Downloaded Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-FP001 Chemical and Biological Approaches Carbohydrate Chemistry Volume 39 Downloaded Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-FP001 View Online View Online Downloaded Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-FP001 A Specialist Periodical Report Carbohydrate Chemistry Chemical and Biological Approaches Volume 39 Editors Amelia Pilar Rauter, Universidade de Lisboa, Portugal Thisbe K Lindhorst, Christiana Albertina University of Kiel, Germany Authors Valquiria Araga˜o-Leoneti, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Sa˜o Paulo, Brazil Binod K Bharati, Indian Institute of Science, Bangalore, India Vanessa Leiria Campo, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Sa˜o Paulo, Brazil Ivone Carvalho, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Sa˜o Paulo, Brazil Dipankar Chatterji, Indian Institute of Science, Bangalore, India Darrell Cockburn, Technical University of Denmark, Lyngby, Denmark Gabriele Cordara, University of Oslo, Oslo, Norway Katalin Czifra´k, University of Debrecen, Hungary N Jayaraman, Indian Institute of Science, Bangalore, India Ana R Jesus, University of Lisbon, Portugal Vladimı´r Krˇen, Academy of Sciences of the Czech Republic, Prague, Czech Republic Ute Krengel, University of Oslo, Oslo, Norway Jian Liu, Eshelman School of Pharmacy, University of North Carolina, USA Kotari Naresh, Indian Institute of Science, Bangalore, India Noe´ On˜a, University of Malaga, Spain Amelia P Rauter, University of Lisbon, Portugal M Soledad Pino-Gonza´lez, University of Malaga, Spain Antonio Romero-Carrasco, University of Malaga, Spain Kristy´na Sla´mova´, Academy of Sciences of the Czech Republic, Prague, Czech Republic La´szlo´ Somsa´k, University of Debrecen, Hungary Arnold E Stuătz, Technische Universitaăt Graz, Graz, Austria Birte Svensson, Technical University of Denmark, Lyngby, Denmark View Online Downloaded Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-FP001 Se´bastien Vidal, Universite´ Claude Bernard Lyon, Villeurbanne, France Shuai Wang, Universite´ Claude Bernard Lyon, Villeurbanne, France Tanja M Wrodnigg, Technische Universitaăt Graz, Graz, Austria View Online If you buy this title on standing order, you will be given FREE access to the chapters online Please contact E-mail: sales@rsc.org with proof of purchase to arrange access to be set up Downloaded Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-FP001 Thank you ISBN: 978-1-84973-587-2 ISSN: 0306-0713 DOI: 10.1039/9781849737173 A catalogue record for this book is available from the British Library & The Royal Society of Chemistry 2013 All rights reserved Apart from fair dealing for the purposes of research or private study for non-commercial purposes, or for private study, criticism or review, as permitted under the Copyright, Designs and Patents Act, 1988 and the Copyright and Related Rights Regulations 2003, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry, or in the case of reproduction in accordance with the terms of the licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page Published by The Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 0WF, UK Registered Charity Number 207890 For further information see our web site at www.rsc.org Downloaded Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-FP001 View Online Preface Downloaded Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-FP007 DOI: 10.1039/9781849737173-FP007 While this volume is mainly dedicated to the investigation and utilisation of carbohydrate-specific enzymes, the reader will also find enzymology and glycobiology combined with glycochemistry, demonstrating how the interdisciplinary approaches taken in the glycosciences contribute to the increasingly important field of glycomics The first chapter of this book is dedicated to the radical bromination of sugars, involving a broad range of substrates and their transformations It highlights the synthetic utility of this type of reactions and, in particular, the uniqueness of carbohydrates as substrates, leading to a wide variety of molecular tools for chemical glycobiology Examples are given of acceptor substrate analogues for glycosyltransferases, inhibitors of glycosidases, compounds that inactivate retaining N-acetylglucosaminidases, amongst many other bioactive compounds that were synthesized via radicalmediated halogenation of carbohydrates While the first chapter is dedicated to synthetic organic glycochemistry, the second illustrates the importance of enzymatic and chemoenzymatic syntheses for the production of the polysaccharide heparin, marketed as anticoagulant agent Recent developments on synthetic glycolipids as ligands and as inhibitors of mycobacterial cell wall components, biosynthesis and functions are described in chapter 3, also focusing on the inhibition of key glycosyltransferases by glycolipids The next chapters deal with carbohydrate-processing enzymes and their inhibitors, most of them small molecule inhibitors Design and synthesis of glycosyltransferase and glycosidase inhibitors is reviewed, paying particular attention to imino sugars and to carbohydrate epoxides as synthetic key intermediates of this important class of therapeutic targets, with applications in the treatment of influenza infection, cancer, AIDS, and diabetes Also an overview on glycosidase metabolic changes in diabetes is presented The deficiency in humans of hexosaminidases causes severe neurodegenerative disorders, including the Alzheimer’s