Cristalización de proteínas en el diseño de fármacos en los últimos 50 años ; protein crystallization for drug design in the last 50 years

ARBOR Ciencia, Pensamiento y Cultura
Enrico A. Stura
CEA, iBiTec-S, SIMOPRO, France Citation/Cómo citar este artículo: Stura, E. A. (2015).
Copyright: 2015 CSIC. This is an open-access article distributed
"Protein Crystallization for Drug Design in the Last 50 Years". under the terms of the Creative Commons Attribution-Non Commercial (by-nc) Spain 3.0 License.
Received: September 12, 2014. Accepted: February 13, 2015.
ABSTRACT: We live in an era where we expect to be able to
RESUMEN: Vivimos en una época en la que esperamos ir al
visit our doctor and obtain a pill to cure any ailment from médico y obtener una pastilla para curar cualquier dolencia que which we suffer. Yet, this is still not the case. Many of the padezcamos; por desgracia, esta expectativa no es real. Aunque current cures are still derived from natural sources although muchos de los remedios en uso provienen de fuentes naturales, new drugs are increasingly the result of intelligent design. In la mayoría de los nuevos medicamentos son el resultado de la this process, X-ray protein crystallography now plays a major investigación científica. En el proceso de diseño y descubrimiento and effective role in the discovery of new treatments. The de fármacos, la cristalografía de proteínas juega un papel central. developments that have made this possible have evolved Los conocimientos que han hecho esto posible han venido during the past fifty years. The methods for crystallizing evolucionando desde hace cincuenta años aproximadamente. macromolecules and determining their structures by X-ray Los métodos de cristalización de macromoléculas y la crystallography have been automated and the speed for X- determinación de sus estructuras a través de la cristalografía de ray data acquisition is several orders of magnitude faster. Fif- rayos X han sido automatizados y miniaturizados y la velocidad ty years ago it took several years to solve a single structure. de la adquisición de datos de difracción ha aumentado en varios Now, several protein–ligand complexes can be determined órdenes de magnitud. Si hace cincuenta años la resolución de una in single day. High-throughput crystallography is considered sola estructura podría llevar varios años, actualmente se pueden to be a great asset to the drug discovery process, providing a determinar las estructuras de varios complejos proteína-ligando fast way to tailor drug candidates to their targets by analys- en un solo día. La cristalografía de alto rendimiento hoy día es ing their binding mode in detail. Crystallization remains the un gran recurso en el proceso del descubrimiento de fármacos main challenge.
pues proporciona una manera rápida y precisa de adaptar los fármacos candidatos a las dianas mediante el análisis de su modo de unión. La cristalización sigue siendo el principal desafío.
KEYWORDS: Drug design, crystallization.
PALABRAS CLAVE: diseño de fármacos, cristalización.

when Dorothy Hodgkin was offered a small sample Protein crystallization pre-dates X-ray crystallogra- of crystalline insulin by Robert Robinson (Howard, 2003). Unfortunately, X-ray crystallography at that phy. Humans have practised this science since 1840 (Giegé, 2013), but other organisms have put protein time could not cope with the complexity of the in- sulin molecule. She was able to grow better crys- crystallization to use much earlier. Bacillus thuring- iensis, known for its insecticidal properties, produc- tals by dialysing concentrated insulin against tap water (containing traces of zinc), but failed to do so es protein crystals during sporulation. Fifty years ago the seminal ideas that would lead to modern protein with distilled water. The 3-dimensional structure of crystallization methods were already in place and as insulin was eventually determined by X-ray crystal- lography in her laboratory in 1969 (Crowfood and or Drug Design in the Las it became understood that it was possible to visu- alize ligands in their binding sites, crystallographic Riley, 1939). Zinc occupies the central position in a pharmaceutical investigations started.
