states. The results suggest that solution scattering data, when combined with the reliable computational method that we developed, show great potential for a better structural description of multidomain complexes in different functional states, and for recognizing structural folds when sequence similarity to a protein of known structure is low.

Simulated x-ray scattering of protein solutions using explicit-solvent models

X-ray solution scattering shows new promise for the study of protein structures, complementing crystallography and nuclear magnetic resonance. In order to realize the full potential of solution scattering, it is necessary to not only improve experimental techniques but also develop accurate and efficient computational schemes to relate atomistic models to measurements. Previous computational methods, based on continuum models of water, have been unable to calculate scattering patterns accurately, especially in the wide-angle regime which contains most of the information on the secondary, tertiary, and quaternary structures. Here we present a novel formulation based on the atomistic description of water, in which scattering patterns are calculated from atomic coordinates of protein and water. Without any empirical adjustments, this method produces scattering patterns of unprecedented accuracy in the length scale between 5 and 100 A, as we demonstrate by comparing simulated and observed scattering patterns for myoglobin and lysozyme.

Biological sensing with magnetic nanoparticles using Brownian relaxation (invited)

Magnetic nanoparticles coated with biochemical ligands are enabling many biological and medical applications. In particular biomagnetic sensors have potential advantages of simplicity and rapidity. We demonstrate a substrate-free biomagnetic sensing approach using the magnetic ac susceptibility of ferromagnetic particles suspended in a liquid. The magnetic relaxation of these particles is mainly due to Brownian rotational diffusion, which can be modified by binding the particles to the intended target. This scheme has several advantages: (i) it requires only one binding event; (ii) there is an inherent check of integrity; and (iii) the signal contains additional information about the target size.

The symmetries of filamentous phage particles.

Abstract The helical symmetries of two classes of filamentous bacteriophage particles are distinctly different. The symmetry of the class I particles is † C 5 S ~2.0 (a 5-fold rotation axis combined with an approximately 2-fold screw axis). The symmetry of the class II particles is C 1 S 5.4 (a one-start helix with 27 subunits equally spaced along five turns). The same basic α-helical interlocking arrangement of the largely α-helical coat protein subunits can be accommodated by the symmetry of the two classes of phage particles. The conservation of this structural pattern reflects intrinsic packing properties of α-helices. The difference between the symmetries of the class I and class II particles suggests that different assembly processes may have evolved to form these structures with very similar protein packing architectures.

Investigating Monoclonal Antibody Aggregation Using a Combination of H/DX‐MS and Other Biophysical Measurements

To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is, dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry and other biophysical measurements was used to make the comparison. The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen/deuterium exchange as compared with its dimer form. In this case, differences observed were located in specific functional regions of the CH 2 domain and the hinge region between CH 1 and CH 2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.