disease Hence a survey of the most efficient and selective inhibitors of these glycosidases, required for the research of their physiological functions, is given in this volume In recent years binding sites of carbohydrate-specific enzymes have been investigated in greater detail, with special focus on surface and secondary binding sites (SBS) SBS, playing several supporting roles in enzyme function, are binding sites that are located on the catalytic domain of a particular enzyme, but separate from the enzyme’s main active site Another chapter is devoted to this interesting area of research that aims to modulate enzymatic behavior without altering the enzyme active site, focusing on SBS potential roles, techniques for SBS study and applications The last but not the least, X-ray crystallography of lectins is the subject of a chapter, emphasizing the characterization of lectincarbohydrate complexes with high precision, and revealing in detail the underlying molecular recognition mechanisms Carbohydr Chem., 2013, 39, vii–viii | vii  c The Royal Society of Chemistry 2013 View Online Volume 39 contains chapters covering chemical, biochemical and biological approaches that demonstrate, in a meaningful way, how interdisciplinary approaches in the glycosciences help to advance and appreciate our understanding of the biological processes involving carbohydrates that may be controlled to promote health and prevent disease Downloaded Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-FP007 Ame´lia Pilar Rauter Thisbe K Lindhorst viii | Carbohydr Chem., 2013, 39, vii–viii CONTENTS Downloaded Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-FP009 Cover Tetrahydropyran-enclosed ball-and-stick depiction of a glucose molecule, and (in the background) part of an a-glycosyl-(1-4)-D-glucose oligosaccharide and a glycosidase, all representative of the topics covered in Carbohydrate Chemistry – Chemical and Biological Approaches Cover prepared by R G dos Santos Preface Ame´lia Pilar Rauter and Thisbe K Lindhorst Radical-mediated brominations at ring-positions of carbohydrates – 35 years later La´szlo´ Somsa´k and Katalin Czifra´k Introduction Radical-mediated brominations Transformations of the brominated compounds Biological effects of and/or studies with compounds obtained via the brominated sugars and their ensuing products Conclusion Acknowledgement References vii 1 16 31 33 33 33 Recent advances in enzymatic synthesis of heparin 38 Ana R Jesus, Ame´lia P Rauter and Jian Liu Introduction Enzymatic synthesis of heparin Conclusions Acknowledgments References 38 43 55 55 55 Carbohydr Chem., 2013, 39, ix–xii | ix  c The Royal Society of Chemistry 2013 Downloaded Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-FP009 View Online Synthetic arabinan, arabinomannan glycolipids and their effects on mycobacterial growth, sliding motility and biofilm formation Binod K Bharati, Kotari Naresh, Dipankar Chatterji and N Jayaraman Introduction Development of synthetic glycolipid inhibitors Biological studies of modified arabinose oligosaccharides Biological studies of iminosugar-arabinan oligosaccharide conjugates Effects of synthetic mannose oligosaccharides on mannosyltransferase (ManT) enzyme Studies of linear and branched arabinan and arabinomannan glycolipids Conclusion and perspectives Acknowledgements References Recent design of glycosyltransferase inhibitors Shuai Wang and Se´bastien Vidal Introduction Inhibitors of galactosyltransferases (GalT) Inhibitors of O-linked N-acetylglucosamine transferase (OGT) Conclusion and perspectives Acknowledgements References 58 58 61 63 65 65 67 74 75 75 78 78 80 92 96 97 97 b-N-Acetylhexosaminidases: group-specific inhibitors wanted Kristy´na Sla´mova´ and Vladimı´r Krˇen Introduction b-N-Acetylhexosaminidases: properties and physiology Inhibitors of b-N-acetylhexosaminidases Conclusions Acknowledgements References 102 Positive attitude, shape, flexibility, added-value accessories or ‘‘just being different’’: how to attract a glycosidase Arnold E Stuătz and Tanja M Wrodnigg Introduction Positive attitude - not always necessary Good shape and flexibility - catering for quite diverse requirements 120 x | Carbohydr Chem., 2013, 39, ix–xii 102 103 105 115 115 115 120 127 130 Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 View Online using the same buffer that was used to dissolve the lyophilized protein This is your ConA working solution for the crystallisation experiment Using a single channel pipette (P2 or P10), transfer a ml drop of the ConA working solution to a silanised cover slip (see Practical Tips and Tricks, note 1); pipette a second ml drop from one of the reservoirs onto the protein drop without mixing or pipetting up and down, which could denature the protein, and try to avoid producing air bubbles (see Practical Tips and Tricks, note 11) Carefully turn around the cover slip and place it on top of the appropriate reservoir Apply delicate pressure (e.g using the tip of the pipette – not with your finger!) to let the oil seal the crystallisation chamber The procedure is repeated until all the 48 wells are completed Carefully check the crystallisation plates to ensure that the wells are correctly sealed (no gaps and no air bubbles trapped between the cover slips and the well’s rim) Place small amounts of plasticine on the four corners of the plate and rest the lid of the plate on these, pressing gently This ensures that the lid is not directly in contact with the cover slips Then, using a microscope, proceed to observe