MOLEC STRU DETER XRAY
MOLEC STRU DETER XRAY BCH 6744
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This 24 page Class Notes was uploaded by Kavon Huel on Friday September 18, 2015. The Class Notes belongs to BCH 6744 at University of Florida taught by Staff in Fall. Since its upload, it has received 24 views. For similar materials see /class/206967/bch-6744-university-of-florida in Biochemistry and Molecular Biology at University of Florida.
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Date Created: 09/18/15
L 2 Sample preparation Before crystallization rst stepmost important Points to consider check list 1 Sequence 2 Stability 3 Solubility 4 Purity 5 Quantity DEEDS Lecture 2 1 Sequence amino acid composition Flexible Glycine Hydrophobic Alanine Valine Phenylalanine Proline Methionine Isoleucine Leucine Cysteine Disul de bridges Lecture 2 2 2 Stability conditionstime Find a stabilization buffer Ideal buffer water let the protein buffer itself Rare to live in ideal world Use the minimum amounts buffers salts etc The protein has to be stable in the crystallization drop until it forms crystals Ideal goal 2 weeks Stability is effected by the environment Lecture 2 3 Solubility The more soluble the protein the more likely it is to crystallize Myoglobin 1955 Kendrew Lsozyme Phillips Hemoglobin Perutz Real world 10mgml And then membrane proteins Lecture 2 4 4 Purity Nothing can be too purelllll 95 try 99 really What it takes Detection SDS gel electrophoresis silver stain Don t run sample off the gel Lecture 2 5 Quantity Crystallography can be soul destroying Need a concentration of 10mgml A trained person A single condition DROP will require a minimum of Zul Sul of sample Hence 100 conditions ONLY DONE ONCE will use up 02 ml of sample 2mg 1mg 50 conditions Robot Requires 06 ml 6 mg to test 96 X 16 conditions 1536 Person 31 ml 307 mg Lecture 2 6 Lyophilization Avoid lyophilization of your protein for storage if you plan to use the protein for crystallization experiments Also don t concentrate it Lyophilization is a not so gentle procedure and can prevent crystallization So don39t go there If you have a lyophilized protein is there hope Should solubilize the protein in water or a stabilization buffer dialyze the protein exhaustively Dialysis is an important step which will help to remove residual nonvolatile buffers and reagents as well as low molecular contaminants Lecture 2 7 Sample aggregation Deterrent to crystallization Detection Dynamic light scattering Native gel electrophoresis silver stain Common Causes Hydrophobic patches on the surface Differently charged isoforms Differently phosphorylated isoforms Mixtures of methylated and nonmethylated samples Glycosylation Electrostatic interactions Lecture 2 Two types of aggregation Heterologous contamination Where the sample is aggregating with other proteins In the case of heterologous contamination further puri cation of the sample should be seriously considered Lecture 2 9 Autologous aggregation Where the protein is aggregating with itself Remedies Molecular biology Manipulate intra and inter molecule interactions by modifying the sample sequence alter add or delete residues Consider a fusion protein Remove Cterminus or Nterminus Truncate domains Remove Histag Chemical additives Manipulate samplesample and sample solvent interactions Detergents Alcohols isopropanol methanol ethanol etc Salts sodium chloride potassium chloride sodium uoride etc Polyols glycerol PEG 400 etc Ligands inhibitors cofactors and metals Lecture 2 10 Other suggestions Use temperature to prevent aggregation 0 C and 60 C Centrifugation or ltration Last hope Mixing the sample with the crystallization reagent allowing the sample to incubate for 15 minutes centrifuging the samplereagent mixture removing the precipitate and setting the drop with the supernatant Lecture 2 ll Proteases Proteases can also be trouble causing cleavage of the sample Talk to Prof Ben Dunn Remedy Add a protease inhibitor Protease contamination can be carried over from isolation and puri cation of the sample Lecture 2 12 Protease Metalloproteases Inhibitor Chelators like EDTA and EGTA bestatin amastatin thiol derivatives hydroxamic acid phosphoramidon Protease Aspartic Acid Proteases Inhibitor Pepstatins and statin derived inhibitors Protease Cysteine Proteases Inhibitor Thiol binding reagents peptidyldiazomethanes epoxysuccinyl peptides cystatins peptidyl chloromethanes Protease Most Inhibitor DEPC Protease Serine Proteases Inhibitor Trypsin inhibitors leupeptin boronic acids cyclic peptides DIFP PMSF Pefabloc aminobenzamidine 34dichloro isocoumarin chymostatin Lecture 2 13 The presence of fungal or microbial contamination in your sample or reagents with subsequent release of proteases from