Re: [ccp4bb] Assays for protein-ligand interaction?
Ho Jun Lee, Have you thought about differential scanning fluorimetry (Thermofluor)? With this biophysical technique you can characterize protein-ligand interactions and screen a wide variety of ligands using minimal concentration of ligand and protein. All you need is a quantitative PCR machine (qPCR). Here is a reference for you https://chemistry.osu.edu/~magliery/pdfs/LavinderMagliery2009JACS.pdf Lorenzo Ihsan Finci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityCollege of Life SciencesBeijing, China Date: Mon, 13 Jan 2014 21:50:07 +0900 From: hojunle...@gmail.com Subject: [ccp4bb] Assays for protein-ligand interaction? To: CCP4BB@JISCMAIL.AC.UK Sorry for the off topic. I'm looking for a way to monitor protein-(potential) ligand interaction. The ligand is small molecule (mw~250) and we're looking for its potential interaction with couple human proteins. (We do not know this small molecule interacts with these human protein or not.) Is there any efficient way to quickly identify whether this ligand interacts with those human protein? We can buy some protein, but the amount of commercially available purified proteins is very little, making them hard to be analyzed by some good methods (e.g. ITC). That would be really great if anyone suggest any idea. Sorry for the off topic question again. Thanks!
Re: [ccp4bb] How to slow down crystallization? Need hep!
Feng Lei, I would personally recommend slowing down the process of nucleation either by lowering the temperature of crystallization or by utilizing Al's oil. These are two powerful ways to slow the overall kinetics of crystallization. Here is a relevant reference for you: http://scripts.iucr.org/cgi-bin/paper?he0181 Sincerely, lorenzo Lorenzo Ihsan Finci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityCollege of Life SciencesBeijing, China Date: Mon, 25 Feb 2013 11:02:03 -0500 From: spartanfeng...@hotmail.com Subject: [ccp4bb] How to slow down crystallization? Need hep! To: CCP4BB@JISCMAIL.AC.UK Hello everyone, I need your suggestion for slowing down crystallization for my protein my protein got hit in PEG/ION #5 ( 0.2 M MgCl2, 20% PEG 3350, pH 5.9), but it crystallize too fast. In 1 hr I can see tons of tiny needles. Can anyone give me some suggestion on how to slow down the process? I used lower conc. of potein, lower conc. of PEG ( 10%), it helped a little bit, giving me small rod crystal. but no improvement after that. Thank you very much for your suggestions
[ccp4bb] Postdoctoral Position in Structural Immunology at Harvard Medical School
Dear All, On behalf of Dr. Jia-huai Wang, I post this message. For inquiries please contact Dr. Wang directly at jw...@red.dfci.harvard.edu Postdoctoral Position in Structural Biology of Immune Receptors: There is an immediate opening for a postdoctoral position in protein crystallography at the Dana-Farber Cancer Institute, Harvard Medical School. The research project is focused on the structural and functional investigation of leukocyte integrins, which are cell surface receptors that play a key role in immunity. The successful candidate should have a Ph.D. in structural biology. In addition to a knowledge of X-ray crystallography, the applicant should also have experience in molecular biology, and protein biochemistry. This includes cloning, protein expression and purification, particularly in eukaryotic systems such as baculavirus. Interested candidates should email a CV and three contacts for reference to Dr. Jia-huai Wang at jw...@red.dfci.harvard.edu. For more information regarding the Wang Laboratory, please see the website: http://wang.dfci.harvard.edu Sincerely, lorenzoLorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China
Re: [ccp4bb] calculation of cavities within crystal protein
Danilo, There are two points that I want you to consider before I attempt to answer your question:1. In terms of capabilities:Voidoo has the capability to detect specific voids or all voids inside a bio-macromolecular complex, as well as certain cavities that are connected to the outside world. It delineates these cavities, meaning that it finds their extent in 3-dimensional space. Secondly, it measures the molecular volumes as well as the volume of the cavity itself. Finally, it has the capability to generate molecular surface plot files which enable the visualization of the cavities. Voidoo will detect voids and invaginations, but it cannot pick pockets, no pun intended :)2. In terms of measuring and displaying cavities, there are three particular modes:a. Vanderwaals cavity, where the cavity comprises the compliment of the Vanderwaals surface of the surrounding atoms.b. Probe-accessible cavity, where the cavity comprises all of the space that can be accessed by the center of the probe sphere.c. Probe-occupied cavity, where the cavity comprises all of the space that can be occupied by