each crystallisation drop and note any ongoing phenomenon inside (see Practical Tips and Tricks, note 12) Keep track of any changes observed in the drops and note them in your logbook; pre-formatted ‘scoring sheets’ to facilitate this task are available on the web pages of many vendors of crystallisation supplies (e.g the ‘‘Crystallisation Scoring Sheet‘‘ available from Hampton Research; see Table 1, link 8) The following time schedule is recommended: observe the drops right after setting up the crystallisation experiment, then a few hours later, then each day for the first week, then once a week for the first month and finally once per month (crystals can form even after a very long time, and these results may give interesting leads) Any phase change (e.g phase separation, formation of spherulites or – in the best case – crystals) is considered a ‘hit’ and provides precious leads to improve the crystallisation conditions (see Practical Tips and Tricks, note 13) It is also recommended to assign a score to each result: useful directions about scoring can be found on the home page of Terese Bergfors (Table 1, link - follow the link to ‘‘Tutorials’’) or in references on the topic.69,70 When carefully comparing the conditions that produced the different hits, potential underlying themes may be revealed that can be exploited in follow-up experiments Some examples of positive and negative outcomes of the crystallisation screen are shown in Fig The remaining ConA solution is stored for future experiments If experiments are planned within a week or two, the sample may be stored at 1C, otherwise it is advisable to prepare 50 or 100 ml aliquots that are subsequently flash-frozen in liquid nitrogen The frozen samples can then be stored at À 80 1C, where they are stable for several months to years (see Practical Tips and Tricks, note and 14) 3.3.2 Refinement of the crystallisation conditions Once a hit has been obtained from the initial screening, it can usually be optimized to get bigger and better diffracting crystals There is again no general strategy: every optimisation screen has to be planned on a case-by-case basis The most straightforward approach 232 | Carbohydr Chem., 2013, 39, 222–246 Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 View Online Fig Different factors affecting a crystallisation experiment The first two pictures show examples of ‘‘bad’’ and ‘‘good’’ precipitate, followed by examples of two events that the experimenter would prefer to avoid, namely a fiber and an air bubble in the crystallisation drop On the second row, the first two pictures show the results obtained with a non-silanised versus a silanised cover slip: no significant difference can be noticed, except for higher spread of the drop on the untreated glass surface The last two pictures on this row show that it is possible to get crystals, even in the presence of high amounts of amorphous precipitate The first two snapshots on the third row show the effect of two different starting concentrations of Concanavalin A (5 mg/ml and 2.5 mg/ml, respectively) on crystal formation If the protein concentration is too low (i.e below the solubility curve in Fig 2), no crystals will form The last two images show the result of crystallisation experiments using chemicals from different sources (#2 in-house, PEG 4000 from Fluka; #2A from MDL) involves varying individual parameters, taking discrete steps in both the positive and the negative direction, or including two or more parameters to design a grid screen Some of the parameters that can be changed are the nature of the buffer and the pH, the concentration and the nature of the precipitant (e.g try ammonium sulphate or tartrate rather than PEG or try varying the nature of the PEG), the presence and concentration of additives (e.g organic solvents, detergents or small amounts of salts) or the counter ions used (e.g try switching from ammonium sulphate to ammonium phosphate or lithium sulphate) Some specific additive screening kits have also been developed and are commercially available from different crystallography suppliers (for a list of crystallography suppliers see Table 1, links 10-12) As an alternative to self-made refinement strategies, there are software tools using statistical methods to help the user in selecting an optimized crystallisation screen, e.g the program XtalGrow (Table 1, link 13) Besides varying the components of the crystallisation solution, it is possible to alter the kinetics of the experiment This can be achieved in a number of different ways, e.g by varying the ratio of protein to reservoir drop volumes (affects both the concentration of the protein and the path by which the equilibrium is reached), by setting up the experiment at a temperature different than room temperature (see Practical Tips and Tricks, note 15; 1C is a good starting point if temperatures other than 20 1C are to be explored) or by uncoupling nucleation and crystal growth The latter can Carbohydr Chem., 2013, 39, 222–246 | 233 View Online Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 be achieved e.g by providing the system with nucleation centres through seeding, by using oils or gels or by setting over crystallisation experiments from higher to lower concentration reservoirs after a defined time period.57,71–75 More complex optimisation strategies and methods are described by Carter and Ries-Kautt.76 Devising a simple PEG/pH grid screen for concanavalin A The initial screening for Con A crystals gave hits for conditions #2, #9, #18, #19, #20, #24, #29, #32, #44, #46 and #47, with the notable case of condition #2, where crystals formed within the first 20 minutes (see Fig for snapshots of some of the hits; Table lists the composition of the crystallisation solutions that led to crystal formation) A closer inspection of the successful conditions revealed that PEG was a suitable precipitant in most of the cases, and that PEGs of different molecular weights gave hits The pH of the buffers varied from 4.6 to 7.5, and often acetate (with different counter ions) was present as an additive The most promising looking crystals had formed in condition #2, which was hence given special weight when designing an optimisation screen Condition #2 contained 0.1 M sodium acetate pH 4.6, 30% w/v PEG 4000 and 0.2 M ammonium acetate Starting from these initial components, a multi-dimensional grid screen was designed, varying Fig Results from the initial crystal screening The figure shows some of the hits obtained from the sparse matrix screening obtained for conditions #2, #18, #20 and #46, in the absence or presence of mannose (labelled ‘‘Man’’) Conditions labelled ‘‘A’’ and ‘‘B’’ differ with respect to the buffer in which Concanavalin A was dissolved (A, 10 mM BisTris propane, pH 7.0; B, 10 mM Tris, pH 7.5) While the protein buffer only has a small effect on the outcome of a crystallisation experiment due to its low concentration compared to the buffer in the reservoir solution (essentially acting as an additive, although there may be additional differences due to statistics), the presence of a ligand can have a much larger effect (here, leading to the formation of different crystal forms) - All pictures have the same magnification 234 | Carbohydr Chem., 2013, 39, 222–246 View Online Table Initial Crystal Screen Hits from the initial screening Position Crystallisation solution Plate #1, A2 SS#02: 0.1 M Sodium acetate pH 4.6, 30% w/v PEG 4000, 0.2 M Ammonium acetate SS#09: 0.1 M Sodium citrate pH 5.6, 20% v/v 2-propanol, 20% w/v PEG 4000 SS#18: 0.1 M Sodium cacodylate pH 6.5, 30% w/v PEG 8000, 0.2 M Sodium acetate SS#19: 0.1 M Sodium cacodylate pH 6.5, 18% w/v PEG 8000, 0.2 M Zinc acetate SS#20: 0.1 M Sodium cacodylate pH 6.5, 18% w/v PEG 8000, 0.2 M Calcium acetate SS#24: 0.1 M Hepes pH 7.5, 30% v/v PEG 400, 0.2 M Magnesium chloride SS#29: 0.1 M Hepes pH 7.5, 1.4 M Sodium citrate SS#44: 2.0 M Ammonium sulphate SS#46: 20% w/v PEG 8000, 0.05 M Potassium dihydrogen phosphate SS#47: 30% w/v PEG 1500 Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 Plate #1, B3 Plate #1, C6 Plate #1, D1 Plate #1, D2 Plate Plate Plate Plate Plate #1, #2, #2, #2, #2, D6 A5 D2 D4 D5 Table Custom-made Optimisation Screen PEG concentration x pH x Ammonium acetate concentration screen Position Crystallisation solution A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6 C1 C2 C3 C4 C5 C6 D1 D2 D3 D4 D5 D6 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 M M M M M M M M M M M M M M M M M M M M M M M M Sodium Acetate pH 4.6, 30% v/v PEG 400, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 1000, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 1500, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 2000, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 6000, 0.2 M Ammonium acetate Sodium Acetate pH 4., 30% w/v PEG 8000, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 26% w/v PEG 4000, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 28% w/v PEG 4000, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 4000, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 32% w/v PEG 4000, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 4000, 0.15 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 4000, 0.25 M Ammonium acetate Sodium Citrate pH 5.5, 26% w/v PEG 4000, 0.2 M Ammonium acetate Sodium Citrate pH 5.5, 28% w/v PEG 4000, 0.2 M Ammonium acetate Sodium Citrate pH 5.5, 30% w/v PEG 4000, 0.2 M Ammonium acetate Sodium Citrate pH 5.5, 32% w/v PEG 4000, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 4000, 0.15 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 4000, 0.25 M Ammonium acetate BisTris pH 6.5, 26% w/v PEG 4000, 0.2 M Ammonium acetate BisTris pH 6.5, 28% w/v PEG 4000, 0.2 M Ammonium acetate BisTris pH 6.5, 30% w/v PEG 4000, 0.2 M Ammonium acetate BisTris pH 6.5, 32% w/v PEG 4000, 0.2 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 4000, 0.15 M Ammonium acetate Sodium Acetate pH 4.6, 30% w/v PEG 4000, 0.25 M Ammonium acetate the precipitant concentration, the pH and the nature of the precipitant (testing PEGs with different molecular weights and keeping in mind that the pH of the solution is not only determined by the buffer, but also by additives such as ammonium acetate); a custom grid screen is reported in Table 3, and Carbohydr Chem., 2013, 39, 222–246 | 235 Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 View Online Fig Results from optimisation The figure shows the outcome of different optimisation strategies based on the hits obtained from the initial screening, taking the most promising condition, #2 (0.1 M acetate pH 4.5, 30% w/v PEG 4000, 0.2 M ammonium acetate), as a standard In the first five pictures, the nature of the PEG was varied (different average molecular weights), while keeping all other parameters constant With lower molecular weight PEGs, nucleation happens statistically less often, leading to a smaller number of large crystals, while the opposite is true for higher molecular weight PEGs A second type of variation concerned the crystallisation