A culture of bacteria growing at 37C was shifted to 25C.

How would you expect this shift to alter the fatty acid composition of the membrane phospholipids? Explain. The shift to the lower temperature would decrease fluidity by enhancing packing of the hydrophobic chains by van der Waals interaction. To prevent this, new phospholipids would be synthesized having shorter chains and a greater number of cis double bonds. The shorter chains would reduce the amount of van der Waals interaction, and the cis double bonds, causing the kink in structure, would prevent packing of the fatty acid tails of the phospholipids.
Effect of incorrect protein folding Protein folding is a process in which a polypeptide folds into a specific, stable, functional, threedimensional structure. It is the process by which a protein structure assumes its functional shape or conformation. But sometimes this process is happened incorrectly, and the scientist call this problem as protein misfolding. The results of protein folding incorrectly are so many diseases happening for human, animals and living things such as Alzheimers disease , Creutzfeldt-Jakob disease, cystic fibrosis and p53-related cancer , Mad Cow disease etc .

Protein folding diseases can be divided into two groups: in the first, excessive quantities of wrongly folded proteins collect in the form of uncontrolled piles of molecular rubbish. This is the group of diseases known as amyloidoses, of which Alzheimers disease is the best-known example. In the other, a small error in the genetic blueprint leads to incomplete folding of a protein, which affects its function. This might, for instance, happen to p53 the malfunctioning of this central tumour suppressor could cause cancer. Amyloidoses The common characteristic of all amyloidoses is the collection of plaques of insoluble protein in the extracellular tissue, which cannot be broken down by enzymes. Their ordered structure gives them crystal-like properties: they are made up of long filaments (fibrils) that are formed from densely packed -pleated sheets of identical proteins. There are about 20 different proteins that can act as the building blocks of these fibrils, each of which is associated with a different disease. In so-called systemic amyloidoses, the precursors of these plaques are transported through the bloodstream from their point of origin to their point of deposition. Localized amyloidoses are of greater clinical significance, as they mainly affect the central nervous system, the extracellular tissue of which is particularly susceptible to damage. Alzheimers disease

One of the main characteristics of Alzheimers disease is the accumulation of plaques of insoluble -amyloid in the brain. It is still not certain whether these plaques are a cause or a consequence of the disease, but there is a lot of evidence for the former being the case. The amyloid plaques are formed by cleavage of amyloid-precursor protein (APP) by two different

enzymatic activities, which release amyloid- peptide fragments that are 40 or 42 amino acids long (Fig. 1). These then form fibrils, which aggregate into insoluble clumps of -amyloid plaques that surround neurons and might cause damage.

Figure 1 | -amyloid and Alzheimers disease. a | -amyloid peptide (A42; red) is released by the cleavage of amyloid precursor protein (purple bar) by -secretase at the amino terminal end of the -amyloid peptide, followed by cleavage involving -secretase at the carboxy terminal. b | A42 is then thought to self associate under certain circumstances to form aggregates called -amyloid in the brain. c | These plaques occur in high density in many parts of the brain in Alzheimers disease and are thought to cause the shrunken appearance of the brain and the clinical symptoms of dementia seen in Alzheimers patients. Part b reproduced with permission from Sisodia, S. S. & St George-Hyslop, P. H. Nature Rev. Neurosci. 4, 281290 (2002) Macmillian Magazines Ltd.

But this cleavage also occurs in healthy individuals and soluble -amyloid proteins are normal constituents of brain tissue. How, then, do the plaques form in Alzheimers patients? It is thought that the misfolding of the protein dramatically alters its properties. In the normal protein, hydrophobic (water-repelling) amino acids bury themselves inside the protein right from the start of folding. However, if the protein folds wrongly, these hydrophobic amino acids are exposed and they rapidly seek out and bind to hydrophobic groups on other protein molecules, forming the insoluble aggregates or plaques that are found in Alzheimers patients. Prion diseases Transmissable spongiform encephalopathies (TSEs), which include mad cow disease (bovine spongiform encephalopathy; BSE) and CreutzfeldJakob disease (CJD) in humans, are special forms of amyloidosis in which the victims brain degenerates to a structure that looks like a porous sponge. These conditions seem to occur when normal human protein particles called

prions misfold. The normal human prion is a component of the membrane of healthy nerve cells (called PrPc), which folds properly, remains soluble and is disposed of without problem. It can, however, misfold in a particular way, which allows it to take on an infectious, incorrectly folded three-dimensional form (called PrPsc), presumably due to a genetic mutation. The infectious prion, which can be transmitted in the diet, triggers a domino effect in healthy prions, forcing them to adopt its incorrectly folded form (Fig. 2).