the probeNow to answer your question:The first step of the cavity detection algorithm is to map the target molecule onto a 3-dimensional grid with a spacing (0.5 - 1 Angstrom). All of the grid points are initially assigned a value of zero. Every subsequent point of the grid whose distance to the nearest atom is less than the sum of the van der Waals radius of that atom and the probe radius, is assigned a value of 1. In order to exclude the outside world, all of the grid points on the faces of the grid are set to zero, and the outside world is zapped.This method is also known as the flood fill algorithm. At the final stage, all grid points are inside a closed cavity and have a value of zero. Voidoo then checks if the cavity has been detected and will delineate the cavity to measure the volume. Then the process of atomic flattening is invoked, which entails multiplying the van der Waals radii of all atoms by a certain flattening factor. The program then goes through iterative rounds of atomic flattening to close off cavities which are in contact with the outside world, and separate cavities which are in contact with each other through small channels. The program will stop after the particular sought after cavity has been found. A detailed answer to your question can be found in the original resource that I sent in my first response:http://journals.iucr.org/d/issues/1994/02/00/gr0263/gr0263.pdfI hope this helps!lorenzoLorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China Date: Fri, 10 Aug 2012 10:39:29 +0200 From: danilo.belv...@ic.cnr.it To: lfi...@hotmail.com Subject: RE: [ccp4bb] calculation of cavities within crystal protein Thanks you for your help Lorenzo. VOIDOO seems to be the proper software for this calculation, and it is suggested by many people. I am not an expert in this field! So, I would have only one thing to ask, because of a doubt arising from what I read in http://binf.gmu.edu/ttaylor/DELAUNAY_PAPERS/chakravarty1.pdf. In the paper, VOIDOO is a grid-based procedure that measures the cavity volume defined by the van der Waals surface of atoms lining the cavity Does this mean that van der Waals surfaces of a protein and the surrounding proteins are considered in the calculation? I hope not too disturbing you. Danilo On Thu, 9 Aug 2012 10:05:19 -0400, Dr. Lorenzo Finci lfi...@hotmail.com wrote: Danilo, The protein cavity can be analyzed utilizing the program Voidoo ( Kleywegt GJ, 1994). This program uses an atomic-flattening algorithm based on a 3-dimensional grid to locate and delineate different cavities. A Van Der Waals cavity can further be generated with a probe radius with a computed cavity grid using the highest number of grid points, and a contact and accessible surface can further be evaluated... Relevant Resources: http://pelican.rsvs.ulaval.ca/mediawiki/index.php/Analysing_protein_cavities_using_VOIDOO http://binf.gmu.edu/ttaylor/DELAUNAY_PAPERS/chakravarty1.pdf I hope this helps! lorenzo Lorenzo Ihsan FInci, Ph.D. Postdoctoral Scientist, Wang Laboratory Harvard Medical School Dana-Farber Cancer Institute Boston, MA Peking University The College of Life Sciences Beijing, China Date: Thu, 9 Aug 2012 13:53:09 +0200 From: danilo.belv...@ic.cnr.it Subject: [ccp4bb] calculation of cavities within crystal protein To: CCP4BB@JISCMAIL.AC.UK Dear all, I am Dr. Danilo Belviso and I am working on a platinum-based inhibitor for matrix-metallo proteasis. I have obtained the crystal structure of the adduct Pt/protein and, for me, would be very interesting to know the cavities of the protein within
Re: [ccp4bb] calculation of cavities within crystal protein
Danilo, The protein cavity can be analyzed utilizing the program Voidoo ( Kleywegt GJ, 1994). This program uses an atomic-flattening algorithm based on a 3-dimensional grid to locate and delineate different cavities. A Van Der Waals cavity can further be generated with a probe radius with a computed cavity grid using the highest number of grid points, and a contact and accessible surface can further be evaluated... Relevant Resources:http://pelican.rsvs.ulaval.ca/mediawiki/index.php/Analysing_protein_cavities_using_VOIDOOhttp://binf.gmu.edu/ttaylor/DELAUNAY_PAPERS/chakravarty1.pdf I hope this helps!lorenzo Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China Date: Thu, 9 Aug 2012 13:53:09 +0200 From: danilo.belv...@ic.cnr.it Subject: [ccp4bb] calculation of cavities within crystal protein To: CCP4BB@JISCMAIL.AC.UK Dear all, I am Dr. Danilo Belviso and I am working on a platinum-based inhibitor for matrix-metallo proteasis. I have obtained the crystal structure of the adduct Pt/protein and, for me, would be very interesting to know the cavities of the protein within the crystal, namely by considering symmetry-related protein molecules around the asymmetric unit. Do you know a software (or server) that carries out this calculation? Best regards Dr. Danilo Belviso
