Blog Intro

This blog shows information about nucleic acids and proteins in soil.
The great challenge in microbial ecology of the inaugural decade of the
twenty-first century is to resolve and then understand the linkage between
microbial community structure and its function. This goal is based on the
fact that the primary value of microbes is their function and the belief that
understanding the composition, relative abundance, biochemical diversity
and dynamics of the microbial community will help us understand and
eventually predict or manage function in microbial communities of importance.
However, microbial communities are very diverse and complex,
and vary at very small scales and in which cells are active. Furthermore,
measurement of microbial activities at the relevant spatial and temporal
scales, especially in soil, is no less challenging. Hence, linking microbial
community structure and function in predictive ways must be considered
the Decadal Grand Challenge! The desire to link function to structure derives from microbial diversity,i.e. that microbes vary enormously in their biochemical potential.
This is understandable. They have been on earth for 3.8 billion years and
have exploited most energy niches and adapted to a tremendous range of
conditions. Their contemporary progeny carry the genetic heritage from
that extensive diversification and selection. The extant diversification represents
a continuum from a range of major features that we first think
about as functional diversity such as differences in energy-producing redox
couples, metabolic pathways, morphological adaptations (e.g. spores, filaments,
motility, surface-to-volume ratio), and tolerance to environmental
stresses such as desiccation, pHand temperature extremes, salt, and growth
kinetics (r and K selected populations). However, diversification has also
produced a number of other variants that can have major effects on function
but may bemore difficult to recognise in community structure studies
such as differences in enzyme kinetics (e.g. Km, Vmax, Ki), gene regulation
(signal transduction, regulatory networks, response time, specificity), enzyme
stability/turnover, rate and mechanisms of genetic change, and gene
redundancy in the same organism (orthologues, paralogues, isozymes).
The combination of these traits in members of the populations can create
large differences in functional diversity, e.g. low Km, psychroactive, with
rapid induction response populations, versus others that vary in these and
other traits. When certain traits of diversity can be associated with a particular
condition, usually one offering a competitive advantage, we then
have a useful linkage of community structure and function.
Given the small size of the organisms and the tendency to attach to surfaces,
individual microbial cells interact with one another only over short
distances controlled by diffusional processes and gradients. Direct competition
among microorganisms in soil is limited by spatial isolation and
resource heterogeneity among various habitats thus resulting in communities
with equally abundant and uniformly distributed bacterial species
.A two-species test of the hypothesis that spatial isolation influences microbial diversity in
soil indicates that spatial isolation, created by low moisture content, plays
an important role in structuring soil microbial communities. Habitats such as soil should also support a large number of different species expressing significant niche overlap.Mechanisticmodels for the differential performance of species over time within communities and experimental data predict that over time the average niche breadth of individualswill decrease resulting in an increased proportion of specialists.The course of succession will be constrained by selective pressures of physical,chemical and biological conditions existing within a given niche. The balancebetween generalistsandspecialists isimportant as anunpredictable extinction of community members dominated by generalists may hamper community function to a greater extent than in a community comprised of
specialists. The large number of organisms capable of performing individual
biochemical reactions enhances the probability of restoring functions
by replacing extinct, diminished or inactive populations .However, the relationship between microbial diversity–community structure and soil processes was found to be non-linear as many processes are carried out in specialised microbial consortia. Small linear changes in
microbial diversitymay result in non-linear changes in processes, especially
if the number of organisms drops below the number of original functional groups of organisms in a sample .The redundancy of function within a community serves to dampen the loss of ecosystem function in two ways: by providing functional redundancy for
the same pathway/enzyme type or by different pathways that achieve the
same processing goal. Diversity seems to contribute beneficially to redundancy
by providingmultiple ecotypes capable of performing an equivalent
function albeit with a different community structure. Under perturbed
conditions, however, significant deviations in functional traits are possible
and, as the perturbation becomesmore severe, the deviationmore likely. In
these cases numerically minor but important populations can bloom and
dominate function .In short, soil microbial diversity is enormous, the environmental responses and interactions are complex, but we are beginning to understand the causes for
some of the simpler cases.

