Solved structures of selected SBPs. Listed are the proteins, the host organism, the substrate, whether the structure was solved in the unliganded-open and/or liganded-closed state, the highest resolution, the biochemically determined affinity, the used substrate concentration during crystallization and the method used: 1) co-crystallization 2) soaking 3) seeding 4) endogenously bound substrates.
1. Introduction
The importance of structural biology has been highlighted in the past few years not only as part of drug discovery programs in the pharmaceutical industry but also by structural genomics programs. Although the function of a protein can be studied by several biochemical and or biophysical techniques a molecular understanding of a protein can only be obtained by combining functional data with the three-dimensional structure. In principle three techniques exist to determine a protein structure, namely X-ray crystallography, nuclear magnetic resonance (NMR) and electron microscopy (EM). X-ray crystallography contributes over 90 % of all structures in the protein data bank (PDB) and emphasis the importance of this technique. Crystallization of a protein is a tedious route and although a lot of knowledge about crystallization has been gained in the last decades, one still cannot predict the outcome. The sometimes unexpected bottlenecks in protein purification and crystallization have recently been summarized and possible strategies to obtain a protein crystal were postulated [1]. This book chapter will tackle the next step: How to crystallize protein-ligand complexes or intermediate steps of the reaction cycle?
A single crystal structure of a protein however, is not enough to completely understand the molecular function. Conformational changes induced by for example ligand binding cannot be anticipated
Within this chapter, the structural conformational changes induced by ligand binding with respect to the methods chosen for the crystallization are described. Here three distinct protein families are exemplarily described: first, where one substrate or ligand is bound, second, a protein with two or more bound substrates and finally, the structures of proteins, in which the product of the reaction cycle is present in the active site.
Specific methods or expressions written in
1.1. General approaches to obtain crystals with bound ligands and how to prepare the ground
Often the knowledge of the structure of a protein or enzyme without bound
In theory there are different approaches to reach this goal even though it can be a difficult task in reality. All of them have in common that the naturally catalysed reaction must not occur. Apart from reporting all possible attempts we would like to give a general overview about several
Possible
(In order to keep it simple and coherent the expression „
first ligand without second ligand
second ligand without first ligand
first ligand with product of the second ligand
product of the first ligand with second ligand
substrate analogue/inhibitor or non-hydrolysable cofactor
application of substances that mimic transition state products (e. g. AlF3 which imitates a phosphate group)
usage of catalytically inactive mutants with bound ligand(s)
creation of an environment (i. g. buffer condition) which shifts the equilibrium constant so that the reaction cannot occur
The most important point concerning preparing
The application of a too low concentration of the ligand can lead to an inhomogeneous protein solution, which means that not all of the protein molecules are loaded with ligand (and this can prevent crystallization). It is also possible, that a low level of
Beyond that all requirements for the protein solution itself remain valid as described in [1] in more detail.
2. Binding protein with one ligand – How to crystallize and what can be deduced from the structure
A typical class of a protein binding one
Structural studies of a substantial number of SBPs revealed a common fold with a bilobal organization connected via a linker region [2]. In the ligand-free, open conformation, the two lobes or domains are separated from each other, thereby forming a deep, solvent exposed cleft, which harbors the substrate-binding site. Upon ligand binding, both domains of the SBP move towards each other through a hinge-bending motion or rotation, which results in the so-called liganded-closed conformation. As a consequence of this movement, residues originating from both domains generate the ligand-binding site and trap the ligand deeply within the SBP [3]. In the absence of a ligand, unliganded-open and unliganded-closed states of the SBP are in equilibrium, and the ligand solely shifts this equilibrium towards the liganded-closed state. This sequence of events has been coined the “Venus-fly trap mechanism” [4-6]; it is supported by a number of crystal structures in the absence and presence of a ligand [7, 8] and other biophysical techniques [3].
For the maltose binding protein (MBP) from
Upon
To fully understand the function as well as the structural changes happening upon
2.1. Crystallization of the open-unliganded conformation (state I)
The crystallization of an open conformation of a rather flexible protein is not straight forward and most of the success came from „trial-and-error“ approaches. After purification of the protein, a reasonable concentration of the protein is taken to set up crystallization trials. Most commonly the
Most of the times the open conformation crystallizes differently from the ligand bound state. This is reflected in the different crystallization conditions as well as in changes of the crystal parameters (unit cell and/or spacegroup). One example is given below for the glycine betaine binding protein ProX.