[ccp4bb] Protein-Protein Interactions
Dear Colleagues, I have a question for all of you bioinformatics oriented structural biologists: How do I predict the sites of protein-protein interactions between two receptors that have been proven to interact biochemically but lack specific details regarding proximity. This is not a straightforward question for me, and I believe it is somewhat complicated. The complicated scenario involves a multitude of different subunits and isoforms. Also, there is not structural data to support all components involved, and thus I presume I should use the sequence based software. I am aware that there are different types of prediction software, either sequence or structure based predictions using different algorithms:http://rosettadesigngroup.com/blog/58/10-protein-protein-interface-prediction-servers/Receptor 1:-Has 5 predicted subunits (Alpha)2-(Beta)2-(Gamma)11. Alpha (6 isoforms)2. Beta (3 Isoforms)3. Gamma (3 Isoforms)Receptor 2:-Is believed to be composed of (Alpha)3-(Beta)21. Alpha (4 isoforms)2. Beta(1 isoform)Any advice or recommendation will be well appreciated! Sincerely, lorenzo Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China
Re: [ccp4bb] off topic Thermal shift assay
Noor, Thank you very much for your inquiry. As we all know, thermodynamic principles of cooperativity and allostery have long been used as a foundation to begin understanding the complex interplay between associated ligand binding events. In principle, the delta Tm shifts that occur when multiple ligands bind to the same protein should further manifest cooperative effects between the inherent binding sites. A unique property attributed to the Thermofluor is that it offers a high throughput approach to the study of allosteric interactions between protein and ligand. In terms of unfolding, Thermofluor has the capability to answer whether the flexibility of the protein is expressed as the number of different stable conformational states in high or low quantities. This is due to the fact that the range of temperature that unfolding occurs is reported by the flexibility of the protein. For example, steep transitions are indicative of highly cooperative unfolding, whereas shallow transitions indicate high flexibility. Multidomain proteins reflect an observed monophasic unfolding transition, and this is what is generally accepted as two-state unfolding. More complex unfolding transitions reflect that unfolding of the domains does not occur in a concerted manner. In order to obtain a detailed understanding of the linkage between ligand binding and protein stability, a concert of biophysical characterization utilizing Thermofluor, ITC, and DSC should be utilized... Refferences:1. Binding Techniques to Study the Allosteric Energy Cycle; Allostery: Methods and Protocols, Methods in Molecular Biology, 2012, Kranz et al2. http://.thermofluor.org3. http://thermofluor.org/resources/Niesen-fingerprinting_Oxford.pdf4. Thermodynamic Stability of Carbonic ANhydrase: Measurements of Binding Affinity and Stoichiometry Using Thermofluor, Biochemistry, 2005, Matulis et al Sincerely, lorenzo Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China Date: Thu, 19 Jul 2012 21:23:59 +0100 From: mohamed.n...@ul.ie To: lfi...@hotmail.com Subject: Re: [ccp4bb] off topic Thermal shift assay Dear Lorenzo a measure of protein cooperatively, Regarding your comment on positive cooperativity, is there any literature on this? I was taught that positive cooperativity for enzyme will require a steady-state kinetic assay. How does this relate to protein unfolding as measured by thermal shift? Sorry for a basic question. Thanks. Regards Mohamed Mohamed Noor Chemical and Environmental Sciences Department University of Limerick Ireland On 19/07/2012 16:25, Dr. Lorenzo Finci wrote:
Re: [ccp4bb] off topic Thermal shift assay
Anita, In terms of the basics:Thermodynamic stability of a protein is related to Gibbs Free Energy of unfolding. The Gibbs Free Energy is made of temperature, enthalpy and entropy (Delta G = Delta H - T Delta S). At Delta G equivalent to zero, the concentration of folded protein is equivalent to the unfolded protein. Thus, proteins are most stable at conditions where their melting temperature (Tm) is highest. Protein solubility and stability are prerequisites for subsequent biochemical and biophysical analysis and characterization. The preparation of concentrated, soluble, and stable protein sample can often be a difficult task as proteins aggregate, precipitate, or denature. Protein stability and solubility is directly correlated to temperature, pH, buffer composition, salt composition, additives, and ligands. The fluorophore ( ANS, SYPRO) has a high quantum yield in a low dielectric medium. Protein unfolding exposes the hydrophobic core corresponding to the low dielectric medium, and the fluorophore is quenched in the solution. Melting a proteins