4. La problemática del saber penal en el ámbito de la intervención delictiva
4.3. La intervención delictiva en los delitos contra la administración pública
4.3.2. Problemas para la determinación de la autoría y participación en los delitos
3.C.1.3.C.1.
3.C.1.IIIIncreasencreasencreasencreases and decreases in the extent of protein s and decreases in the extent of protein s and decreases in the extent of protein s and decreases in the extent of protein
crystallization
crystallization
crystallization
crystallization on ‘active’ on ‘active’ on ‘active’ on ‘active’ and ‘inactive’ and ‘inactive’ and ‘inactive’ oil/waterand ‘inactive’ oil/wateroil/water interfacesoil/water interfaces interfaces interfaces....
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with
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3.C.1.1.
3.C.1.1.
3.C.1.1.
3.C.1.1.
The protein hydration shellThe protein hydration shellThe protein hydration shellThe protein hydration shell
The interaction between a protein and the surrounding solvent environment is
highly complex24. This complexity is often attributed to the distribution of
hydrophobic and hydrophilic patches on the protein surface, and how these patches interact with surrounding water molecules in order to produce fully hydrated protein
structures24. Proteins are not geometrically spherical; they do however possess a
complicated and irregular surface geometry24-25. Due to surface irregularities, solvent
accessible protein surfaces will not be structurally or energetically homogeneous.
Under correct solution conditions the inhomogeneous structural and energetic protein surface features may produce areas which initiate weak anisotropic protein-
protein interactions26. These weak anisotropic protein-protein interactions are typical
The protein surface, when in contact with an aqueous solution, is hydrated, and generally water will form one or more distinct, dynamic hydration shells around
a particular protein25. These hydration shells consist of water which is different, both
structurally and dynamically, from water found in bulk. In particular, the first of these hydration layers is said to extend approximately 3 Å from the protein surface
and has a water density of 10-15 % in excess of that found in bulk28-29. Of this first
hydration shell water, about 5-30 % of the water molecules display rotational and translational diffusional times that are significantly reduced (in the order of tens of picoseconds) in comparison to other ‘water’ found within the first hydration shell,
and may be termed ‘long residence time’ water25.
In particular, it is the diffusional component, perpendicular to the protein
surface, which is dramatically reduced25. ‘Long residence time’ water is said to occupy
various preferential locations on a protein surface, with water which displays the
longest residence times of all, found within deep protein surface cavities25.
At this juncture it would be prudent to make the reader aware of a debate within the literature, regarding the classification of water found on the surface of
hydrated proteins30. Crystallographers usually term protein surface water, found via
diffraction methods in crystal structures as either ‘free’ or ‘bound’, with ‘bound’ water referring to water that is found at high frequency at one site on the protein surface
within the crystal structure30. It has been argued, maybe correctly, that diffraction
data does not give any indication of the rates of the molecular movement of water,
Chapter 3 Page 162
Furthermore, it is suggested that the term ‘bound’ insinuates a largely
exothermic water adsorption process at the protein surface30. Additionally, it is
presented that water molecules are not ‘bound’ on the protein surface in either a thermodynamic or kinetic sense, and that ‘in the absence of co-solvents every exposed hydration site is populated with water’ which is free to reversibly exchange with bulk water.
One may therefore find it more appropriate to think in terms of short and long-residence time water instead of ‘free’ and ‘bound’ water which gives no weight
to the strength of the protein-water interaction30. Although, it may be said that
viewed with a sufficiently low temporal resolution, the dynamics of long residence time water could be considered immobile and hence bound in some sense.
For a protein to achieve and maintain successful folding, the folded protein must maintain favourable interactions between its amino acid side chains and the
surrounding solvent environment25. The enforcement of favourable protein-solvent
interactions leads to the well known hydrophobic effect31, in which non-polar
residues which do not contribute significantly to the aqueous solvent interaction are sequestered to the protein interior whilst polar residues which interact favourably
with the solvent, are found in abundance on the protein surface25, 31. However, not all
hydrophobic residues are found buried within the protein interior. Indeed, some are found on the protein surface, and are said to have hydration shells which display
The formation of ‘ordered’ hydration structures (polygonal or clathrate-like) around hydrophobic residues or hydrophobic areas is also a contentious issue with
some papers presenting evidence for order33-34, whilst others present evidence against
some form of geometrical ordering35. These opposing views of hydrophobic solvation
appear to be the result of interpretations based on either dynamic or structural arguments.