Misfolding and cancer Whereas too much of an incorrectly folded protein can cause amyloidoses, another group of protein folding diseases is caused by lack of a correctly folded protein. This form of protein folding defect is thought to be involved in diseases such as cystic fibrosis, but mainly affects a protein called p53, which occupies the most important position in the bodys cancer resistance network. Normally, the p53 system is switched off or, at most, is in stand-by mode. It is activated inside a cell if the cell becomes excessively stressed or damaged, which can lead to genetic mutations in DNA that can cause the uncontrolled division and proliferation of cells that is the hallmark of tumour formation. p53 is so good at its job that even a single break in the DNA strand is enough to activate it. It rushes into the cell nucleus and induces the production of other proteins that stop uncontrolled cell division or trigger the programmed death of a cell (Fig. 3.

This tumour-suppressing function of p53 is so important that the protein has been described as the guardian of the genome. So, it is no surprise that faults in the p53 gene can be disastrous. Even a mutation in one of the letters (nucleotides) in the gene can be enough to lead to the expression of p53 proteins that do not fold correctly. Half crippled, they cannot carry out their job properly, so the damage to the DNA that would normally be repaired goes unnoticed, allowing the abnormal cell to grow in an uncontrolled manner. This type of mutation in p53 is thought to occur in 50% of all cases of cancer and as many as 95% of all cases of lung cancer. What is the difference between parallel and antiparallel -sheets? How do the R-groups protrude from the sheets? Beta sheets are parallel if the polypeptide strands run in the same direction, N-terminus to Cterminus. The N-terminus of one beta strand will be opposite the N-terminus of the other beta strand. Beta sheets are anti-parallel if the polypeptide strands run in opposite directions. The Nterminus of one beta strand will be opposite the C-terminus of the other beta strand. R groups protrude from the top and bottom of the beta sheet. The R groups on one side of a beta-sheet tend to be either all hydrophilic or all hydrophobic. The hydrophilic side of the sheet will be on the surface of the protein and be exposed to the polar H2O solvent. The hydrophobic side of the sheet will be buried in the protein and will only rarely be exposed to the polar H2O solvent.

Why hydrophobic interaction is the major contributor to the stability of protein structure ?

Hydrophobic interaction are a major driving force in protein folding. Hydrophobic interactions are also important in the formation of secondary structure elements such as -helices and sheets. The peptide backbone is relatively hydrophillic because of the C=O and N-H groups of each peptide bond. However in the centre of the protein these tend to be in a hydrophobic environment. One way to reduce the hydrophilicity of the peptide backbone is to for hydrogen bonds between the peptide atoms. The -helix and -sheet are two structures which maximise the hydrogen bonding between the peptide bonds of the backbone and reduce its hydrophilicity. When unfolded, all polar/hydrophillic sidechains can interact via H-bonds with water. When the protein folds, they must H-bond to each other and exclude much of the water. All groups capable of forming a hydrogen bond MUST, hence H-bonding in the backbone (C=O to N-H) by way of helices and sheets is an efficient way of ensuring maximum H-bonding. Sidechains can either accept (as in C=O) or donate (as in N-H, or O-H) an H-bond. The capacity of proteins to form hydrogen bonds is an important determinant of protein stability. Hydrogen bonds can be between backbone groups, as in helices and sheets; between side chains, such as serine or threonine O-H groups and carbonyl carbons of side chains (-C=O); and between backbone groups and side chain groups. The hydrophobic effect is considered to be the major driving force for the folding of globular proteins. It results in the burial of the hydrophobic residues in the core of the protein. It is exemplified by the fact that oil and water do not mix and was described well by G. S. Hartley in 1936 . "The antipathy of the paraffin chain for water is, however, frequently misunderstood. There is no question of actual repulsion between individual water molecules and paraffin chains, nor is there any very strong attraction of paraffin chains for one another. There is, however, a very strong attraction of water molecules for one another in comparison with which the paraffin-paraffin or paraffin-water attractions are slight." The thermodynamic factors which give rise to the hydrophobic effect are complex and still incompletely understood. The free energy of transfer of a non-polar compound from some reference state, such as an organic solution, into water, Gtr, is made up of an enthalpy, H, and entropy, -T S, term.

At room temperature, the enthalpy of transfer from organic solution into aqueous solution is negligible; the interaction enthalpies are the same in both cases. The entropy however is negative. Water tends to form ordered cages around the non-polar molecule and this leads to a decrease in entropy. At high temperatures (~ 110C) these cages are no longer any stronger than bulk water, and the entropy contribution tends to zero. The enthalpy of transfer, however, is now positive (unfavourable). Because the temperature dependence of entropy and enthalpy are not the same, there is some temperature at which the hydrophobic effect