Analysis of spatial distribution of bacteria at microhabitat levels revealed that 80% of the bacteria in fertilised soils were located in micropores of
stable soil aggregates suggesting that these habitats offer the most favourable conditions for microbial growth (water and substrate availability, gas diffusion, predation protection). Nunan
showed that the degree of randomness in the distribution of bacteria is greater in the subsoil than in topsoil suggesting that bacteria have a hot-spot distribution in bulk soil, with the hot spots being smaller in the subsoil than in the topsoil. Sessitsch showed that microbial
diversity increases with decreasing particle size and that particle size has a more notable impact on microbial diversity and community structure than does bulk pH and the amount and type of organic substrate. Other investigations of different soil types found that the type and amount of
available organic substrates strongly influence the abundance of microbial groups and their functional diversity in soil ecosystems .An emerging theme is that structure–function interactions and vice versa are not easily predicted as either can produce positive, neutral or negative effects on the other depending on the environmental context. In this respect, ecological responses tomultiple stressors over long time scales have yet to be thoroughly explored.

Protein Purification(PP 1)

There are various methods that separate proteins according to distinct chemical and physical properties. Proteins can be purified by sequentially applying these techniques. Before purifying a protein one needs to consider how much protein is needed, how pure it needs to be, whether the purified protein needsto be in its native configuration, or express an activity.

These considerations will be determined by the ultimate use for the purified protein. If the purified protein is going to be used in functional and structural studies,isolating the protein in its native and active configuration will be important. However, if the goal is to determine a partial amino acid sequence then concerns about denaturation are minimal.

The optimal method to purify a protein is empirically determined by carrying out smallscale pilot studies. After the optimal conditions for a particular purification step is worked out, then the procedures can be scaled up. The order in which the steps are carried out will also affect the final outcome. In general, though, it is best to start with high capacity methods that are easily and quickly carried out and then proceed to high resolution/low capacity methods.

Capacity refers to how much total protein can be readily accommodated and
resolution refers to the ability to separate the protein of interest from contaminating proteins. Generally, as the resolution of the technique increases,
the time required to carry out the technique increases and the protein capacity decreases. One exception is that gel filtration is not usually ahigh resolution technique, as well as being low capacity and relatively difficult to carry out.

The capacity and resolution of affinity chromatography depends on the ligand. Some ligands afford both high capacity and high resolution. The resolution of any of the chromatographic technique can be substantially enhanced through the use of HPLC. However, the capacity of HPLC columns is much lower so that it may not be useful until later in the purification protocol.

PP 1 - MICROSEQUENCING AND PEPTIDE MAPPING

A partial amino acid sequence can provide information about a proteins
used in the design of recombinant DNA probes. It is possible to obtain partial sequence data on as little as 10-100 pmoles of protein, which corresponds to 0.5-5 mg of a 50 kDa protein.

The N-terminus of proteins can be sequenced following separation by gel electrophoresis.This obviates the need to purify the protein to near homogeneity and allows one to take advantage of the high resolution of SDS gel electrophoresis. The requirements are sufficient protein which does not co-migrate with contaminating proteins .Proteins are electrophoretically transferred from the gels to a membrane support. The protein of interest is detected by staining and the band excised. Following extensive washing in water, the protein is sequenced directly from the membrane.

In some cases the N-termini of proteins are 'blocked' or internal sequence information is wanted. In such cases, proteins can be cleaved at specific residues through the use of proteases or chemical means. For example, CNBr cleaves proteins on the C-terminal side of cysteine residues. Many protease recognized specific amino acid residues and reproducibly cleaveproteins at specific sites. The resulting polypeptides are then isolated , absorbed onto glass fiber filters as a solid support, and the N-termini sequenced.

Peptide mapping. It is also possible to use site specific proteases to compare proteins and determine if they are related. The general procedure is to digest a protein with a protease and characterize the pattern of peptides formed. Each protein will exhibit a unique pattern of peptides. Such procedures are known as 'protein fingerprinting' or 'peptide mapping'. The peptides can be analyzed by HPLC, thin-layer chromatography or gel electrophoresis.

In two-dimensional peptide mapping the peptides are separated by TLC using
voltage electrophoresis followed by separation by high-voltage electrophoresis in a second dimension. Another form of peptide mapping involves excising a protein band from the gel and loading it on a second gel of higher acrylamide concentration. A protease is added to the well and the protein of interest is partially digested during the re-electrophoresis.

Proteins will exhibit a distinct pattern of smaller polypeptides. This method, often referred to as the Cleveland method, is rapid and easy to carry out and allows proteins to be directly compared in neighboring lanes. The pattern of polypeptides can be used to assess whether two proteins are similar or different.

Amino Acid Structure

Proteins start out life as a bunch of amino acids linked together in a headto-tail fashion—the primary sequence. Methylene groups (–CH2–) may be important, but keeping track of them on an individual basis is just too much to ask. Organize the amino acids based on the functional group of the side chain.Remember a few of the amino acids by functional groups. The rest are hydrophobic.