unit of six insulin molecules (hexamer). The insulin hexamer is not a crystallization artefact, but this is Protein crystals are themselves drugs (Figure 1). the form produced and stored in the body. It is in- Insulin is important in the treatment of diabetes, active but has long-term stability. The monomer is and to slow down the release of this hormone it is the active form. The hexamer serves to keep the delivered in crystalline form. NPH insulin, a suspen- highly reactive monomeric insulin protected, yet sion of crystalline zinc-insulin combined with a pos- available through hexamer-monomer conversion. itively charged polypeptide, was created in 1936 Insulin can aggregate and form fibrillar interdigitat- by Nordisk, but the role of zinc, as an additive, to ed β-sheets. This can cause injection amyloidosis, induce protein crystallization was not known until and prevents the storage of insulin for long periods much later. The insulin crystal story began in 1934 (Ivanova et al., 2009).
Figure 1. Crystals are drugs. Slow release insulin is designed with the addition of specific bulky hydrophobic
groups. These insulin crystals are more stable. In addition to zinc stabilization (A), the covalently linked litho-
cholyl group form specific van der Waals and hydrogen-bonding interactions with neighbouring molecules (B)
to strengthen the crystalline network and slow down dissolution of crystals. Lithocholic acid acylated insulin has affinity for circulating serum albumin to ensure slow absorption into the blood stream and prolongation of its half-life. The engineered insulin retains its affinity for its insulin receptor. (From PDB entry: 1UZ9; Whittingham et al., 2004).
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Amyloid fibril formation and deposition can lead salting-out). Transthyretin is a tetrameric protein in to diseases, including spongiform encephalopathies, dynamic equilibrium with a monomeric form that, Alzheimer's and familial amyloidotic polyneuropa- like insulin, has a tendency to aggregate. To prevent thies. In familial amyloidotic polyneuropathy (FAP), FAP, various potential inhibitors are being studied the amyloid fibrils are mostly constituted by variants to understand the structure–activity relationship of transthyretin (TTR) (Quintas, Saraiva and Brito, (SAR) (Nencetti and Orlandini, 2012). The relation- 1997). Protein instability, leading to aggregation can ship between the chemical or 3D structure of a mol- be a problem in protein crystallization for X-ray crys- ecule and its biological activity. The objective is to tallography, but not always. Transthyretin, previously understand which chemical groups are responsible known as prealbumin, is easily crystallized. The first for its effect on the target protein. To achieve such crystals were obtained fifty years ago by Purdy et an understanding, 222 transthyretin structures, from al. (1965) and by Haupt and Heide (1966) from 55% human and other species, complexed and uncom- saturated ammonium sulphate. The crystallization plexed have been deposited in the Protein Data Bank of insulin and transthyretin characterise the varia- (PDB) (Berman et al., 2007). The inhibitors stabilize tion in solubility of proteins, with respect to the salt the tetrameric form shifting the equilibrium away concentration in which they are bathed (salting-in/ from the amyloidogenic monomer.
Figure 2. Crystals and quaternary structure of transthyretin (TTR) a protein that transports thyroid hormones. The
morphology of transthyretin crystals is highly variable although the arrangement of the molecules in the lattice remains constant (A-C). The crystals shape depends on the rate of growth of the crystals and not on the contacts
that the molecules make with one another. Four TTR molecules form a tetramer (D) and the tetramer packs
together with other tetramers always in the same manner. To reliably grow crystals streak seeding can be used. The crystals grow in a straight line (E) where the seeds have been deposited by the cat whisker. TTR is studied to
develop amyloid inhibitors. Such inhibitor binds in the tetramer cavity in two independent binding sites (F). (From
PDB entry: 4PM1; Ciccone et al., 2015).