buffer, where acetate pH 4.6 was exchanged for either citrate pH 5.5 or BisTris pH 6.5, here shown for the complex with mannose The result is a change in crystal morphology, as visible on the last three snapshots on the second row On the third row, either the cation or the anion of the additive (ammonium acetate) was varied, in an extension of the original optimisation protocol This gave mixed results: improvement in some cases (e.g magnesium acetate and ammonium formate), and heavy precipitation in others (e.g calcium acetate) Further optimisation should build on all the obtained information, testing different combinations of PEGs, buffers and additives, preferentially at different temperatures, keeping in mind that X-ray diffraction remains the ultimate test of crystal quality Final optimisation may further include seeding techniques and other crystallisation methods - All pictures within a group are shown to scale a series of snapshots of the results of the optimisation screening is displayed in Fig Experimental protocol Prepare stock solutions for each of the components used in the optimisation screen in Table or, alternatively, for a screen of your own design The concentration of the stock solutions should be twice (or more) their maximum working concentration Usually, 0.5 M stock solutions are prepared for the buffers and 50% w/v stock solutions for PEG (Z1000) (see Practical Tips and Tricks, notes 3-5, 16 and 17) Moreover, all the solutions should be prepared with ultra-pure water (see Practical Tips and Tricks, note 3) and either passed through a 0.22 mm filter or complemented with mM sodium azide to prevent bacterial and fungal growth Prepare a pipetting scheme for each of the reservoir solutions in the optimisation screen (final volume: ml), using your stock solutions and pure water as ingredients Then prepare a 24-well tissue culture plate as described in 3.3.1 Add to each well the calculated amounts of ultra-pure water and stock solutions to a final volume of ml (see Practical Tips and Tricks, note 18) 236 | Carbohydr Chem., 2013, 39, 222–246 Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 View Online Mix the content of the wells thoroughly by pipetting up and down or put the plate on an orbital shaker at low speed for minutes or until complete mixing is ensured (particularly important when using very viscous solutions like PEG; visually check that there is no partial phase separation due to incomplete mixing) Proceed as with the initial screening, depositing a ml drop of protein solution and thereafter a 1ml drop of the reservoir solution onto the cover slip, then seal the well and check the plate under the microscope 3.4 Practical tips and tricks Although untreated glass microscope cover slips can be used for the crystallisation experiment, silanised cover slips are usually preferred since they allow the formation of a perfectly round drop, avoiding the solution to spread on the glass surface and maximizing the area of the liquid/air interface To keep costs down, cover slips can be silanised in your lab, just keep in mind that there are potential health hazards connected to handling silanisation reagents (use a fume hood!) It is nevertheless possible to use normal glass cover slips and some people even prefer them for initial screens The results are shown in Fig During crystal screening, a large number of conditions are to be explored, with different buffers, pH, precipitants, etc Therefore it is generally advisable to keep the solution in which the protein is stored as simple as possible, preferably consisting only of a suitable buffer at low concentration (e.g 10 mM), in order to allow ‘‘out-buffering’’ by the buffers used for crystallisation (typically 100 mM) The protein storage buffer is chosen to maximize activity and long-term stability, based on existing biochemical data Here we chose BisTris propane pH 7.0 and, in an alternative experiment, Tris pH 7.5 To increase protein solubility, it can sometimes be necessary to add some salt (typically 0.1 M sodium chloride) It is generally advisable to prepare stock solutions for crystallisation using chemicals of very high purity (p.a grade) and ultra-pure water (doubly distilled or ‘Type 1’ water: purified by reverse phase chromatography, desalted and sterile-filtered) The preparation of the buffers may affect the crystallisation experiments and should therefore be carefully considered and noted (e.g it can make a difference if the pH is adjusted with hydrochloric acid, introducing chloride into the solution, or with the conjugated acid/base) Different vendors of crystallisation supplies use different methods to adjust the pH, which may account in part for different crystallisation results obtained under nominally identical conditions PEG stock solutions are highly viscous 50% w/v stock solutions are best prepared directly in low 50 ml measuring cylinders, into which a magnetic stirring bar is placed at the bottom The weighed, solid PEG is added before the pure water and the sodium azide stock solution Mixing occurs first manually, sealed with parafilm, and thereafter on a magnetic stirrer (several hours or over night) More water is gradually added before the magnetic stirring bar is removed with a magnet and thoroughly rinsed to ensure correct concentration – PEGs with average molecular weights Carbohydr Chem., 2013, 39, 222–246 | 237 Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 View Online of o1000 are liquids and hence prepared on a v/v basis Stock solutions are not required, as the