Russell E. Jacobs

Nuclear magnetic resonance microscopy with 4‐μm resolution: Theoretical study and experimental results

Nuclear magnetic resonance(NMR) microscopy with 4‐μm resolution, a step closer to the 1‐μm resolution with which in vivo cellular imaging would be possible is described. An analysis of the ultimate resolution and voxel size dependent signal‐to‐noise ratio (SNR) in NMR microscopy is presented and experimentally verified. For microscopic scale objects (<1‐mm diameter), the SNR based on the geometrical scale factor (s) is found to be proportional to s n where n<2, rather than n=3 as previously supposed. This comes about because of a drastic reduction in sample noise coupled with a significant sensitivity gain realized in small diameter radio-frequency coils. A new pulse sequence which reduces both diffusion dependent resolution degradation and signal attenuation is presented. The selection of optimal bandwidth and acquisition time for maximal SNR is discussed. Experimental results obtained on both a 2.0‐T whole‐body system and a 7.0‐T small bore system adapted for microscopy indicate the potentials of 4‐μm resolution microscopy with the existing magnets.

Partial specific volumes of lipid and water in mixtures of egg lecithin and water

We describe in this paper measurements of the partial specific volumes of lipid (upsilon L) and water (upsilon W) in mechanical mixtures of egg yolk lecithin and water over a range of hydrations (5-55 wt% water) that includes the region at which excess water appears. upsilon L and upsilon W are found to be very nearly 1 cm3/g over the entire range. The water activities of the mixtures were also determined and found to be the same as for lipid deposited as oriented multilayers on solid substrates.

Statistical distribution of hydrophobic residues along the length of protein chains. Implications for protein folding and evolution.

We consider in this paper the statistical distribution of hydrophobic residues along the length of protein chains. For this purpose we used a binary hydrophobicity scale which assigns hydrophobic residues a value of one and non-hydrophobes a value of zero. The resulting binary sequences are tested for randomness using the standard run test. For the majority of the 5,247 proteins examined, the distribution of hydrophobic residues along a sequence cannot be distinguished from that expected for a random distribution. This suggests that (a) functional proteins may have originated from random sequences, (b) the folding of proteins into compact structures may be much more permissive with less sequence specificity than previously thought, and (c) the clusters of hydrophobic residues along chains which are revealed by hydrophobicity plots are a natural consequence of a random distribution and can be conveniently described by binomial statistics.

The evolution of proteins from random amino acid sequences. I. Evidence from the lengthwise distribution of amino acids in modern protein sequences

SummaryWe examine in this paper one of the expected consequences of the hypothesis that modern proteins evolved from random heteropeptide sequences. Specifically, we investigate the lengthwise distributions of amino acids in a set of 1,789 protein sequences with little sequence identity using the run test statistic (ro) of Mood (1940,Ann. Math. Stat.11, 367–392). The probability density ofro for a collection of random sequences has mean=0 and variance=1 [the N(0,1) distribution] and can be used to measure the tendency of amino acids of a given type to cluster together in a sequence relative to that of a random sequence. We implement the run test using binary representations of protein sequences in which the amino acids of interest are assigned a value of 1 and all others a value of 0. We consider individual amino acids and sets of various combinations of them based upon hydrophobicity (4 sets), charge (3 sets), volume (4 sets), and secondary structure propensity (3 sets). We find that any sequence chosen randomly has a 90% or greater chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. We regard this as strong support for the random-origin hypothesis. However, we do observe significant deviations from the random expectation as might be expected after billions years of evolution. Two important global trends are found: (1) Amino acids with a strong α-helix propensity show a strong tendency to cluster whereas those with β-sheet or reverse-turn propensity do not. (2) Clustered rather than evenly distributed patterns tend to be preferred by the individual amino acids and this is particularly so for methionine. Finally, we consider the problem of reconciling the random nature of protein sequences with structurally meaningful periodic “patterns” that can be detected by sliding-window, autocorrelation, and Fourier analyses. Two examples, rhodopsin and bacteriorhodopsin, show that such patterns are a natural feature of random sequences.