2.2. Crystallization of the substrate bound closed conformation (state II)
The vast majority of
The first method is
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BtuF |
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vitamin B12 | Y | Y | 2 | 15 nM | 5 mM | 1 | [18] |
Lbp |
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zinc | - | Y | 2.45 | ~10 µM | - | 4 | [19] |
GGBP |
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D-glucose | Y | Y | 1.9 | 0.5 µM | 3 mM | 1 | [20] |
MBP |
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Oligo-sacharide | Y | Y | 1.67 | 0.16 µM | 2 mM | 1 | [21] |
RBP |
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D-ribose | Y | Y | 1.6 | 0.13 µM | 1 mM | 1 | |
OppA |
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Oligo-peptide | Y | Y | 1.3 | 0.1 µM | 0.5-5 mM | 1 and 4 | [22] |
ProX |
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glycine betaine, proline betaine | Y | Y | 1.8 | 50 nM | 1 mM | 1 | [23] |
PotD |
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spermidine | - | Y | 1.8 | 10 nM | n.n | 2 | [24] |
SiaP |
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sialic acid | Y | Y | 1.7 | 58 nM | 5 mM | 1 | [25] |
UehA |
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ectoine | - | Y | 2.9 | 1.1 µM | 10 mM |
1 | [26] |
ChoX |
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choline | Y | Y | 1.8 | 2.7 µM | 2 mM | 1 and 3 | [14, 15] |
2.2.1. Co-crystallization to obtain the ligand bound structure
The method of
2.2.2. Ligand soaking to obtain the ligand bound state
The second method, which can be used to obtain a
2.2.3. Seeding – A method to obtain the ligand bound state with unusual substrates
In some cases the
2.2.4. Endogenously bound ligands
During purification of some proteins with high
2.3. Crystallization of the closed-unliganded state (state III)
The intermediate states of SBPs have been crystallized as well, although only a couple of structures have been reported. This energetically unfavorable state has been crystallized not on purpose in most cases. The choline binding protein ChoX from
2.4. Crystallization of a semi-open or semi-closed state (state IV)
During our efforts to solve the crystal structure of the choline-binding protein ChoX from
The structure revealed that the domain closure upon
2.5. State I-IV: What do they tell about conformational changes
The unliganded substrate binding proteins are thought to fluctuate between the open and closed state. The angle of opening varies between 26° up to 70° as observed in several open-unliganded structures, suggesting that the extent of opening is likely influenced by crystal packing. This has been observed very nicely for the ribose binding protein of which three different crystal structures have been described. Here the opening of the two domains varies between 43° and 63°. This suggests that the opening can be described as a pure hinge motion. The variation of the degree of opening has been elucidated by NMR in solution for the maltose binding protein MalF. Here 95 % of the protein adopts an open conformation fluctuating around one state with different degrees of opening.
2.5.1. Open and closed - An overall structure view
As an example for the closing movement observed when comparing the open-unliganded and closed-liganded structure the glycine betaine (GB) binding protein ProX from
In Figure 2 the opening and closing of the glycine betaine binding protein ProX from
Figure 2 highlights the open conformation (green), which is in equilibrium with the closed state although only a small percentage will be present in the closed unliganded state. Upon the addition of glycine betaine a stable closed conformation is reached and the equilibrium is shifted towards this state. Besides the crystal structure of the
2.5.2. Open and closed - An active site view
A closer look at the binding site or the amino acids involved in
The binding site is located in the cleft between domains I and II and can be subdivided into two parts, one binding the quaternary ammonium head group and the other binding the carboxylic tail of these compounds. The quaternary ammonium head group is captured in a box formed by Asp109 and the four tyrosine residues Tyr63, Tyr111, Tyr190, and Tyr214 being oriented almost perpendicular to each other. The tyrosine side chains provide a negative surface potential that is complementary to the cationic quaternary ammonium head group of GB. The carboxylic tail of GB is pointing outward of this partially negatively charged environment forming interactions with Lys13 (domain I), Arg149 (domain II), and Thr66 (domain I), respectively. Furthermore the structure was solved at a resolution sufficient to locate water molecules. An important water molecule was observed, which was held in place by residues Tyr111 and Glu145, and stabilizes domain closure. Here it is important to mention that this water molecule was not observed in the open unliganded structure and its importance would therefore be easily overlooked when no comparison between the two states were possible.