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Protein solubility in water is highly variable and proline isomerase FKBP. The successful structure de- the salting-in/salting-out concept applies only to termination of the FKBP/FK506/calcineurin complex soluble proteins. Crambin, a polypeptide from the encourages others to follow the pathway. Amgen, seeds of Crambe abyssinica, is not soluble in water. another biotechnology start-up focuses on erythro- It was dissolved in ethanol and crystallized in 1965 poietin (EPO), a glycoprotein hormone that controls by adding water. It is resistant to denaturing agents red blood cell production. In 1998 they solve the like 3M urea, and only partially affected by guani- structure of the EPO complex with the extracellular dinium: It showed some solubility in 8 M guanidine ligand-binding domain of its receptor (EPO binding hydrochloride (Teeter and Hendrickson, 1979). Urea protein EBP) (Syed et al., 1998), but they were pre- and guanidine are now used to refold proteins ex- ceded by a team at The Scripps Research Institute or Drug Design in the Las pressed in Escherichia coli and found in inclusion (TSRI). The TSRI team succeeds, two years earlier, bodies (Palmer and Wingfield, 2004), using similar to determine the structure of the complex between concentrations of these agents. High-level expres- EBP and an EPO mimetic peptide (EMP) discovered sion of many recombinant proteins in Escherichia by Affymax (Livnah et al., 1996). The synthetic pep- coli has revolutionized protein crystallization. Highly tide, attached to polyethylene glycol, is approved the aggregated protein (inclusion bodies) are formed in FDA in 2012, and enters the market in 2013, only to the bacteria cytoplasm when high-level expression be recalled almost immediately, after 3 deaths, as is induced. It is recovered from cell lysates by low 0.02% of patients suffer from severe hypersensitivity speed centrifugation and the protein extracted from reactions. Successes dominate over failures. Struc- the washed pellets with guanidine·HCl in a soluble tural studies have resulted in anti-HIV drugs that but unfolded form that needs to be re-folded into target the viral reverse transcriptase, integrase and its native and biologically active form. Currently the protease; and anti-cancer drugs aimed at various ty- PDB contains 105,465 structures, of these 67,321 rosine kinases.
(64%) were expressed in Escherichia coli. In the 1970s, insulin and recombinant DNA technology al- lows the birth of two biotech companies, Genentech and Biogen. The challenge is to insert the DNA se- The immunosuppressant drug, rapamycin, pro- quence for human insulin into bacteria and let the duced by the bacterium Streptomyces hygroscopicus, bacteria produce the hormone.
and FK-506, produced by S. tsukubaensis, are used to prevent organ rejection in transplantation. Their The use of polyethylene glycol (PEG) to obtain crys- action prevents activation of T cells and B cells by tals for X-ray data analysis starts in 1975 with deoxy- inhibiting the production of interleukin-2 (IL-2). FK- hemoglobin crystals diffracting to 3.5 Å. Previously 506 gives the name to a family of FK-binding proteins it had mainly been used for fractional precipitation (FKBP). FK-506 binds to FKBP12 thus reducing the (Ward et al., 1975). The steric exclusion mechanism peptidyl-prolyl isomerase activity of this immunophi- of this precipitant was correctly identified as being lin. Most crystals of FKBPs have been obtained only in similar to that of dextran reported by Torvard Lau- complex with the immunosuppressant drugs.
rent (Laurent, 1963). Size exclusion chromatography, using gels, typically made of dextran (Sephadex) and In "The Billion Dollar Molecule: One Company´s of other polymers, has become important as a pro- quest for the perfect drug", Barry Werth describes tein purification method prior to crystallization. PEG the first few years of Vertex in its quest to create is the single most successful precipitant, with cur- drugs by rational drug design. Werth renders both rently 38,976 macromolecular structures deposited the science and the intricacies of the business deals at Vertex. The narrative offers an insight at a critical moment in the history of science when under the im- While Genentech focuses mainly on proteins and pulse of Joshua Boger, a researcher that leaves Merck antibodies as their products, Vertex Pharmaceuticals to found a new company, the focus of scientific re- was founded in 1989 to pioneer an explicit strategy search also shifts from screening soil samples and of rational drug design rather than combinatorial insect secretions to a new world where proteins and chemistry. The aim is to understand the molecular their inhibitor complexes can be crystallized and from mechanisms of action of natural immunosuppres- their structure using computing power new drugs sants, FK506 and rapamycin, that act on peptidyl can be designed. The founding of Vertex's is vision- ARBOR Vol. 191-772, marzo-abril 2015, a222. ISSN-L: 0210-1963