1.HYDROPHILIC (POLAR):-

CHARGED POLAR:-

• Acidic (–COO) and basic (–NH3) amino acidside chains have a charge at neutral pH and strongly “prefer” to be on the exterior, exposed to water, rather than in the interior of the protein.The acidic amino acids, Asp and Glu, are really bases (proton acceptors). Lys, Arg, and His are considered basic amino acids, even though they have a proton at neutral pH. The same argument applies: Lys, Arg, and His are such good bases that they have already picked up a proton at neutral pH.

• Charged groups are usually found on the surface of proteins. It is very difficult to remove a charged residue from the surface of a protein and place it in the hydrophobic interior, where the dielectric constant is low. This is termed a salt bridge.

NEUTRAL POLAR:-  

•  These side chains are uncharged, but they have groups (–OH, –SH, NH, C“O) that can hydrogen-bond to water. In an unfolded protein, these residues are hydrogen-bonded to water. They prefer to be exposed to water, but if they are found in the protein interior they are hydrogen-bonded to other polar groups.

2.HYDROPHOBIC (APOLAR):-

•  Hydrocarbons do not have many groups that can participate in the hydrogen-bonding network of water. They’re greasy and prefer to be on the interior of proteins . Note that a couple of the aromatics, Tyr and Trp, have O and N, and Met has an S, but these amino acids are still pretty hydrophobic. The hydrophobic nature usually dominates; however, the O, N, and S atoms often participate in hydrogen bonds in the interior of the protein.

Interaction of Amino Acids

         A few basic interactions are responsible for holding proteinstogether. The properties of water are intimately involved in these interactions.The dielectric constant is a fundamental and obscure property of matter that puts a number on how hard it is to separate charged particles or groups when they’re in this material. In water, charge is easy to separate .This dipolar nature of water makes it easy for it to dissolve ionic material.  There are two kinds of Interactions. They are

1.HYDROPHILIC INTERACTION:-

• The properties of water dominate the way we think about the interactions of biological molecules. That’s why many texts start with a lengthy, but boring, discussion of water structure, and that’s why you probably do need to read it. 
• Basically, water is a polar molecule. The H—O bond is polarized the H end is more positive than the O end. This polarity is reinforced by the other H—O bond. Because of the polarity difference, water is both a hydrogen-bond donor and a hydrogen-bond acceptor.

Water does two important things:

1. It squeezes out oily stuff because the oily stuff interferes with the interaction of water with itself 
2.  It interacts favorably with anything that can enter into it hydrogen-bonding network.

2.HYDROPHOBIC INTERACTION:-

• As hydrophobic surfaces contact each other, the ordered water molecules that occupied the surfaces are liberated to go about their normal business. The increased entropy  of the water is favorable and drives  the association of the hydrophobic surfaces.

•  Putting a hydrophobic group into water is difficult to do. Normally, water forms an extensive hydrogen-bonding network with itself. The water molecules are constantly on the move, breaking and making new hydrogen bonds with neighboring water molecules. Water has two hydrogen bond donors  and two hydrogen bond acceptors , so a given water molecule can make hydrogen bonds with neighboring water molecules in a large number of different ways and in a large number of different directions.

• When a hydrophobic molecule is dissolved in water, the water molecules next to the hydrophobic molecule can interact with other water molecules only in a direction away from the hydrophobic molecule.  In this case, organization means restricting the number of ways that the water molecules can be arranged in space. The increased organization of water that occurs around a hydrophobic molecule represents an unfavorable decrease in the entropy of water.

Secondary Structure of Amino Acids:-

Secondary structure is not just hydrogen bonds.It also includes the following

Alpha Helix: Right-handed helix with 3.6 amino acid residues per turn. Hydrogen bonds are formed parallel to the helix axis.
Beta Sheet: A parallel or antiparallel arrangement of the polypeptide chain. Hydrogen bonds are formed between the two (or more) polypeptide strands.
Beta Turn: A structure in which the polypeptide backbone folds back on itself. Turns are useful for connecting helices and sheets.

Secondary structure exists to provide a way to form hydrogen bonds in the interior of a protein. These structures  provide ways to form regular hydrogen bonds. These hydrogen bonds are just replacing those originally made with water. As a protein folds, many hydrogen bonds to water must be broken. If these broken hydrogen bonds are replaced by hydrogen bonds withinthe protein, there is no net change in the number of hydrogen bonds Because the actual number of hydrogen bonds does not change as the secondary structure is formed, it is often argued that hydrogen bonds don’t contribute much to the stability of a protein.