pure liquid serves as the stock Not all proteins retain their fold and activity after the freezing/thawing process; keep that in mind and check activity (if possible) for different storage conditions and time intervals In some cases, a cryoprotectant (like glycerol) may need to be added before freezing, although it might get in the way of protein-ligand interfaces and should therefore be carefully considered Sugars are also known as cryoprotecting agents, hence freezing the lectin with sufficient amounts of its sugar ligand may protect the protein from damage and provide ready-made material for crystallisation screens There are, however, also proteins that cannot be stored without significantly losing activity In this case, the protein needs to be freshly prepared before setup Different sealants have different advantages and disadvantages and affect the outcome of the crystallisation experiment Vacuum grease, for example, offers real air-tightness, while oils allow a certain degree of evaporation, and hence may be preferable for initial screening since a larger range of concentrations can be sampled over time User preferences are often also influenced by practical handling (oil with a paint brush, grease with stoppers or gloved fingers) It is generally advisable to apply oil after filling the reservoirs to avoid leakage from the well’s rim to the underlying reservoir solution However, it is important to keep in mind that in the presence of reservoir solutions with highly volatile components (e.g methanol, ethanol or other organic solvents), the time interval between the reservoir deposition and the sealant application should be as short as possible In these cases inverting the order of the steps or waiting with the addition of the volatile component until after greasing is advisable As noted in Jancarik and Kim,52 10 mg/ml is a reasonable protein concentration to start screening If the majority (W80%) of drops contain precipitate after one week, this suggests that the starting protein concentration was too high: try to halve the starting protein concentration On the other hand, if the majority of the drops remain clear, the protein concentration might be too low: try to double the starting concentration (here, a more concentrated stock solution comes in handy) Concentration can also be achieved easily in the crystallisation drop, by mixing the protein and reservoir solutions e.g in a 2:1 instead of a 1:1 ratio and subsequent equilibration 10 If the available protein material is scarce, it can be a good idea to apply the so-called ‘dilution method’, i.e diluting protein solution and reservoir drops to be combined in the crystallisation drop.77 This allows the handling of very small amounts of protein, while the drop volumes remain reasonable (i.e allowing accurate pipetting and avoiding problems with evaporation), when no nanoliter crystallisation robot is available While the starting concentration is lower than in a standard experiment, which may hamper the formation of crystal nuclei, the final concentration after equilibration is the same 11 Besides hindering drop observation under the microscope, air bubbles have been reported to promote protein denaturation and thus failure of 238 | Carbohydr Chem., 2013, 39, 222–246 Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 View Online the crystallisation experiment It is hence recommended that air bubbles are removed or popped using a fine pipette tip (e.g a gel loading tip) or a syringe needle When the first option is chosen (air bubble removed with pipette), this small additional droplet may be placed next to the ‘‘mother drop’’ for comparison If sufficient protein sample is available, duplicating crystallisation drops can be a generally advisable strategy (better statistics) For an example of a crystallisation drop containing an air bubble covered with a skin of denatured protein see Fig 12 Be careful: some microscopes have warm light sources, which may heat up the crystallisation experiments under observation The formed water vapour condenses on the cover slip, diluting the crystallisation drop and, in the worst case, dissolving the crystals 13 Watch out – your beautiful crystals may in fact be salt crystals Possible methods to assess the nature of a crystal include a) testing them for X-ray diffraction (contrary to protein crystals, the diffraction pattern of salt crystals displays only few, strong and widely separated spots), b) moving them around in the drop (protein crystals are usually lighter than salt crystals due to their high solvent content; see Matthews78), c) staining them blue, by adding a protein dye to the crystallisation drop (e.g using the IZIT dye, sold by Hampton Research, although this does not always work and in addition sacrifices the crystals), d) washing them in mother liquor and then dissolving them in SDS sample buffer for SDS PAGE analysis, e) inspecting the crystallisation reservoir (if similar crystals are present in the reservoir, it is likely that the crystals contain salt) and, f) as a last resort, crushing them (protein crystals fall apart easily, while salt crystals pose much more resistance and make a clicking sound when the crushing tool snaps on the cover slip) 14 While flash-freezing in liquid nitrogen is recommended, usually not much harm is done if the aliquots are placed in the freezer directly Thawing frozen protein for subsequent experiments is best done quickly in your hand (before storing the sample on ice) 15 Variations in temperature can have a profound