The superposition of the open-unliganded form and the closed-liganded form of ProX allowed an unambiguous identification of residues of domain II that are involved in ligand binding. They show virtually the same orientation in the open and closed forms (see Figure 3). Residues Tyr63, Tyr214, Lys13, and Thr66 superimpose very well. Only the main chain carbonyl of Asp109 from domain I is slightly out of place compared to the closed form because of the enormous main chain rearrangement between Asp110 and Tyr111 upon domain closure. The residues contributed by domain II behave quite differently. Tyr111 and Tyr190 are not only moved as parts of domain B but they undergo a major conformational change to adopt the conformation of the closed-liganded binding site. The side chain conformation of Arg149 shows only small changes between the open and closed conformations although it undergoes a large movement as part of domain II.
Recently, another structure of ProX was solved in the liganded but open conformation [29]. This conformation represents a state of which only very few structures are known. In other words, the protein has a ligand bound and is on its way to close up the binding site. This structure provided an even more detailed picture on the function of ProX and finally highlighted the crucial role of Arg149. In addition to the direct interaction with GB and residues that are part of the substrate-binding pocket (Tyr111, Thr66), Arg149 is a major determinant in domain-domain interactions in the closed structure. As such, Arg149 interacts with Val70 (domain I) and Asp151 (domain II), thereby acting as a linking element between the two domains enforcing stable domain closure. These interactions complement those mediated by Pro172 of domain II, where Pro172 interacts via its Cα-atom and a water molecule with Glu155 of domain II. Together, this provides a further explanation for the crucial role of Arg149 for the stability of the liganded-closed state, which has been observed in mutagenis studies. Here, the binding
In summary, the “Venus fly trap” model describes the opening and closing of SBPs. Here the equilibrium between these two conformations is shifted towards the closed state upon
3. Protein with multiple ligands – How to crystallize the different ligand bound intermediate states
Besides proteins that bind one substrate, a large number of enzymes are binding two or more substrates and convert these into a product. Here, the crystallization of the
Most of these proteins are enzymes. In reactions mediated by enzymes, the molecules at the beginning of the process, called
Below the structural studies of the octopine dehydrogenases (OcDH) from
This enzyme has been chosen due to the fact that three
In 2007 Mueller and co-workers achieved cloning and heterologously expression of this enzyme using
3.1. The crystallization of apo-enzyme and the binary complex
Parallel to the biochemical characterization, the crystallization of the enzyme was started. Due to the two-domain structure OcDH can adopt multiple conformations in solution, which prevents crystal formation. However, purified OcDH-His5 yielded small crystals that appeared to be multiple on optical examinations (Figure 5 A). They diffracted to a resolution of 2.6 Å. However the diffraction showed multiple lattices in one diffraction image and could not be used for structure determination (Figure 5 A) [34]. All attempts to improve these crystals using for example
This highlights the importance to verify the
The structure of the OcDH-NADH
In summary, the apo-state of multiple
3.2. The crystallization of the ternary complexes CI and CII
OcDH catalyzes the condensation of L-arginine with pyruvate to form octopine under the oxidation of NADH. Biochemical analysis as well as the crystal structure revealed that NADH is the first
So the next step was to determine the structure of the OcDH in the presence of the second and third
Since crystallization was not successful the next step was to use
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OcDH-NADH | 99.8, 99.8, 126.5 |
OcDH-NADH/L-arginine | 95.9, 95.9, 117.9 |
OcDH-NADH/pyruvate | 95.0, 95.0, 120.2 |
The change in unit cell parameters suggested that a conformational change occurred during the
A comparison of the two
This ordered sequence of
The crystal structures of the different states of OcDH, delivered snapshots elucidating for the first time the precise and very distinct binding order [35]. Unfortunally the crystals with the endproduct octopine did not diffract X-ray with a resolution and quality high enough for structure determination. The same hold true for a complex with all three substrates present at once. This is likely due to the fact that the immediate condensation occured and the product was formed.
To show how proteins can be crystallized with their enzymatic endproducts we chose another enzyme family as example and will describe the different procedures during the next paragraphs.