Preparation of multilamellar vesicles of defined size-distribution by solvent-spherule evaporation

A novel method of preparing multilamellar vesicles is described. The process involves dispersing in aqueous solutions small spherules of volatile hydrophobic solvents in which amphipathic lipids are dissolved. The lipids form vesicles when the solvents are evaporated in the proper manner. The resulting vesicles have been characterized morphologically with microscopy and electron microscopy. The method yields multilamellar vesicles with a defined size distribution which can be adjusted by varying the duration of mechanical agitation of the spherules and by varying the concentration of amphipathic lipids in the solvents. This is the first fundamentally new method of multilamellar vesicle preparation since Banghams report in 1965 (Bangham, A.D., Standish, M.M. and Watkins, J.C. (1965) J. Mol. Biol. 13, 238-252).

Observations concerning topology and locations of helix ends of membrane proteins of known structure.

SummaryHydropathy plots of amino acid sequences reveal the approximate locations of the transbilayer helices of membrane proteins of known structure and are thus used to predict the helices of proteins of unknown structure. Because the threedimensional structures of membrane proteins are difficult to obtain, it is important to be able to extract as much information as possible from hydropathy plots. We describe an “augmented” hydropathy plot analysis of the three membrane proteins of known structure, which should be useful for the systematic examination and comparison of membrane proteins of unknown structure. The sliding-window analysis utilizes the floating interfacial hydrophobicity scale [IFH(h)] of Jacobs and White (Jacobs, R.E., White, S.H., 1989.Biochemistry28:3421–3437) and the reverse-turn (RT) frequencies of Levitt (Levitt, M., 1977,Biochemistry17:4277–4285). The IFH(h) scale allows one to examine the consequences of different assumptions about the average hydrogen bond status (h=0 to 1) of polar side chains. Hydrophobicity plots of the three proteins show that (i) the intracellular helix-connecting links and chain ends can be distinguished from the extracellular ones and (ii) the main peaks of hydrophobicity are bounded by minor ones which bracket the helix ends. RT frequency plots show that (iii) the centers of helices are usually very close to wide-window minima of average RT frequency and (iv) helices are always bounded by narrowwindow maxima of average RT frequency. The analysis suggests that side-chain hydrogen bonding with membrane components during folding may play a key role in insertion.

Recent progress in NMR microscopy towards cellular imaging

Recent advances in NMR microscopy based on fundamental physical parameters and experimental factors are discussed. We consider fundamental resolution limits due to molecular diffusion and the experimental system bandwidth, as well as practical resolution limits arising from poor signal-to-noise ratio due to small imaging voxel size and finite line broadening due to signal attenuation brought about by diffusion. Several microscopic imaging pulse sequences are presented and applied to elucidating cellular imaging problems such as the cell lineage patterns in Xenopus laevis embryos. Experimental results obtained with 7.0 T NMR microscopy system are presented.

Three-dimensional microscopy toward in Vivo cellular imaging using NMR tomography

Recent advances in NMR microscopy are discussed on the basis of the fundamental physical parameters and experimental parameters obtained recently. We consider the fundamental resolution limit due to molecular diffusion and experimental bandwidth and the practical resolution limit arising from signal-to-noise ratio and line-broadening factor. Several microscope imaging pulse sequences are presented and applied to elucidate cellular imaging such as the cell lineage patterns in Xenopus laevis embryos. Both basic resolution studies and some applications of the NMR microscopy to the cellular imaging are discussed. Experimental results obtained with a 7.0-T NMR microscopy system developed at the University of California, Irvine, are presented.

Finite element analysis of gradient coil deformation and vibration in NMR microscopy

Resolution degradation due to gradient coil deformation and vibration in NMR microscopy is investigated using finite element analysis. From the analysis, deformations due to the Lorentz force can be as large as 1-10 mum depending on the gradient strength and coil frame material. Thus, these deformations can be one of the major resolution limiting factors in NMR microscopy. Coil vibration, which depends on the input current waveform and resolution degradation due to time-variant deformation and time-invariant deformation are investigated by numerical simulations.