is strongest, and the effect decreases at temperatures above and below this temperature. The decrease in the strength of the hydrophobic effect with decreasing temperatures is probably the major cause of cold-denaturation in proteins. The contribution of the hydrophobic effect to globular protein stability has been estimated empirically both by measuring the thermodynamics of transfer of model compounds (e.g. blocked amino acids, cyclic peptides...) from organic solvents to water, and by site directed mutagenesis studies on proteins. The number arrived at is usually given as a function of the change in the solvent accessible non-polar surface area upon going from the unfolded to the folded state. The model compound studies predict that the hydrophobic effect of exposing one buried methylene group to bulk water is 0.8 kcal/mol (in Pace, 1995). The site directed mutagenesis studies yielded a larger number with greater statistical variation: the average hydrophobic effect estimated by SDM for a buried methylene group is about 1.3 kcal/mol. However, when the SDM results for methylene were plotted against the size of the cavity created by the residue substitution, and extrapolated to zero, the result at zero cavity size is 0.8 kcal/mol - in agreement with the value found for the transfer of model compounds from octanol to water (Pace et al., 1996 and references therein). In the SDM studies, cavities created by residue substitution have an additional destabilizing effect: the loss of favourable VDWs interactions (as compared to the wild-type). Thus, the "hydrophobic effect" measured by SDM includes both an entropic component due to solvent ordering and a (primarily) enthalpic component due to loss of VDWs contacts within the protein. Such an SDM study of T4 lysozyme replaced the 80% buried Ile3 residue by Val (Eriksson et al, 1992): the loss of this methyl group gave rise to a decrease in stability of 0.6 kcal/mol (corrected to 100% burial). This is smaller than expected (c.f. 0.8 kcal/mol for methylene) and suggests that the mutation introduced some smaller stabilizing influence, perhaps such as the alleviation of strain within the protein. In barnase, 15 mutants were constructed in which a hydrophobic interaction was deleted (V10A, V36A, V45A, I4A, I25A, I51A, I55A, I76A, I109A, I4V, I25V, I51V, I55V, I76V & I109V). The finding was a strong correlation between the degree of destabilization (which ranges from 0.60 to 4.71 kcal/mol) and the number of methyl or methylene side chain groups surrounding the methyl or methylene group that was deleted (r = 0.91) (Serrano et al, 1992). See figure below.

Correlation between the number of side chain methylene and methyl groups, in a radius of 6 of the group deleted from wild-type, and the changes in the free energy of unfolding for mutations of hydrophobic residues in barnase. (Taken from Serrano et al, 1992. with permission)

The average free energy decrease for removal of a completely buried methylene group was found to be 1.50.6 kcal/mol. This is additive, such that Ile or Leu to Ala can destabilize a protein by up to 5 kcal/mol. (Remember that many mesophilic proteins are stable by <10 kcal/mol, so two deletions such as this would be enough to destabilize a protein completely). The figure below shows the backbone of barnase with the hydrophobic side-chains coloured blue. Those parts of the side-chains deleted in the mutagenesis experiments have been coloured green and given dot surfaces to show the potential cavities left behind.
Calculation of PI Values of Amino Acids For an amino acid with only 2 pKa values, the pI is simply the average of the 2. For an amino acid with 3 pKa values, the pI is the average of the 2 "like" functional groups. When I say "like" here, I mean in terms of charge. Amine functional groups are + charged in the acid form and neutral in the base form. Whereas, carboxylic acids, alcohols, and sulfhydryl (-SH) groups are neutral in the acid form and charged in the base form. Some examples: for histidine you'd take the average of the pKas of the alpha amino group and the R group as they're both amines and are "alike" in terms of charge. For aspartic acid you'd take the average of the pKas of the carboxylic acid of the backbone and the carboxylic acid of the R group as they are "alike" in terms of charge. For tyrosine you'd take the average of the pKas of the carboxylic acid of the backbone and the R group (an alcohol) as they are "alike" in terms of charge. Usually this reduces down to taking the average of the 2 pKas that are closest to each other but if you look at the pKas you'll notice that tyrosine is a big exception to this line of thinking. As far as calculating pI for di/polypeptides, this is more complex. What I usually do is list all the pKas of all the potentially charged groups. Then I'll draw the structure at pH=0 and it'll have some positive charge at this pH. Then I'll raise the pH and everytime I cross a pKa value it'll get less positively charged. Once you get an overall charge of zero then the 2 pKa values providing the boundaries to that structure are the 2 pKas you'll take the average of to get the pKa. I attached an image of a dipeptide (histidine, aspartic acid). I originally drew the predominant structure at pH=0 and gave you all the pKa values. I then provided the structure that would be neutral (no overall charge). The last pKa we crossed as we made the solution more basic was the R group of aspartic acid (3.86) and the next pKa value we'd cross would be the R group of histidine (6.0) and so we can see that the neutral species is only the predominant species between a pH of 3.86 and pH 6.0 and the pI is therefore the average of these 2 (pI=4.93).

For

histidine

pKa1=2.3,

pKa2=6.0

and

pKa3=9.6.

At the pI the net charge of the molecule is zero. To find the pI, average the two pKa values on either side of the neutral form of histidine.

(pKa2 + pKa3)/2 = pI (6 + 9.6)/2 = 7.8