4. Enzymatic products in protein structures – How to crystallize this rather unfavored states
The state found to be important within an enzyme reaction cycle is supposedly the product bound state. After the reaction occurs the product is still sitting within the protein and will be released. Often these product have a low(er)
Examples of prosperous structure determination however are the shikimate dehydrogenase (SDH or AroE) of
Shikimate-/quinate dehydrogenases belong to the superfamily of NAD(P)-dependent (nicotinamide adenine dinucleotide phosphate) oxidoreductases whereas SDHs catalyse the reversible reduction of 3-dehydroshikimate to shikimate under oxidation of NAD(P)H (reduced form of nicotinamide adenine dinucleotide phosphate) and QDHs the oxidation of quinate to 5-dehydroquinate with reduction of NAD(P), respectively. The overall fold consists of a N-terminal or
4.1. Shikimate dehydrogenase from Aquifex aeolicus
Crystals of the native (apo-)
There were eight (apo) and four (
The open conformation therefor represents the protein structure in state 9.) (or 1.), respectively) in Figure 7, the closed conformation correlates to state 6.) in that scheme.
4.2. Shikimate dehydrogenase from Thermus thermophiles
In case of
Evaluation of the complex structures revealed an open and a closed conformation of the two domains but neither the binding of shikimate nor NADP+ seem to induce that conformational change. Shikimate could bind to the closed as well as to the open form, whereas NADP+ was found only in closed conformation. As described for
4.3. Bifunctional dehydroquinase-shikimate dehydrogenase (Ath DHQ-SDH) from Arabidopis thaliana
Remarkable are the
For the shikimate-tartrate complex crystals they used the
Not only that the closed conformation of the enzyme after binding of both products could be demonstrated (Figure 10) but also the activity of that ternary complex was proven as the oxidation of shikimate was evidenced by the generation of dehydroshikimate, – the product of the DHQ moiety – found in the DHQ site [43].
The structures of the
4.4. Shikimate dehydrogenase from Helicobacter pylori
Recently three different catalytic states of the
However, the structure is ideally suited to visualize structural changes during
In the
4.5. Quinate dehydrogenase from Corynebacterium glutamicum
Last but not least the bacterial quinate dehydrogenase of
For growing the crystals of the apo-form the protein solution was mixed with the reservoir solution and a NADH solution (2 µg/ml) in a drop ratio 1:1:1. The reduced cofactor could not be detected in the electron density due to the very low concentration [46].
For the
To obtain the
Crystals of the binary and ternary complexes were different in shape compared to the crystals of the
The crystals of all three
By comparing the overall structures of all these states an open, a semi-open and a closed conformation of the enzyme (Figure 14) was observed. Surprisingly, the apo-structure of the
Within the
4.6. Insights into the structural changes during catalysis and elucidation of substrate and cofactor specificity, using the example of Cgl QDH
4.6.1. Structure overview of C. glutamicum QDH
All
The 282 residues in the QDH molecule form two structural domains (Figure 15): the N-terminal or catalytic domain (residues 1 to 113 and 256 to 283), which binds the
4.6.2. Description and analysis of QDH active site
The strict specificity for NAD(H) is determined by the negatively charged aspartate residue 158, the neutral Leu159 and the bulk side chain of Arg163, which would result in steric hindrance with the additional phosphate group in the NADP(H) molecule.
After quinate binding a slight closure of the N- and C-terminal domain of
In comparison to the
In the case of shikimate binding a somewhat different situation was observed. In principle the above mentioned residues except Thr19 are involved in shikimate binding (Figure 17 C), but only eight polar interactions are achieved (compared to eleven when QA is bound), from which some are furthermore weaker pronounced: Thr19 is not involved in polar contacts to SA, Thr69 has contact only to the hydrogen group of C3, Asn94 is about 0.2 Å farer apart from the hydrogen atom of C4 and has no contact to the OH-group of C5. Remarkable is the appearance of an alternative side chain conformation of Lys73, as evidenced by the excellent electron density in this region. The first conformation of the Lys73 side chain in the crystal exhibits the sprawled conformation as found for the quinate binding; the second conformation reveals an angled rotamer as it occurs in apo-
4.6.3. Substrate and cofactor specificity and discrimination
All results of the structural analysis are also in excellent agreement with the findings of the kinetical assays. The higher
A further occasion for the lower affinity and catalytic efficiency regarding shikimate results from the appearance of an alternative conformation of the Lys73 side chain (Figure 17 C), which leads to a loss of an important hydrogen bond. At last we compared the substrate binding residues of
5. Conclusions
Crystal structures of proteins and enzymes are important to fully understand the mechanism and mode of action. Although the crystallization of a large number of proteins was successful and delivered valuable information the goal must be to fully understand the function. When crystallizing a protein a snapshot of the protein in a certain conformation is observed in the electron density. It is known that proteins are flexible and can obtain several states in solution.