effect on crystallisation Therefore many crystallography laboratories have temperature-controlled rooms (usually set to a standard temperature of 20 1C) If you not have such a room at your disposal, it is advisable to take detailed notes of the temperature and possible temperature variations (e.g crystals appearing after a heat wave may suggest that a higher temperature is needed for nucleation) 16 PEGs have average molecular weights and each sample is unique, especially those from different vendors Therefore testing of different PEGs may include testing of PEGs of the same molecular weight, but from different vendors 17 PEG solutions are prone to light-induced oxidation, which leads to significant reduction of the pH over time, especially within the first few weeks This should be kept in mind when experiments cannot be reproduced It may be worthwhile to always use either old or new PEG stocks, and to generally store PEG solutions in the dark 18 It is difficult to accurately pipette very viscous solutions and liquids, such as PEG, with standard pipettes Here, positive displacement pipettes such as the Distriman pipette from Gilson can make life significantly easier Carbohydr Chem., 2013, 39, 222–246 | 239 View Online Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 Alternatively, if such a pipette is not available, we recommend cutting off the pipette tips with a clean scissor 3.5 Ligand complexes While the 3D-structures of lectins are of considerable interest, especially if a lectin exhibits a new fold, there is no doubt that the ultimate goal of lectin structural analyses is to characterize in detail their molecular recognition mechanisms After all, it is the molecular interactions of lectins with their carbohydrate ligands that determine their biological functions Moreover, lectins are widely used as specific biomolecular probes and tools79, e.g for blood typing, which may be further optimized on the basis of a detailed structural characterisation The sample preparation, crystallisation and crystallographic analysis of protein-ligand interactions has been the subject of a number of publications.80–83 There are two different technical approaches to the problem: soaking and co-crystallisation 3.5.1 Soaking lectin crystals with sugar ligands Protein crystals are characterized by a very high solvent content (in average ca 50%, ranging from ca 30–80%)78, with relatively wide solvent-filled channels that allow the diffusion of small molecules in the crystal It is due to this high solvent content that proteins usually retain their activity in a crystal, allowing biochemically meaningful structure-function analysis By soaking the protein crystal in a solution of a potential (or known) ligand for a few minutes to several days, it is possible for small ligands to diffuse into the crystal and bind specifically to the protein Soaking of crystals has many applications, for example the possibility to screen a high number of different ligands without screening for new crystallisation conditions, which can be useful for drug discovery84, and the possibility to prepare heavy atom derivatives for MIR (or MAD) phasing Our focus is on the soaking of carbohydrate ligands or sugar mimics into lectin crystals In principle, the procedure is simple: The sugar ligand can either be added to the crystal drop directly in solid form or dissolved in the mother liquor (the equilibrated solution, in which the crystal resides; e.g taken from the reservoir solution) Alternatively, the protein crystal can be transferred to a new drop containing the ligand, either with a small loop (handmade or commercially available) or with a glass or quartz capillary After the transfer, the experiment should be well sealed, so that the drop does not dry out (and the crystals lose diffraction power) – These tasks are particularly challenging despite their apparent simplicity: protein crystals are very fragile entities and easily crack, dissolve or lose diffraction power even without showing any outer signs of disturbances Moreover, the binding of the sugar ligands may involve sites that are inaccessible due to crystal contacts Binding to these sites will then necessarily interfere with crystal packing, causing the crystal to crack or even to dissolve Nevertheless, in spite of these challenges, soaking is often the method of choice for small ligands, especially if the apo-crystals can be easily obtained or if the ligands are very expensive or difficult to synthesize 240 | Carbohydr Chem., 2013, 39, 222–246 Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 View Online 3.5.2 Co-crystallisation For larger ligands (trisaccharides or larger), soaking is often impossible, because ligand binding usually interferes with crystal contacts For very large ligands, also the solvent channels can be too narrow for effective diffusion In this case, co-crystallisation is a good alternative As the name of the method suggests, and in contrast to soaking, during a co-crystallisation experiment the ligand is already present in the crystallisation drop from the very start of the crystallisation experiment The concentration of the ligand is usually chosen in a molar ratio of 2:1 to the protein concentration However, for weakly binding ligands, such as those of many lectins, it is recommended to increase this ratio (in some cases to up to 20:1 or higher) Protein-ligand complexes often crystallise in conditions that differ greatly from those of the protein apo form, such that the co-crystallisation of a protein with a ligand