ary, but the idea that the new drugs would be with- helps crystallization because all the molecules can out side effects because of the precision of the de- shift in synchrony from one form to another, avoid- sign, is still to be realised. The book describes Boger's ing heterogeneity that would make crystallization first target molecule, FKBP, important in preventing more difficult. In absence of a cooperative process, the host's body from rejecting transplanted organs. heterogeneity is inevitable and the binding affinity of The contrast between real science and the necessity the ligand for the protein becomes important as it of fund raising sees Boger going out to raise money, will determine how many of the protein molecules Vertex's researchers hunkered down in the laboratory are complexed and how many are not. Higher affinity benches to isolate and analyse FKBP, in a race against ligands ensure that a higher portion of the protein a tough team composed of Prof. Stuart L. Schreiber molecules will be complexes. For low affinity ligands at Harvard and Manuel Navia, the crystallographer. the ligand is added in excess, up to ten times more Manuel's parents went to the U.S. from Cuba, worked compared to the protein. This strategy is not effec- really hard and a strong value system that he tried to tive to crystallize complexes of two or more proteins. emulate. The advantage of academic science, where In my laboratory we have investigated the use of bi- collaborations are possible, contrasts with the world functional inhibitors to bring together two proteases of business where scientists need to hide proprietary to change the manner in which crystallization occurs results instead of presenting them at conferences or (Antoni et al., 2013).
publishing them open to the criticism of reviewers. The book is warmly recommended as a riveting tale Ideas that the use of PEG instead of salts like am- of human endeavour that shows how scientific antag- monium sulphate would help maintaining complexes onism can drive discovery. In this historical review we have been abandoned as it has been realised that at are more concerned on how crystallization methods high salt concentrations, AS is as effective as PEG.
have evolved in the past 50 years than in discussing academic in contrast to industrial research. In crystallization, the inhibitors, substrates, modu- Administration of folic acid (vitamin B9) worsens lators and other ligands, be they other proteins or leukemia. This led to the development of folic acid small-molecules are the most important considera- analogues, including methotrexate (MTX), to inhibit tion in crystallization (Dale, Oefner and D'Arcy, 2003). folic acid metabolism. MTX is used in the treat- The various complexes have a different likelihood for ment of cancer and autoimmune diseases. It acts crystallization and different complexes will be able by binding to dihydrofolate reductase (DHFR). MTX to select a different polymorph (Vera et al., 2013) was originally synthesised by an Indian biochemist depending on how the ligand is able to change the and in 1947. Sidney Farber and co-workers showed properties of its target. One of the most fundamental that the molecule could induce remission in chil- changes that ligands can cause is the bringing togeth- dren with acute lymphoblastic leukemia. The first er of more that one molecule, in a manner that does crystal structures of DHFR with MTX were deter- not occur spontaneously. Even single atoms, like zinc, mined for bacterial enzymes in 1982. In 1989, the in the case of insulin, can bring together six or more crystal structure of the chicken enzyme was solved protein molecules. This has been recognized to be and one year later the first complex of human DHFR critical in the crystallization of insulin. A single atom, with the folate and 5-deazafolate were solved. The oxygen makes the difference between oxy- and de- first crystal structure of human DHFR with MTX was oxy- haemoglobin. When oxygen binds to the iron obtained six years later, in 1995. The enzyme was a complexed at the centre of the plane of the porphy- MTX-resistant mutant with a single point mutation rin ring it causes the iron atom to move back. This in the drug binding pocket. The long delay between triggers as series of cooperative changes that result the demonstration of the effectiveness of the ligand in a large scale movement of the whole assembly of and the first crystal structure of the human enzyme the four molecules that constitute the heamoglobin complex gives an idea of the difficulty of obtain- tetramer. The change in a single haemoglobin mol- ing suitable crystals to carry out drug design. There ecule is transmitted to the other three monomers was great interest in DHFR and MTX on behalf of in the tetramer, so that these too adopt a similar pharmaceutical companies. The human DHFR-MTX conformation in their hemes to facilitate the bind- structural studies saw the involvement of research- ing of oxygen to these sites. A cooperative process ers from Gensia Pharmaceuticals. Agouron Pharma- ARBOR Vol. 191-772, marzo-abril 2015, a222. ISSN-L: 0210-1963
ceuticals was founded in 1978 with the aim to find DRUGS FROM STRUCTURAL STUDIES
selective inhibititors for DHFR and thymidate syn- AIDS was first clinically observed in 1981 in the thetase (TS). Hoffmann-La Roche had interests in an- United States. In 1983, when the causative agent tibacterial agents that target bacterial dihydrofolate was identified the crystallization field was mature reductases. The difficulties associated with protein to face the challenge to design molecules for anti- crystallization became evident. Dr. Villafranca from retroviral therapy.