Within that book chapter we explained the importance to acquire structural information of different catalytical states of proteins or enzymes, to fully understand how the protein behaves during catalysis or how the substrate bound state differs from the apo-enzyme.
The open and closed structures of the substrate binding protein ProX as apo-protein or with different substrates bound revealed enormous conformational changes during ligand binding and clearly visualzes how flexible a protein can be and elucidates the side chain movements within the substrate site upon ligand binding.
All described crystallization trials of the different transition states of the OcDH showed impressively that protein crystallization is a trial and error approach and that knowledge of the protein (especially the kinetical parameters beside others) is the essential thing to be successful. At best and as recompenses for ones effort one will achieve important insights that clearly explains the catalytic mechanism.
Last but not least the different structural information of the enzymes of the shikimate dehydrogenase family could bring to light how substrate and cofactor specificity and discrimination can be achieved throught detailed analysis of apo-, binary and ternary structure information about involved amino acids in substrate and cofactor binding.
So with these three examples the difficulties in crystallization on one hand and on the other hand the beauty of looking at proteins at work is shown.
PDB entries used
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ChoX | 2RF1 | Crystal structure of ChoX in an unliganded closed conformation |
3HCQ | Structural analysis of the choline binding protein ChoX in a semi-closed and ligand-free conformation | |
2REJ | ABC-transporter choline binding protein in unliganded semi-closed conformation | |
2RIN | ABC-transporter choline binding protein in complex with acetylcholine | |
2REG | ABC-transporter choline binding protein in complex with choline | |
ProX | 1SW1 | Crystal structure of ProX from |
1SW4 | Crystal structure of ProX from |
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1SW2 | Crystal structure of ProX from |
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3MAM | A molecular switch changes the low to the high affinity state in the substrate binding protein |
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1SW5 | Crystal structure of ProX from |
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CenDH | 1BG6 | Crystal structure of the N-(1-D-carboxylethyl)-L-norvaline dehydrogenase from |
OcDH | 3C7C | A structural basis for substrate and stereo selectivity in octopine dehydrogenase (OcDH-NADH-L-arginine) |
3C7D | A structural basis for substrate and stereo selectivity in octopine dehydrogenase (OcDH-NADH-pyruvate) | |
3C7A | A structural basis for substrate and stereo selectivity in octopine dehydrogenase (OcDH-NADH) | |
AroE | 2HK8 | Crystal structure of shikimate dehydrogenase from |
2HK9 | Crystal structure of shikimate dehydrogenase from |
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1WXD | Crystal structure of shikimate 5-dehydrogenase (AroE) from |
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2D5C | Crystal structure of shikimate 5-dehydrogenase (AroE) from |
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2EV9 | Crystal structure of shikimate 5-dehydrogenase (AroE) from |
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3PHG | Crystal structure of the shikimate 5-dehydrogenase (AroE) from |
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3PHH | Crystal structure of the shikimate 5-dehydrogenase (AroE) from |
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3PHI | Crystal structure of thesShikimate 5-dehydrogenase (AroE) from |
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DHQ-SDH | 2GPT | Crystal structure of |
2O7Q | Crystal structure of the |
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2O7S | Crystal structure of the |
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QDH | 3JYO | Quinate dehydrogenase from |
3JYP | Quinate dehydrogenase from |
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3JYQ | Quinate dehydrogenase from |
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2NLO | Crystal structure of the quinate dehydrogenase from |
Glossary
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Enzymes that require a cofactor but do not have one bound are called |
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The dissociation constant is commonly used to describe the |
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A binary complex refers to a protein complex containing two different molecules which are bound together. In structural biology, the term binary complex can be used to describe a crystal containing a protein with one small molecule bound, for example the cofactor or the substrate; or a complex formed between two proteins. |
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Co-crystallization means that the protein solution is mixed with one or more ligand prior to the crystallization. Often the protein-ligand mixture is preincubated before setting up the crystallization drops. |
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A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations. Cofactors are either organic or inorganic. They can also be classified depending on how tightly they bind to an enzyme, with loosely bound cofactors termed coenzymes and tightly-bound cofactors termed prosthetic groups. Examples of widespread cofactors are ATP, coenzyme A, FAD, and NAD+, vitamins or metal ions. |
