Schedule for CMC Workshop and mini-Symposium now available

During the week of the Membrane Protein Structural Dynamics Consortium (MPSDC)’s Frontiers in Membrane Protein Dynamics conference, a “hands-on” Workshop specifically dedicated to Membrane Protein Modeling will be held at The University of Chicago on Tuesday, May 1st. This Workshop is designed to provide an introduction to the most important computational tools and techniques used in molecular dynamics simulations of membrane proteins, including utilization of the programs NAMD and VMD.

The Workshop is co-organized by the Computational Modeling Core (CMC) of the MPSDC and the Theoretical and Computational Biophysics Group (TCBG) of the University of Illinois at Urbana-Champaign.

Register for the CMC Membrane Protein Workshop (required) »

The CMC Workshop will be followed by a one day, open-to-all mini-symposium on the new development of computational tools presented by the members of the CMC. If you are not already attending the CMC Workshop, please fill out this form.

View the schedule for the mini-symposium »
Read more »

Flexibility of the Flexizyme

By Robert K. Nakamoto
Department of Molecular Physiology and Biological Physics, University of Virginia

There is almost nothing more useful to the biophysicist (except perhaps a talented grad student or post-doc) than to have probes in known positions within the structure of a protein. Spectroscopic and biochemical approaches use such probes to monitor the dynamics, environment and conformational shifts that occur during function. Probe placement is most commonly done by site-directed mutagenesis to introduce a unique cysteine and chemically modifying the purified protein with a sulfhydryl-reactive label. In recent years, investigators have developed new methods to incorporate custom-made amino acids directly into polypeptide chains by expanding the genetic code. In principle, amino acids with side chains of any chemical design can be incorporated into the growing polypeptide chain by the ribosome if the acyl-tRNA charged with the unnatural amino acid can be made. Usually a tRNA is used that recognizes one of the non-sense codons (UAG, AAG or UGA) or a codon not otherwise used in the coding sequence of the open reading frame. Of course, there are many limitations to this process but the main one is acylation of the 3’ base of the tRNA with the amino acid. This reaction is done in the cell by the amino acid-specific aminoacyl-tRNA synthetase. To broaden the genetic code, investigators have developed methods to “evolve” orthogonal synthetases to shift the specificities to their custom amino acid of choice (see Liu & Shultz for a review). Here, I describe a new and highly flexible method for synthesizing the acyl-tRNA with a wide range of unnatural amino acids using the “Flexizyme”.

Developed and optimized in the laboratory of Professor Hiroaki Suga at the University of Tokyo, the “Flexizyme” approach is based on the use of a ribozyme to incorporate unnatural amino acids in proteins. A Nature Protocols paper (Goto et al.) describes the entire system and provides a detailed list of reagents and instructions. Though highly flexible in many ways, it is important to highlight that the system only works in cell-free translation systems.

(click to enlarge)

From Flexizymes for genetic code reprogramming
Yuki Goto, Takayuki Katoh & Hiroaki Suga
Nature Protocols 6, 779–790 (2011)

Like in most systems designed to incorporate unnatural amino acids (see Liu & Shultz) a charged suppressor tRNA is used to integrate a designed amino acid into the nascent polypeptide chain. However, rather than acylating the tRNAsup with an aminoacyl-tRNA synthetase specifically evolved to accept only a given unnatural amino acid, the Flexizyme system acylates the tRNAsup with a ribozyme. This avoids altogether the difficult and somewhat daunting process of evolving an orthogonal tRNA synthetase to recognize each unnatural amino acid. The Suga laboratory has developed a set of Flexizymes capable of charging a tRNA with a diverse array of unnatural amino acids. Because the system is used only in vitro, it also avoids the additional worry of importing the unnatural amino acids into a cell, which can have toxic effects.

A key part of the system is the acyl-donor substrate, which is used by the Flexizyme to acylate the tRNAsup. Because the reaction is primarily driven by the nature of the leaving group, the amino acid part of the substrate can vary. Suga and his colleagues have developed a set of three Flexizymes optimized to work with four different leaving groups. The Flexizyme system is particularly attractive for incorporating spectroscopic probes such as fluorescence or nitroxide spin labels at user-defined positions. Normally, investigators try to create a background devoid of cysteines and unique sulfhydryls are introduced to provide specific reactive positions. The Flexizyme system abrogates the potential problems that replacing native Cys can have on protein stability and function, or that a given Cys may be inaccessible and unreactive.The investigator must determine which donor-leaving group/Flexizyme combination works best with their specific unnatural amino acid. For example, aromatic side chains generally work best with the cyanomethyl ester-leaving group and the “eFx” ribozyme, while non-aromatic side chains would use the 3,5-dinitrobenzyl ester with the “dFx” ribozyme (see Goto et al. for sequences and structures). The Flexizymes and tRNAs are easily made in the laboratory and the chemistries to synthesize the acyl-donor substrates are relatively straightforward. Assays to determine the efficiencies of the transfer reactions are described by Murakami et al.

In a very nice example, Öjemalm et al. used the Flexizyme system to create substrates to study the influence of polypeptide hydrophobicity on insertion into the endoplasmic reticulum membrane via the Sec61 translocon. The Flexizyme was used to charge the tRNAsup with a series of unnatural amino acids with linear aliphatic side chains up to 10 carbons in length.

