1. Quantitative analysis of carrier parameters demonstrates that with decreasing substrate concentration the optimal strength of substrate-carrier interaction which maximizes the flux across the membrane increases and requires less fine-tuning than at higher concentrations of the substrate. Many of the nutrients that a cell needs for its functioning, such as sugars, amino acids, nucleotides, or organic bases, require specialized transporters to cross the cell membrane. The rapid growth of available information characterized as transporter explosion by Uhl and Hartig, has led to creation of the transporter classification system, with division of all transporters into channels and carriers. Channel proteins are mainly considered to function as selective pores which do not need conformational rearrangements at each substrate translocation event. We focused on optimization of carrier-facilitated transport. A carrier transfers substrates via a mechanism that includes at least four steps: i), Binding of the substrate to the carrier on one side of the membrane;ii), carrier conformational change leading to substrate transition to the other side;iii), dissociation of the substrate, and iv), return of the carrier to its initial conformation/position in the membrane. In the human genome there are 43 distinct families of transport systems that comprise >300 isoforms of individual solute carriers. Although the majority of these transport systems is responsible for uptake of specific substrates, a substantial number of transporters are used for uptake of the same solute, and often have an overlapping expression of multiple isoforms that exists in the same cell type. So, the naturally arising question is: Why are there so many transporter isoforms? Here we offer a possible answer by analyzing the carrier facilitated transport with the focus on the optimal efficiency of the transporter. Analytical expressions are derived for the optimal values of i), the dissociation rate constant, and ii), the ratio of the forward and backward rates of the carrier conformational transitions, which maximize the flux. We demonstrate that at lower substrate concentrations stronger substrate binding is required, and that the deviations from optimal interaction become more critical as the substrate concentration increases, i.e., higher concentrations necessitate more precise tuning. Thus, uniporters designed to transport the same molecule in the same cell have to be optimized with different amino-acid sequences, with onegene coding for a uniporter protein that functions most efficiently at high solute concentrations, whereas another gene is coding for the one that is most efficient at low concentrations. Although quantitative analysis of optimization of carrier-facilitated transport was conducted almost 30 years ago for the liquid membranes of extraction technology, to the best of our knowledge it has never been applied to biological carriers. The existence of multiple transporter isoforms that carry the same molecule is well documented for almost any important substrate. Although this variety of isoforms may seem redundant and, in principle, could be explained by the lack of strong evolutionary pressures to decrease the size of the genome, our analysis offers a different possibility. We have demonstrated that transporter efficiency is fine-tuned to specific ranges of substrate concentration, so that different isoforms might be tailored accordingly to adjust their amino-acid composition for the optimal strength of substrate/transporter interactions and the transition rates between different conformations. 2. Negative staining is a pivotal technique for visualizing the averaged structure of protein molecules, counting among its successes the organization of acetylcholine receptors and structural proteins in ribosomes, the structure and flexibility of myosin molecules, and changes in the organization of influenza hemagglutinin with pH. Averaging images of negative stained CaMKII has provided images at a molecular level comparable to those from cryomicroscopy. Averaged images of negative stained bacterial flagellar filaments show individual α-helices. Detailed structure has been less forthcoming when negative staining has been applied to complex structures not amenable to averaging or viruses. In particular, overlap of small components of complex structures, piled layer upon layer, obscures their structure. However, it has been shown that application of single axis tomography to paramyxovirus negatively stained with phosphotungstic acid reveals individual spikes on the surface of the virus. This stain has been used to make three dimensional reconstructions of single synthase molecules. Application of tomography to negatively stained material encounters the limitation that the large electron doses needed to collect a dual axis tomographic series can degrade the negative stain, inducing aggregation. Furthermore, heavy-metal stains, when thick enough to envelop larger structures, can form layers so dense they compromise visualization of the finest specimen details. Here, we report that methylamine tungstate (NanoW) has an electron scattering cross section and resistance to electron irradiation that make it suitable for dual axis tomography of Influenza A virus. The present findings open the way to using negative stain tomography to realize fine molecular detail in individual viral spikes imaged near focus. While breaks in the viral envelop incident to negative stains do not appear to affect the distributions or structure of individual spikes, negative staining itself can distort proteins. Indeed, the structure of influenza A virus depends on the species of negative stain used, raising the issue of how real are the structures revealed by negative stain tomography. For instance, influenza A virus manifests much less pleiomorphism when negative stained with uranyl acetate than with phosphotungstic acid. The numbers and distribution of spikes on the surface of the virus can be compared with results with other techniques. Cryomicroscopy of influenza virus, which is expected to present a realistic picture of the numbers and distribution of spikes, provides sufficient detail after averaging, to compare with the distributions and sizes of the two types of spikes seen in negative stain tomography. The ratio of the small type to the large type of spike in samples from the equator of the negatively stained virus is eight to one. For comparison, counts of spikes on two influenza A viruses viewed by cryomicroscopy yielded ratios of eight to one for one virus and six to one for the other. Thus methylamine tungstate (NanoW) has a density and resistance to electron irradiation that makes it suitable for dual axis tomography of Influenza A virus. The structure of individual spikes on the surfaces of the virus are consistent with their distribution and atomic structure from cryomicroscopy and X-ray crystallography. Negative stain tomography thus opens the door to examining conformations of individual protein molecules, and provides a useful complementary tool for elucidating structures.

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