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In chemistry, biochemistry, and pharmacology, a dissociation constant |
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In biochemistry, Michaelis-Menten kinetics is one of the simplest and best-known models of enzyme kinetics. The Michaelis constant |
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Binding of ligands to proteins frequently causes changes to their three-dimensional structure. Exampes of this include the binding of substrates, inhibitors, cofactors or allosteric modulators to enzymes or of hormons to receptors. If this structural change has an effect on the environment of an intrinsic or extrinsic fluorophore in the protein, this can result in measurable changes in the fluorescence spectrum. Provided that the fluorophore has a unique location in the protein, such changes of fluorescence at a particular wavelength can be used to determine the dissociation constant ( |
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This technique is useful for protein concentrations in the range of mg/ml. A typical experiment involves measurement of heat change as a function of addition of small quantities of a reagent to the calorimeter cell containing other components of the system under investigation. For example, this reagent could be a protein ligand or substrate/ inhibitor of an enzyme. At the beginning of the experiment, there is a large excess of protein compared to ligand. This means that Δ |
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In biochemistry a ligand is a substance (usually a small molecule), that forms a complex with a biomolecule to serve a biological purpose. In the context of this chapter ligand is used as a more general expression for substrate, product or cofactor. |
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Ligand soaking means the addition of ligands into the mother liquid with preformed crystals. The idea is that the ligand diffuses into the crystals and binds at the active site. This technique was initially used for the incorporation of heavy atoms into protein crystals for phasing purposes. |
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During Macro Seeding the protein crystal is replaced into a freshly made mother liquid which allows the further enlargement of the crystals size. In Micro Seeding a suspension of microcrystals is prepared by either resuspending or crushing a protein crystal cluster or single crystals. These seeds are then used (often streaked through a new droplet of precipitant and fresh protein) to serve a crystallization starting point. |
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Microbatch is a method in which the molecule to be crystallized is mixed with the crystallizing agents at the start of the experiment. The concentration of the ingredients is such that supersaturation is achieved immediately upon mixing, thus the composition and the volume of a trial remain constant and crystals will only form if the precise conditions have been correctly chosen. |
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Occupancy means the degree of protein molecules in solution or in a crystal with bound ligand. If every second protein has attached a ligand the occupancy is 50 %. |
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In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate(s). In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or more products, which are then released from the active site. The active site is now free to accept another substrate molecule. In the case of more than one substrate, these may bind in a particular order to the active site, before reacting together to produce products. |
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SPR is an optical technique which depends on changes in refractive index or mass changes near metal surfaces. When two surfaces, one a metal and the other a dielectric material are exposed to a beam of plane-polarized light of wavelength, λ, a longitudinal charge density wave (a surface plasmon) is propagated along the interface between them. This only happens when one of the surfaces is a metal and works best with silver, gold, copper and aluminium. This is because metals contain free oscillating electrons called plasmons. When light traveling through an optically dense medium such as glass arrives at an interface with a lower optical density (e.g. liquid), it is reflected back into the more optically dense medium, a phenomenon called total internal reflectance. Any process altering |
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A ternary complex refers to a protein complex containing three different molecules which are bound together. In structural biology ternary complex can be used to describe a crystal containing a protein with two small molecules bound, for example cofactor and substrate; or a complex formed between two proteins and a single substrate. |
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Two of the most commonly used methods for protein crystallization fall under the category of vapor diffusion. These are known as the hanging drop and sitting drop methods. Both entail a droplet containing purified protein, buffer, and precipitant being allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. Initially, the droplet of protein solution contains an insufficient concentration of precipitant for crystallization, but as water vaporizes from the drop and transfers to the reservoir, the precipitant concentration increases to a level optimal for crystallization. Since the system is in equilibrium, these optimum conditions are maintained until the crystallization is complete [51]. |
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