However, hydrogen bonds that form after the protein is already organized into the correct structure may form more stable hydrogen bonds than the ones to water. Hydrogen bonding does contribute somewhat to the overall stability of a protein; however, the hydrophobic interaction usually dominates the overall stability.Small peptides generally do not form significant secondary structure in water . For small peptides that do not form stable secondary structure, there are often other favorable interactions within the peptide that stabilize the formation of the helix or sheet structure.

The stability of secondary structure is also influenced by surrounding structures. Secondary structure may be stabilized by interactions between the side chains and by interactions of the side chains with other structures in the protein. For example, it is possible to arrange the amino acid sequence of a protein or peptide into a helix that has one face that is hydrophobic and one that is hydrophilic. The peptide backbone spirals up and around the cylinder.

In an unfolded protein, water makes hydrogen bonds to all the donors and acceptors. As the protein folds and some polar groups find themselves inside, many of the hydrogen bonds with the solvent are replaced by hydrogen bonds between the different donors and acceptors in the protein. Because hydrogen bonds are
being replaced rather than gained or lost as the protein folds, there is not a large net stabilization of the protein by the hydrogen bonds.

Temperature Sensitive Mutations

Intoroduction:-

 These are mutations that decrease the stability of a protein so that the denaturation temperature is near 40°C. A single methylene group (–CH2–) involved in a hydrophobic interaction may contribute as much as -1.5 to -2 kcal/mol to the stability of a protein that is only stable by -10 kcal/mol. A single hydrogen bond might contribute as much as -1.5 to -3.5 kcal/mol. If a mutation disrupts interactions that stabilize the protein, the protein may be made just unstable enough to denature near body temperature. It might strike you as strange that we were talking earlier about how hydrogen bonds didn’t contribute much to the net stability of proteins and now I’m telling you they contribute -1.5 to -3.5 kcal/mol. Both statements are more or less right.

What we’re talking about now is messing up a protein by changing one amino acid for another by mutation. Here we’re destroying an interaction that’s present in the intact, folded protein. For any hydrogen-bonded group in the folded protein, there must be a complementary group. A donor must have an acceptor, and vice versa. Making a mutation that removes the donor of a hydrogen bond leaves the acceptor high and dry, missing a hydrogen bond.

Structure:-

 In the unfolded protein, the deserted acceptor can be accommodated by water; however, in the folded protein the loss of the donor by mutation hurts. It costs a hydrogen bond when the protein folds. The result: a loss in stability for the protein. Loss in stability means that the protein will denature at a lower temperature than before. Temperature-sensitive mutations usually arise from a single mutation’s effect on the stability of the protein.

 Temperature-sensitive mutations make the protein just unstable enough to unfold when the normal temperature is raised a few degrees. At normal temperatures , the protein folds and is stable and active. However, at a slightly higher temperature  the protein denatures and becomes inactive. The reason proteins unfold over such a narrow temperature range is that the folding process is very cooperative—each interaction depends on other interactions that depend on other interactions.

Analaysis:-

 For a number of temperature-sensitive mutations it is possible to find a seond mutation in the protein that will suppress the effects of the first mutation. For example, if the first mutation decreased the protein stability by removing a hydrogen-bond donor, a second mutation that changes the acceptor may result in a protein with two mutations that is just as stable as the native protein. The second  mutation is called a suppressor mutation.

Ligand Binding

The specificity of the interaction between a protein and a small molecule or another protein is also a compromise. We’ve just said that charge–charge and hydrogen-bond interactions don’t contribute a lot to the stability of a protein because their interaction in the folded protein simply replaces their individual interaction with water. The same may be said of the interaction between an enzyme and its substrate or one protein and another. However, there is a huge amount of specificity to be gained in these kinds of interactions. For tight binding, the protein and its ligand must be complementary in every way—size, shape, charge, and hydrogen-bond donor and acceptor sites.

Both the protein and the ligand are solvated by water when they are separated. As the two surfaces interact, water is excluded, hydrogen bonds are broken and formed, hydrophobic interactions occur, and the protein and ligand stick to each other. As in protein folding and for the same reasons, the hydrophobic interaction provides much of the free energy for the association reaction, but polar groups that are removed

ASSOCIATION:-
The association of two molecules uses the same interactions that stabilize a protein’s structure: hydrophobic interactions, van der Waals interactions, hydrogen bonds, and ionic interactions. To get the most out of the interaction, the two molecules must be complementary.