Agouron was a co-organizer of the fifth Interna- tional Conference on the Crystallization of Biological The inhibition of the HIV viral protease is regard- ed as major success of structure-based drug design. Macromolecules (ICCBM-5) in San Diego, in 1993. Dr. D'Arcy from Hoffmann-La Roche was there too. The protease inhibitors are highly effective against or Drug Design in the Las The National Aeronautics and Space Administration the virus and since the 1990s have been a key com- (NASA), interested in crystallization in microgravity, ponent of anti-retroviral therapies for AIDS. Nelfi- was a sponsor of the meeting.
navir (Viracept) (Figure 3), a protease inhibitor, was developed by Agouron Pharmaceuticals as part of Growing crystals in space was impractical, and re- a joint venture with Eli Lilly and Company. Saquina- searchers believed that convection free systems could vir was developed by Roche, Ritonavir (Norvir) by be developed to obtain the same effect on earth. A AbbVie, Inc. Other companies focused on inhibiting system where silica-gel methods can be used in both HIV's reverse transcriptase. AZT, a nucleoside-based vapour diffusion sitting drop and liquid-liquid diffu- thymidine-analogue inhibitor, works by selectively sion was proposed as an alternative to micrograv- inhibiting transcription. Resistance against nucleo- ity during the ICCBM-5 conference by Bob Cudney tides developed, so non-nucleoside reverse-tran- (Cudney, Patel and McPherson, 1994). Bob Cudney is scriptase inhibitors were developed. To improve the the current president of Hampton Research, a crys- crystals of a clinically relevant double mutant HIV-1 tallization supply company. Until the introduction of reverse transcriptase in complex with ATP and of gels, three classical crystallization methods had the non-nucleoside inhibitor HBY-097 (Das et al., dominated the field. These were batch, dialysis and 2007), streak seeding was used (Stura and Wilson, vapour diffusion. The major advances consisted in 1990). One of the problems with protein crystalli- the miniaturization of each of these methods. In the zation is that as the crystal growth conditions are batch method, the protein is mixed with the precipi- optimized, nucleation can fail. Seeding is used to tant, nucleation takes place on mixing and the crys- stimulate nucleation. Various seeding techniques tals are left to grow. By changing the temperature in can be used (Stura and Wilson, 1991). In microseed- a controlled manner, the degree of supersaturation ing crystals are added to the protein solution before can be changed. The miniaturization of this method, full equilibration of the protein precipitant drop. for crystallization under oil suitable for crystallization This method can fail because the seeds dissolve or with the IMPAX robot made by Douglas Instruments, too many seeds are added. Macroseeding where revived the method that was no longer used (Chay- large seeds are added after equilibration, solves en, Shaw Steward and Baldock, 1994). A generaliza- both problems encountered with microseeding, but tion of crystallization with oils was later proposed by it is time consuming and manipulation errors can Naomi Chayen during ICCBM-7 in Granada, Spain, also lead to failure.