The Flexizyme system is particularly attractive for incorporating spectroscopic probes such as fluorescence or nitroxide spin labels at user-defined positions. Normally, investigators try to create a background devoid of cysteines and unique sulfhydryls are introduced to provide specific reactive positions. The Flexizyme system abrogates the potential problems that replacing native Cys can have on protein stability and function, or that a given Cys may be inaccessible and unreactive. Furthermore, there is a wider choice of labels with the Flexizyme and the investigator can use the proper probe to monitor specific properties such as backbone dynamics or local environments, or to impart desired modifications into the structure.

In combination with robust cell free synthesis systems such as those developed by Volker Dõtsch and colleagues (see Klammt et al. for a review, and the Membrane Protein Expression and Purification Core page of this website for detailed protocols), the Flexizyme system can be a powerful tool for the efficient production of the modified protein in biochemical amounts. Obviously, the system is potentially limited by the ease with which the in vitro synthesized polypeptide can be folded into its native conformation. This can be particularly difficult with integral membrane proteins, since folding must be done in detergents, lipids or a complex combination of both. Recent successes are indeed encouraging and the advantages provided by combining the two approaches represent an untapped resource in the analysis of the structure and dynamics of membrane proteins.

References

Goto, Y., Katoh, T. and Suga, H. (2011) Flexizymes for genetic code reprogramming. Nature Protoc. 6, 779-790.

Klammt, C., Löhr, F., Schäfer, B., Haase, W., Dötsch, V., Rüterjans, H., Glaubitz, C. and Bernhard, F. (2004) High level cell-free expression and specific labeling of integral membrane proteins. Eur. J. Biochem. 271, 568–580.

Liu, C. C. and Schultz, P. G. (2010) Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413-444.

Murakami, H., Ohta, A., Ashigai, H. and Suga, H. (2006) A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nature Meth. 3, 357-359.

Öjemalm, K., Higuchi, T., Jiang, Y., Langel, Ü., Nilsson, I., White, S. H., Suga, H. and von Heijne, G. (2011) Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum. Proc. Natl. Acad. Sci. U.S.A. 108, 359-364.
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Frontiers in Membrane Protein Dynamics full schedule announced

Click to enlarge poster or download a pdf for 8×11″ printing.

The Membrane Protein Structural Dynamics Consortium (MPSDC) will be holding its first Frontiers in Membrane Protein Dynamics conference on Thursday, May 3rd and Friday, May 4th.

This conference consists of scientific sessions and poster presentations, and will feature both Consortium members and external invitees.

Attendance is open to the public and will take place within the context of our 3rd Annual MPSDC Meeting, where all members, NIH representatives and our External Advisory Committee will participate.

The MPSDC’s Computational Modeling Core will host a NAMD/VMD workshop and a mini-symposium prior to the conference, which will take place on the University of Chicago campus. For more information on either of these events, click here »

We are currently booked over capacity for the conference, and are therefore no longer taking registrations. Please inquire at mpsdc_admin@bsd.uchicago.edu if you have any questions.
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MPSDC-funded poster author wins Biophysical Society 2012 Student Research Achievement Award

Thomas Chew with the award-winning poster. Click to enlarge.

The MPSDC had a strong showing at the Biophysical Society’s 56th Annual Meeting this year, with members collaborating in a total of 72 posters and presentations taking place in various subgroups, platforms, symposia, mini-symposia, and workshops. Now, we are proud to announce that one of our posters was selected as one of the thirteen 2012 Student Research Achievement Award Winners.

Thomas Chew, the first author of the poster, is an undergraduate student at the University of California, San Diego. He has performed summer research projects in the laboratory of Merritt Maduke at Stanford University.

The winning poster is entitled Structural Investigations of CLC-ec1, A Large Integral Membrane Protein, Using Solution-State NMR and Nanodisc Techology and features Thomas Chew, Sherwin J. Abraham, Shelley M. Elvington, and Merritt Maduke as authors. The poster was selected by the National Institute of General Medical Sciences as the sole NIGMS Poster Winner.

Thomas Chew at the February 27 Awards Ceremony together with the other winners and Steve Block. Click to enlarge.

Thomas was recognized and received a monetary award at the Awards Ceremony on February 27, preceding the 2012 National Lecture delivered by Steve Block. He was also selected to attend the NIGMS/NIH’s 50th anniversary symposium in October 2012.

Congratulations, Thomas and Merritt!

Read the Biophysical Society’s press release »




Computational Modeling Core to host Workshop and mini-Symposium on May 1st and 2nd

During the week of the Membrane Protein Structural Dynamics Consortium (MPSDC)’s Frontiers in Membrane Protein Dynamics conference, a “hands-on” Workshop specifically dedicated to Membrane Protein Modeling will be held at The University of Chicago on Tuesday, May 1st. This Workshop is designed to provide an introduction to the most important computational tools and techniques used in molecular dynamics simulations of membrane proteins, including utilization of the programs NAMD and VMD.

The Workshop is co-organized by the Computational Modeling Core (CMC) of the MPSDC and the Theoretical and Computational Biophysics Group (TCBG) of the University of Illinois at Urbana-Champaign.

Register for the CMC Membrane Protein Workshop (mandatory) »

The CMC Workshop will be followed by a one day, open-to-all mini-symposium on the new development of computational tools presented by the members of the CMC. If you are not attending the CMC Workshop, please fill out this form.

A schedule and more details for both of these events is now available »

CMC Membrane Protein Modeling Workshop

Tuesday, May 1st
8:30 am – 5:00 pm
University of Chicago
Knapp Center for Biomedical Discovery (KCBD)
Room 3200
(directions)

CMC Mini-Symposium

Wednesday, May 2nd
8:30 am – 3:00 pm
University of Chicago
Gordon Center for Integrative Science (GCIS)
Room W301
(directions)

View the schedule »

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