internal amino acid side chains is a first order phase transition associated with absorption heat. The Differential Scanning Fluorimetric (DSC, or Thermofluor) assay gives a direct measurement of a proteins melting temperature (Tm).In terms of Applications:The Thermofluor assay is used for the optimization of solution for protein stabilization. This is utilized for protein preparations and biochemical assays, crystallization, as well as optimization of ligand binding affinity, including drug screening, a measure of protein cooperatively, and to probe function. In terms of Extrinsic Dyes:SYPRO Orange is by far the most popular, along with ANS as they both are utilized to bind to the hydrophobic core of the soluble protein. CPM has been recently utilized to study membrane proteins as it binds to buried cysteines. I would look at the paper by Stevens et al 2008, and for high throughput, I would look at the utilization of CPM in the presence of detergents by Fan et al 2011. In terms of limitations, there are advantages and disadvantages:Advantages include the small of quantity of protein needed, the low concentration needed, the reproducibility, and the possibility of simultaneous screening of multiple conditions. For disadvantages, the interactions between the dye chosen and other compounds may mask stabilization or cause artifacts, as well as difficult interpretation effects of oligomeric proteins that show multi-phasic unfolding characteristics. Also, most dyes are not applicable to conditions comprising hydrophobic additives such as detergents that are necessary for membrane proteins (Hence, applications utilizing CNS). I hope this helps, good luck with your studies!Sincerely,lorenzoLorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China Date: Thu, 19 Jul 2012 11:00:28 -0400 From: jubo...@jhsph.edu Subject: Re: [ccp4bb] off topic Thermal shift assay To: CCP4BB@JISCMAIL.AC.UK Yes that is very true. But I assume true membrane proteins exclude high throughput :-) However the cytoplasmic part might be fine in this case and then I would just go for Sypro Orange. Jürgen On Jul 19, 2012, at 10:39 AM, Edwin Pozharski wrote:My understanding is that the advantage of the thermofluor assay is that you can test many conditions rapidly unless of course you have some kind of high throughput CD spectrometer in mind. If you have a CD available (not the one with music on it) you don't need a dye just sufficient protein and you can thermal denature your protein assuming it contains some secondary structure elements. Jürgen On Jul 19, 2012, at 4:26 AM, anita p wrote: Hi All, I want to use a thermofluor for the thermal shift assay. My proteins are cytoplasmic truncations of membrane protein. I have read about ANS, sypro-orange and CPM. Which is the once that is popularly used by the crystallographers for condition optimization for crystallization ?? I have read that it sypro orange is not good for hydrophobic proteins and CPM can't be used with DTT or bME in the buffer. I am a bit confused . Please help thanks in advance Anita .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu -- Edwin Pozharski, PhD University of Maryland, Baltimore .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office:
Re: [ccp4bb] Disulphide bonds and closed conformation
Jan, I agree that the DTNB assay (Ellman's Reagent) can be used to quantify exposed Cysteines. Alternatively, you could do site directed mutagenesis and mutate significant amino acids responsible for the conformational change from the open to the closed state, with hopes to lock the protein in the closed conformation. However, this may prove to be tricky. If the protein is indeed an enzyme, you can also attempt to crystallize the protein with a transition state analog inhibitor, thus inhibiting turnover and locking the protein in the closed conformation Good luck!lorenzo Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China Date: Wed, 11 Jul 2012 11:12:17 -0400 From: kellydaugh...@gmail.com Subject: Re: [ccp4bb] Disulphide bonds and closed conformation To: CCP4BB@JISCMAIL.AC.UK Jan,If the Cys residues are accessible, you could try DTNB to quantify the number of Cys, thus determining if they are reduced or bridged. http://en.wikipedia.org/wiki/Ellman's_reagent Kelly*** Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 *** On Wed, Jul 11, 2012 at 11:08 AM, Jan Rashid Umar jan...@googlemail.com wrote: Dear all, I am working on a protein where I have to stabilize the closed conformation of the protein using disulphide bond. The strategy to design the cysteine mutants is based on the molecular dynamic simulations, and accordingly the residues were chosen. The ultimate goal is to trap the ligand in closed conformation of protein and crystallize it. I am facing few issues: Is there some reliable assay that can check the formation of disulphide bonds in protein. Additionally, does anybody knows another method(s) that can be used to trap a closed conformation. I look forward for your suggestions and discussions on this issue. Thanks very much! Jan