in 1998 (Chayen, 1999). Dialysis was miniaturized Streak seeding where crystals are taken from an more than forty years ago with microdots of 5-20 µL existing crystal with a cat whisker and dispersed in capacity made in plexiglas. New set-ups have been a non-equilibrated or equilibrated experiment in proposed using agarose gels to carry out dialysis ex- straight line is fast. Since it is easily repeated if it fails, periments in capillaries (Thiessen, 1994). Dialysis and it is a practical method to find the best conditions to batch account for only 60 and 766 structures in the grow crystals. It can also be used to grow crystals in PDB, respectively, while vapour diffusion counts for a space group different from that of the seed crys- 66,887 entries. José Antonio Gavira has promoted tals. This application is called: epitaxial jumps (Stura, the use of free-interface diffusion (Otálora et al., Charbonnier and Taussig, 1999). The field has still 2009). Triana Science & Technology, Granada, Spain many challenges. Over 50% of all modern medicinal markets a kit to use the technique. The method ac- drug targets are membrane proteins, proteins that counts for only 12 PDB database entries.
interact with biological membranes (Figure 4).
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Figure 3. From crystals to drugs. The path towards a drug starts with the conception of an inhibitor (A) which
ignores the actual three dimensional structure of the compound once in the active site of the enzyme (B). Before arriving at a formulation for the patient (C) it passes through a crystal structure. The results are often shown in a simplified representation that shows only the secondary structure of the protein (D) and not all the atoms (E). HIV protease inhibitors, including Viracept, are one of the great successes of structure-based drug design. (From the PDB entry: 1OHR; Kaldor et al., 1997).
To crystallize these proteins, they must be extract- tallization experiments using the vapour diffusion ed from the membrane using a detergent, and then technique as for soluble proteins. The method has in- the crystallization can proceed in a manner analo- creased the number of membrane proteins that have gous to that used for soluble proteins. The problems been crystallized.
arise when the protein is unhappy in the detergent. The in meso method for crystallizing membrane pro- Even if progress has been spectacular in the past teins is growing in popularity (Li et al., 2014). fifty years, the rapid progress is likely to continue in the next fifty years. A crystal ball would be useful to The method involves both lipids and detergents. predict the future of this science.
The lipids are manipulated to generate a continuous hard gel called lipid cubic phase (LCP). The protein solubilized in detergent is added to the LCP so that it can migrate into the lipid phase. The protein incor- I am grateful to Natalia Stura for help with the Span- porated into the LCP is dispensed and used in crys- ish abstract.
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Figure 4. Membrane proteins. Membrane proteins are important targets for drug design. G-protein-coupled
receptors (GPCR) belong to the bateriorhodopsin (BR) family. They are of great interest to the pharmaceuti- cal industry because they act as sensors to activate signal transduction pathways and cellular responses. Seven transmembrane helices characterize GPCR. BR was the first membrane protein to be crystallized. Initially as two dimensional crystals, but now after crystallization using lipidic cubic phases a three dimensional structure at atomic resolution has been determined. The transmembrane helices are surrounded by lipids (green) (A). In the
centre there is a retinol (vitamin A) molecule (cyan). In mammals retinol is transported by retinol binding protein which in plasma is found complexed with transthyretin (C). (From PDB entries: 1C3W and 1QAB; Luecke et al.,
1999; Naylor and Newcomer, 1999).