must be treated like the crystallisation of a completely different protein This is because of the different molecular surfaces of protein-ligand complexes compared to the protein alone, resulting in different crystal contacts Consequently, co-crystallisation necessitates the screening of initial crystallisation conditions from scratch For this tutorial, we recommend setting up a ConA-mannose screen, according to the Experimental Protocol in Section 3.3.1., using a molar ratio of ConA to mannose of 1:6 The rationale for this choice is the simulation of the trimannoside core with three mannose residues, increased by a factor of two to enhance binding The results of the screen are shown in Fig Co-crystallisation of a lectin with a sugar ligand can both be easier or more difficult compared to the crystallisation of the apo protein: While the binding of the sugar ligand can stabilize disordered loops, facilitating crystallisation, flexible ligands may also contribute to disorder (increasing the entropy), and hence interfere with crystal formation 3.6 Testing crystals for diffraction Once single crystals have been obtained, they need to be tested for X-ray diffraction This is the ultimate test of crystal quality (see Fig 1), as the appearances can sometimes be deceiving For example, the most promising looking crystals from our optimisation screening were those obtained with low molecular weight PEGs as precipitants (see Fig 7), although they were not the best diffracting Protocols for crystal mounting, X-ray data collection, processing and structure determination can be found in Doublie´.85 Alternatively, it may be a good idea to undertake the crystal structure analysis together with specialists Local experts can be found through the European Macromolecular Crystallographers List or through the World Directory of Crystallographers (Table 1, links 14 and 15) Laboratories with special interest in lectins, like us, will be especially happy to help you to proceed Should you decide to move forward on your own and mount the crystals, you may like to consider using ‘FedEx-crystallography’ services and phasing portals offered by some synchrotrons (such as sending your crystals to the synchrotron by courier and receiving the X-ray data - or sometimes even the solved structures - in return86–89; see also Table 1, link 16) Carbohydr Chem., 2013, 39, 222–246 | 241 Published on 17 June 2013 on http://pubs.rsc.org | doi:10.1039/9781849737173-00222 View Online 3.7 No success? In spite of all efforts, not all proteins can be crystallised in their native state; some of them require some sort of modification The main suspect in preventing crystallisation is usually the presence of flexible elements, like flexible parts of the protein (loops or unstructured parts like the N- or Ctermini) or N- or O-glycans, as mentioned in Section 3.1 While deglycosylation or limited proteolysis prior to the crystallisation experiments can remove flexible regions, they sometimes result in sample heterogeneity (thus worsening the situation) or they are simply not sufficient by themselves In such a case, one may consider recombinant DNA technologies Apart from the obvious advantage of reproducible production of large amounts of protein, DNA constructs offer the unique opportunity to engineer the sequence of the protein at will, e.g to delete flexible regions in a defined manner or to introduce mutations in the protein sequence in order to increase or decrease protein solubility, improve crystallisation or to add different fusion tags to be exploited in affinity chromatography (like the popular hexahistidine or ‘His’-tag).14,90,91 Alternatively, glycosylation can be simplified or completely removed by expression in other organisms like Escherichia coli92,93, yeast94, insect and mammalian cells (e.g CHO-Lec cells)95–97, heterologous plants98 or even in a cell-free system99 (for a comparison of different methods see Oliveira et al.100) A further advantage of expressing the protein under controlled conditions is the possibility to substitute methionine residues with seleno-methionines, which can be exploited when phasing the protein structure, as mentioned in the Introduction.101,102 Leads for the design of suitable DNA constructs can be obtained from the inspection of crystallisation or structure databases Possible strategies involve making truncated mutants (especially relevant for multi-domain proteins), mutating residues that confer a high degree of entropy to the protein surface (Surface Entropy Reduction strategy), or adding solubility tags.103 Some exploratory tools for designing mutants with enhanced crystallisability are available online, e.g the SERp server (Table 1, link 17) A promising (and less labour-intensive) alternative to ‘surface-entropy-reduction mutagenesis’ is chemical engineering, such as the reductive methylation of surface lysines.104 With this short introduction to the world of X-ray crystallography and, in particular, protein crystallisation, we hope to have inspired you to crystallise a lectin of your choice and provided you with useful tips for crystal optimisation and trouble-shooting Acknowledgements We would like to thank the two project students in our group, Tina Bryntesen and Alexander Thiemicke, for testing the ConA crystallisation protocol presented here This work was supported by grants from the Norwegian Research Council [grants no 171631/V40 and 183613/S10 (FUGE-GlycoNor)] as well as by the University of 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Lindhorst,