these organisms Remedy Include sodium azide or thymol Deterrent to microbial growth which prevents the possibility of microbial agents growing and secreting proteases into the sample solution Typically add lmM sodium azide Note Sodium azide and thymol can sometimes bind the sample are toxic and in some cases do not live well with heavy atoms Our Lab Rather clean workspace sterile ltered samples and reagents good technique prevents microbial contamination Lecture 2 l4 However Proteolytic modi cation of proteins can be a tool for making small active fragments of proteins which might have enhanced solubility characteristics compared to the native protein which in turn might make the protein more amenable to crystallization Lecture 2 15 Nucleases Problems Modi cation of size charge or hydrophobicity partial or total loss of activity or utter destruction of nucleic acids Dif cult to detect even on overloaded electrophoresis gels Damage can be during puri cation concentration and storage Remedies Include a nuclease inhibitor in the prep or sample to protect the sample from ribonuclease and deoxyribonucleases Inhibitors RNasin from Promega ribonucleaosidevanadyl complexes and DEPC Inhibitors of deoxyribonucleases include DEPC and chelators such as EDTA or EGTA Lecture 2 l6 Reducing Agents Oxidation can lead to nonspeci c aggregation heterogeneity inactivity or denaturation of protein Remedy Reducing agents are substances that cause other chemical species to be reduced or gain electrons In order for reducing agents to cause the gaining of electrons on some other chemical species they must undergo oxidation and prevent the oxidation of free sulihydryl residues cysteines in proteins Also glove box Examples Dithiothreitol DTT betamercaptoethanol betame and Tris2CarboxyethylPhosphine Hydrochloride TCEPHCl Concentration range of 1 to 10 mM in the crystallization drop Lecture 2 l7 Crystallisation improvements of samples examples Site directed mutagenesis Recombinant DNA technologymeans to make speci c changes in a protein sequence Systematic approach of improving crystallisability by changing hydrophobic to hydrophilic residue eg HIV integrase Davies Science 1994 255 19811986 Alterations of protein and RNA sequence to obtain a more stable complex eg U1A proteinRNA complex Outbridge JMB 1995 249409423 Lecture 2 18 Fusion proteins Carrier protein introduced into an internal position of a target protein Carrier protein soluble single compact domain crystallisable N and C close together no disulphide bonds easily cloned and eXpressed larger than target protein eg Introduction of the E coli Cytochrome b562 into lactose permase membrane protein Prive Acta Cryst D 1994 50375379 Lecture 2 l9 Complexes with antibodies Proteins of interest are complexed with Fab fragments Concept same as fusion proteins eg P24Fab complex Kovari Structure 19953z12911293 Odd result Improvement of crystal lattice contacts by mutations eg Glutathione reductase Ecoli Schulz Acta Cryst D 1994 50 228231 Double mutation gtgtCrystals grew 40 times faster than for native protein Lecture 2 20 Finally questions to ponder about YOUR sample C Does a similar sample exist and has it been crystallized 0 Does the sample contain free cysteines Q Does the sample contain additives such as sodium azide ligands inhibitors or substrates 0 Is the protein glycosylated C Is the protein phosphorylated C Is the protein Nterminal methylated C At what temperature is the protein stable 0 How does sample solubility and stability change with temperature 0 How does sample solubility and stability change with pH C Does the sample bind metals 0 Is the protein sensitive to proteolysis Q What class of proteinNA am I working with antibody Virus enzyme membrane protein Lecture 2 21 Q What has been the most successful approaches with my class of protein C What is the source of the sample 0 How was the sample puri ed and stored before it arrived into my hands 0 What is in the sample besides the sample buffer additives etc 0 Is the sample pooled puri cation aliquots or a single batch Q How much sample do I have and how much more is available 0 How pure is the sample 0 How homogeneous is the sample 0 Does anyone possess any solubility information on this sample C What is unique about this protein 0 What is necessary chemically and physically to maintain a stable active sample Lecture 2 22 Last thought Never never never see protein puri cation as a separate event from crystallization It should be seen as a continuous process Crystallization is the nal step in puri cation Lecture 2 23 Assignment In half a page note form Think about the questions above and write a pro le that might help you crystallized YOUR sample Lecture 2 24
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