ation for Drug Design in the Las ARBOR Vol. 191-772, marzo-abril 2015, a222. ISSN-L: 0210-1963
Antoni, C., Vera, L., Devel, L., Catalani,
Giegé, R. (2013). A historical perspective on son, I. A. (1996). Functional mimicry of M. P., Czarny, B., Cassar-Lajeunesse, protein crystallization from 1840 to the a protein hormone by a peptide agonist: E., Nuti, E., Rossello, A., Dive, V. and present day. FEBS Journal, 280, pp. 6456– the EPO receptor complex at 2.8 Å. Sci- Stura, E. A. (2013). Crystallization of ence, 273, pp. 464–71. bi-functional ligand protein complexes. Journal of Structural Biology, 182, pp. Haupt, H. and Heide, K. (1966). Crystalliza- tion of prealbumin from human serum. Naylor, H. M. and Newcomer, M. E. (1999). Experientia, 22, pp. 449–451. The structure of human retinol-binding protein (RBP) with its carrier protein Berman, H., Henrick, K., Nakamura, H. and transthyretin reveals an interaction with Markley, J. L. (2007). The worldwide Howard, J. A. K. (2003). Dorothy Hodgkin the carboxy terminus of RBP. Biochem- Protein Data Bank (wwPDB): ensuring and her contributions to biochemis- istry, 38, pp. 2647-2653. a single, uniform archive of PDB data. try. Nature Reviews Molecular Cell Bi- Nucleic Acids Research, 35, pp. D301– ology, 4, pp. 891-896. Nencetti, S. and Orlandini, E. (2012). TTR fibril formation inhibitors: is there a Ivanova, M. I., Sievers, S., Sawaya, M. R., Wall, SAR? Current Medicinal Chemistry, Chayen, N. E., Shaw Stewart, P. D. and J. S. and Eisenberg, D. (2009). Molecular 19, pp. 2356–2379. Baldock, P. (1994). New develop- basis for insulin fibril assembly. Proceed- ments of the IMPAX small-volume ings of National Academy of Sciences of automated crystallization system. the USA, 106, pp. 18990–18995. Otálora, F., Gavira, J. A., Ng, J. D., García-Ruiz, Acta Crystallographica, D50, pp. J. M. (2009). Counterdiffusion meth- ods applied to protein crystallization. Kaldor, S. W., Kalish, V. J., Davies, J. F. 2nd., Progress in Biophysics and Molecular Shetty, B. V., Fritz, J. E., Appelt, K., Bur- Biology, 101, pp. 26–37. Chayen, N. E. (1999). Crystallization with gess, J. A.,, Campanale, K. M., Chir- oils: a new dimension in macromolecu- gadze, N. Y., Clawson, D. K., Dressman, lar crystal growth. Journal of Crystal B. A., Hatch, S. D., Khalil, D. A., Kosa, M. Palmer, I. and Wingfield, P. T. (2004). Prepa- Growth, 196, pp. B., Lubbehusen, P. P., Muesing, M. A., ration and extraction of insoluble (in- Patick, A. K., Reich, S. H., Su, K. S. and clusion-body) proteins from Escherichia Tatlock, J. H. (1997). Viracept (nelfina- coli. Current Protocols in Protein Sci- Ciccone, L., Tepshi, L., Nencetti, S. and Stu- vir mesylate, AG1343): a potent, orally ence, Chapter 6, Unit 6.3. ra, E. A. (2015). Transthyretin complexes bioavailable inhibitor of HIV-1 protease. with curcumin and bromo-estradiol: Journal of Medicinal Chemistry, 21, pp. evaluation of solubilizing multicompo- Purdy, R. H., Woeber, K. H., Holloway, M. nent mixtures. New Biotechnology, 32, T. and Ingbar, S. H. (1965). Preparation of Crystalline Thyroxine-binding Preal- Laurent, T. C. (1963). The interaction be- bumin from Human Plasma. Biochem- tween polysaccharides and other istry, 4, pp. 1888–1895. Crowfood, D. and Riley, D. (1939). X-Ray macromolecules. 5. The Solubility of Measurements on Wet Insulin Crystals. Proteins in the presence of dextran. Nature, 144, pp. 1011–1012. Biochemical Journal, 89, pp. 253–257.