Re: [ccp4bb] recommendations_on_purification
Petros, It has indeed been speculated that high concentrations of Magnesium and/or other metals present in the cell lysate effect the binding of the Histidine-tag, and thus specific conditions for binding and elution need to be optimized for specific elution of your target protein. I believe that the standard recommendations when using a Nickel column to bind to your Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted unspecific binding protein, to try manipulating the binding by lowering the pH of the buffers, and by determining the specific concentration of . Alternatively, you can also try using another metal column such as Cobalt (Talon) with a higher affinity for the Histidine tag.Sincerely, lorenzo Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China Date: Mon, 26 Mar 2012 15:04:43 +0300 From: peg...@pasteur.gr Subject: [ccp4bb] recommendations_on_purification To: CCP4BB@JISCMAIL.AC.UK Dear all, I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris, using the standard minimal medium described in the invitrogen manual (plus PTM1). Following collection of the culture medium, I am having problems with purification of the protein as only a small fraction (~10%) binds to the Ni-NTA beads even after extensive buffer exchange (when expressed in full BMGY media this is not observed). Could this be attributed to metal ions still present in my sample? Is it likely to be due to poor protein quality in this medium? Or any other suggestions? Thanks in advance Petros
Re: [ccp4bb] recommendations_on_purification
Etros, It has indeed been speculated that high concentrations of Magnesium and/or other metals present in the cell lysate effect the binding of the Histidine-tag, and thus specific conditions for binding and elution need to be optimized for specific elution of your target protein. I believe that the standard recommendations when using a Nickel column to bind to your Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted unspecific binding protein, to try manipulating the binding by lowering the pH of the buffers, and by determining the specific concentration of Imidazole your protein elutes at by using a gradient before progressing to a step-wise elution. Alternatively, you can also try using another metal column such as Cobalt (Talon) with a higher affinity for the Histidine tag.Sincerely, lorenzoLorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China Date: Mon, 26 Mar 2012 15:04:43 +0300 From: peg...@pasteur.gr Subject: [ccp4bb] recommendations_on_purification To: CCP4BB@JISCMAIL.AC.UK Dear all, I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris, using the standard minimal medium described in the invitrogen manual (plus PTM1). Following collection of the culture medium, I am having problems with purification of the protein as only a small fraction (~10%) binds to the Ni-NTA beads even after extensive buffer exchange (when expressed in full BMGY media this is not observed). Could this be attributed to metal ions still present in my sample? Is it likely to be due to poor protein quality in this medium? Or any other suggestions? Thanks in advance Petros
[ccp4bb] Protein Biochemist - Peking University Beijing, China
Protein Biochemist Position in Structural Neuroscience There is an immediate opening for an enthusiastic and experienced protein biochemist to join the international structural biology laboratory of Jia-Huai Wang based at Peking University in Beijing, China. The research is focused on the structural and functional investigation of cell surface receptors in the nervous system and their role in axon guidance. The project is the close collaborative efforts between Professor Jia-huai Wang's structural biology lab and Professor Yan Zhang's neuroscience lab. The successful candidate will have a Ph.D. in Biochemistry or related discipline, with 3-5 years postdoctoral experience. The candidate will be responsible for the optimization of cloning, expression, and purification of protein for X-ray crystallographic analysis. Extensive expertise with multiple expression systems including E.coli, insect, and mammalian systems (HEK293/CHO) is absolutely required, and a proficiency in molecular biology and protein biochemistry is also required to help develop strategies to clone, express and purify difficult target proteins. Although not a requirement, a preference will be given to those applicants who also have a strong background in X-ray crystallography. Interested candidates can email a CV, three contacts for reference, as well as an email address and a telephone number to Dr. Jia-huai Wang at jw...@red.dfci.harvard.edu. For more information regarding the Wang Laboratory, please see the website:http://wang.dfci.harvard.edu