Consider what happens when a nonoptimal ligand binds to the protein. The binding of this modified ligand is much weaker not because it’s not the right size to fit into the protein-binding site, but  because the complementary group on the protein loses a favorable interaction with water that is not replaced by an equally favorable interaction with the ligand

As with the formation of secondary structure, the multiple, cooperative hydrogen bonds that can be formed between the ligand and the protein may be stronger and more favorable than hydrogen bonds that the ligand might make to water. Hydrogen bonding may, in fact, make some contribution to the favorable free energy of binding of ligands to proteins.

Functional roles of lipids in membranes

Introduction and overview:-

Lipids as a class of molecules display a wide diversity in both structure and biological function. A primary role of lipids in cellular function is in the formation of the permeability barrier of cells and subcellular organelles in the form of a lipid bilayer. Although the major lipid type defining this bilayer in almost all membranes is glycerol-based phospholipid, other lipids are important components and vary in their presence and amounts across the spectrum of organisms, Sterols are present in all eukaryotic cytoplasmic membranes and in a few bacterial membranes.

The ceramidebased sphingolipids are also present in the membranes of all eukaryotes. Neutral glycerol-based glycolipids are major membrane-forming components in many Grampositive bacteria and in the membranes of plants while Gram-negative bacteria utilize a glucosamine-based phospholipid  as a major structural component of the outer membrane. Additional diversity results in the variety of the hydrophobic domains of lipids. In eukaryotes and eubacteria these domains are usually long chain fatty acids or alkyl alcohols with varying numbers and positions of double bonds.

In the case of archaebacteria, the phospholipids have long chain reduced polyisoprene moieties, rather than fatty acids, in ether linkage to glycerol. If one considers a simple organism such as Escherichia coli with three major phospholipids and several different fatty acids along with many minor precursors and modified products, the number of individual phospholipid species ranges in the hundreds. In more complex eukaryotic organisms with greater diversity in both the phospholipids and fatty acids, the number of  individual species is in the thousands.

If one or two phospholipids are sufficient to form a stable bilayer structure, why is the above diversity in lipid structures present in biological membranes. The adaptability and flexibility in membrane structure necessitated by environment is possible only  with a broad spectrum of lipid mixtures. The membrane is also the supporting matrix for a wide spectrum of proteins involved in many cellular processes. Approximately 20-35% of all proteins are integral membrane proteins, and probably half of the remaining proteins function at or near a membrane surface.

Therefore, the physical and chemical properties of the membrane directly affect most cellular processes making the role of lipids dynamic with respect to cell function rather than simply defining a static barrier. In this chapter, the diversity in structure, chemical properties, and physical properties of lipids will be outlined. Next, the various genetic approaches available to study lipid function in vivo will be summarized. Finally, how the physical and chemical properties of lipids relate to their multiple functions in living systems will be reviewed.

DNA Displacers:-

Nucleic acids such as DNA and RNA constitute a second important group of biological macromolecules besides proteins. While proteins have for some time now been isolated  preparatively in fairly large scales, DNA purification has only recently entered that realm.

Compared to proteins, which are constructed from more than 20 amino acids of varied hydrophobicity and which in addition can contain complex sugars, lipids, or even metal ion, the structure of the DNA molecule is much simpler. From a chromatographic point of view, DNA can be considered as a very large linear polyanion with a very homogeneous charge distribution. Both anion exchange and hydroxyapatite chromatography can be used for its purification.

Nevertheless, DNA also presents some specific challenges to large-scale preparation. Concentrated DNA solutions are very viscous. Especially in elution chromatography, this causes problems since the peak maximum should stay below the viscosity/concentration limit. As a result only low concentration preparations can be obtained. In this context, displacement chromatography has the theoretical advantage of allowing to keep the concentration in the entire zone just below the viscosity/concentration limit. The overall concentration of the “product fraction” will be higher in this case.

According to a recent publication, the separation of protein and plasmid DNA as well as  that of DNA and  lipopolysaccharides is possible by displacement chromatography. Linear  polyacrylic acid was successfully used as displacer. A particularity was the fact that conventional stationary phase materials based on porous anion exchanger or hydroxyapatite beads could not be used for the separation of the plasmid DNA from a standard protein . The use of a monolithic column, on the other hand, yielded promising results. In this case protein and DNA capacities were comparable. Further research is obviously necessary. However, the investigation of DNA displacement also offers some principal opportunities, for example, to elucidate the effect of size and rigidity, or charge density and chemistry for the competitive adsorption of linear polyelectrolytes.