Time resolved measurements36 have shown that hydrophobic hydration shells
can display liquid-like structural characteristics but dynamically the hydration shells are better described as ‘ice-like’, with ‘ice-like’ dynamics being more prevalent at
higher solute concentration35. Although no ‘cage’ or ‘iceberg’ like structures are
readily seen, water orientational dynamics are slow enough around hydrophobic moieties to warrant description as ‘long residence time’ water as well as displaying some kind of structure on an appropriate time scale.
Water surrounding polar residues can exchange freely with bulk and display fast bulk-like orientational dynamics and thus water molecules in the vicinity of polar or charged residues have ‘short residence’ time on the protein surface and display bulk-like structural characteristics.
Chapter 3 Page 164
Since proteins have evolved almost exclusively in an aqueous environment, one may find it surprising that proteins can function and even exist in a variety of solvents with physical properties either very similar or remotely different to that of
water37. Most of the work conducted in this area has centred on enzyme catalysis in
nonaqueous solvents38 and forms the basis of the field described as ‘nonaqueous
enzymology’. Because of the different physical and chemical properties of these nonaqueous solvents, protein hydration mechanisms and protein stability within such
solvents is, in some cases, very different from that found in water37. For example,
earlier studies by Parker39et al. using sensitive NMR techniques in conjunction with
deuterated water, compared the degree of hydration of subtilisin Carlsberg (an endopeptidase) in air, and within various polar and nonpolar solvents. They found that ‘strongly bound water’ adsorption was hardly affected by nonpolar solvents
whilst ‘loosely bound water’ was significantly reduced39. Parker’s39 work illustrates
that when immersed within nonpolar solvents (viz. hexane, toluene and benzene)
water is preferentially localised in the most polar regions on the enzyme surface
whilst in nonpolar regions it becomes reduced or even stripped off40.
Additional evidence for this difference in protein hydration within
nonaqueous solvents is provided by work by Gorman40et al. who found that when
three different enzymes (chymotrypsin , subtilisin Carlsberg and horse radish
peroxidise) were hydrated with tritiated (T O2 ) water, the enzymes lost the most
water in solvents of moderate to high polarity, whilst in nonpolar solvents it was
found that water desorption was minimal. The study40 also found that both solvent
dielectric and a measure of the saturated molar solubility of water in the given solvent
was a good indicator of the solvents ability to allow T O2 desorption from the enzyme.
Gorman40et al. conclude by stating that ‘water stripping in nonaqueous environments
These experimental findings, just described, are substantiated via molecular
modelling studies of protein hydration within nonpolar solvents by Soares41et al. who
found that nonpolar solvents enhanced the formation of ‘large water clusters’ that are tightly bound to the enzyme surface, whereas water in polar organic solvents is fragmented in ‘small clusters’ loosely bound on areas of the enzyme surface . The
Soares41et al. study also found that water was localised preferentially on the protein
surface in similar regions regardless of the solvent used.
In summary, one may say that within an aqueous environment, hydrophilic and charged residues have preferential hydration and ‘protein water’ exchange with bulk water is extremely rapid. Hydrophobic residues, on the other hand (within an aqueous solvent), experience hydration which is characterised by slow dynamics and slow water exchange with bulk. This ‘slowdown’ may cause a modicum of
‘structuring’ of the hydration water around hydrophobic residues to occur by virtue
of slow hydrogen bond dynamics25.
By placing the same protein in a nonaqueous environment, one of low polarity for example, we find that the situation reverses, in that water around hydrophilic residues now becomes ‘long residence time’ water whilst water around hydrophobic residues becomes desorbed from the protein surface. The ability of the hydrophilic residues to retain water appears to be a function of solvent polarity and the higher the solvent polarity the more likely it is for solvent molecules to exchange or replace with strongly held water molecules on the hydrophilic patches on the protein surface. Since proteins require a certain critical amount of water to be present on the surface in order to maintain native structure, high polar solvents can acts as denaturants by
Chapter 3 Page 166