Quintas, A., Saraiva, M. J. and Brito, R. M. (1997). The amyloidogenic po- Luecke, H., Schobert, B., Richter, H. T., tential of transthyretin variants cor- Cudney, R., Patel, S., and McPherson, A. Cartailler, J. P. and Lanyi, J. K. (1999). relates with their tendency to aggre- (1994). Crystallization of macromole- Structure of bacteriorhodopsin at 1.55 gate in solution. FEBS Letters, 418, pp. cules in silica gels. Acta Crystallograph- Å resolution. Journal of Molecular Biol- ica, D50, pp. 479–483. ogy, 291, pp. 899-911. Stura, E. A. and Wilson, I. A. (1990). Analyti- Dale, G. E., Oefner, C. and D'Arcy, A. (2003). Li, D., Howe, N., Dukkipati, A., Shah, S. cal and production seeding techniques. The protein as a variable in protein T. A., Bax, B. D., Edge, C., Bridges, A., Methods, 1, pp. 38–49. crystallization. Journal of Structural Bi- Hardwicke, P., Singh, O. M. P., Giblin, ology, 142, pp. 88–97. G., Pautsch, A., Pfau, R., Schnapp, G., Wang, M., Olieric, V. and Caffrey, M. Stura, E. A. and Wilson, I. A. (1991). Applica- (2014). Crystallizing Membrane Pro- tions of the streak seeding technique in Das, K., Sarafianos, S. G., Clark, A. D., Boyer, teins in the Lipidic Mesophase. Experi- protein crystallization. Journal of Crystal P. L. Hughes, S. H. and Arnold, E. (2007). ence with Human Prostaglandin E2 Syn- Growth, 110, pp. Crystal structures of clinically relevant thase 1 and an Evolving Strategy. Crystal Lys103Asn/Tyr181Cys double mutant Growth & Design, 14, pp. 2034–2047. HIV-1 reverse transcriptase in com- Stura, E. A., Charbonnier, J. and Taus- plexes with ATP and non-nucleoside sig, M. J. (1999). Epitaxial jumps. inhibitor HBY 097. Journal of Molecular Livnah, O., Stura, E. A., Johnson, D. L., Mid- Journal of Crystal Growth, 196, pp. Biology, 365, pp. 77–89. dleton, S. A., Mulcahy, L. S., Wrighton, N. C., Dowr, W. J., Jolliffe, L. K. and Wil- ARBOR Vol. 191-772, marzo-abril 2015, a222. ISSN-L: 0210-1963
Syed, R. S., Reid, S. W., Li, C., Cheetham, Thiessen, K. J. (1994). The use of two novel deoxyhemoglobin a crystals grown J. C., Aoki, K. H., Liu, B., Zhan, H., Oss- methods to grow protein crystals by from polyethylene glycol solutions. lund, T. D., Chirino, A. J., Zhang, J., Finer- microdialysis and vapor diffusion in Journal of Molecular Biology, 98, pp. Moore, J., Elliot, S., Sitney, K., Katz, B. A., an agarose gel. Acta Crystallograph- Matthews, D. J., Wendoloski, J. J., Egrie, ica, D50, pp. 491–495. J. and Stroud, R. M. (1998). Efficiency of Whittingham, J. L., Jonassen, I., Havelund, signalling through cytokine receptors depends critically on receptor orienta- Vera, L., Antoni, C., Devel, L., Czarny, B., S., Roberts S. M., Dodson E. J., Verma Cassar-Lajeunesse, E., Rossello, A., Dive, C. S., Wilkinson A. J. and Dodson G. tion. Nature, 395, pp. 511–516. V. and Stura, E. (2013). Screening Us- G. (2004). Crystallographic and solu- ing Polymorphs for the Crystallization tion studies of N-lithocholyl insulin: a Teeter, M. M. and Hendrickson, W. A. (1979). of Protein–Ligand Complexes. Crystal new generation of prolonged-acting or Drug Design in the Las Highly ordered crystals of the plant seed Growth & Design, 13, pp. 1878-1888. human insulins. Biochemistry, 25, pp. protein crambin. Journal of Molecular Biology, 127, pp. Ward, K. B., Wishner, B. C., Lattman, E. E. and Love, W. E. (1975). Structure of ARBOR Vol. 191-772, marzo-abril 2015, a222. ISSN-L: 0210-1963


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Egg Thaw Cycle Orientation Visit us online at  Copyright 2008 – 2013 NYU Fertility Center – rev. 06/05/2013 Meet Our Physicians Dr. Frederick Licciardi Dr. James Grifo Dr. Nicole Noyes Dr. Alan Berkeley Dr. Lisa Kump-Checchio Dr. M. Elizabeth Fino Dr. David Keefe


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