Assignment: Structure of Coelenterate Fluorescent Proteins Template

Assignment: Structure of Coelenterate Fluorescent Proteins Template

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1

. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are ≈25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.
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The red fluorescent protein cloned from Discosoma coral (DsRed or drFP583) (1) holds great promise for biotechnology and cell biology as a spectrally distinct companion or substitute for the green fluorescent protein (GFP) from the Aequorea jellyfish (2). GFP and its blue, cyan, and yellow variants have found widespread use as genetically encoded indicators for tracking gene expression and protein localization and as donor/acceptor pairs for fluorescence resonance energy transfer (FRET). Extending the spectrum of available colors to red wavelengths would provide a distinct label for multicolor tracking of fusion proteins, and together with GFP (or a suitable variant) would provide a FRET donor/acceptor pair that should be superior to the currently preferred cyan/yellow pair (3). However, the evolution of DsRed from a scientific curiosity to a generally applicable and robust tool has been hampered by several critical problems, including a slow and incomplete maturation and obligate tetramerization (4). Most previous attempts to address the rate and/or extent of maturation of DsRed (5, 6), including the commercially available DsRed2 (CLONTECH), have provided only modest improvements. However, an engineered variant of DsRed, known as T1 (see Fig. 1A), has recently become available and effectively solved the problem of the slow maturation (7).

Another approach to overcoming these shortcomings has been to continue the search for DsRed homologues in sea coral and anemone, an approach that has yielded several red-shifted tetramers (8, 9). The more fundamental problem of tetramerization, however, has yet to be overcome. The only published progress toward decreasing the oligomeric state of a red fluorescent protein involved an engineered DsRed homologue, commercially available as HcRed1 (CLONTECH), which was converted to a dimer with a single interface mutation (10). Although HcRed1 has the additional benefit of being 35 nm red-shifted from DsRed, it is limited by a rather low extinction coefficient (20,000 M−1⋅cm−1) and quantum yield (0.015) (11).

Important information for writing discussion questions and participation

Welcome to class

Hello class and welcome to the class and I will be your instructor for this course. This is a -week course and requires a lot of time commitment, organization, and a high level of dedication. Please use the class syllabus to guide you through all the assignments required for the course. I have also attached the classroom policies to this announcement to know your expectations for this course. Please review this document carefully and ask me any questions if you do. You could email me at any time or send me a message via the “message” icon in halo if you need to contact me. I check my email regularly, so you should get a response within 24 hours. If you have not heard from me within 24 hours and need to contact me urgently, please send a follow up text to

I strongly encourage that you do not wait until the very last minute to complete your assignments. Your assignments in weeks 4 and 5 require early planning as you would need to present a teaching plan and interview a community health provider. I advise you look at the requirements for these assignments at the beginning of the course and plan accordingly. I have posted the YouTube link that explains all the class assignments in detail. It is required that you watch this 32-minute video as the assignments from week 3 through 5 require that you follow the instructions to the letter to succeed. Failure to complete these assignments according to instructions might lead to a zero. After watching the video, please schedule a one-on-one with me to discuss your topic for your project by the second week of class. Use this link to schedule a 15-minute session. Please, call me at the time of your appointment on my number. Please note that I will NOT call you.

Please, be advised I do NOT accept any assignments by email. If you are having technical issues with uploading an assignment, contact the technical department and inform me of the issue. If you have any issues that would prevent you from getting your assignments to me by the deadline, please inform me to request a possible extension. Note that working fulltime or overtime is no excuse for late assignments. There is a 5%-point deduction for every day your assignment is late. This only applies to approved extensions. Late assignments will not be accepted.

If you think you would be needing accommodations due to any reasons, please contact the appropriate department to request accommodations.

Plagiarism is highly prohibited. Please ensure you are citing your sources correctly using APA 7th edition. All assignments including discussion posts should be formatted in APA with the appropriate spacing, font, margin, and indents. Any papers not well formatted would be returned back to you, hence, I advise you review APA formatting style. I have attached a sample paper in APA format and will also post sample discussion responses in subsequent announcements.

Your initial discussion post should be a minimum of 200 words and response posts should be a minimum of 150 words. Be advised that I grade based on quality and not necessarily the number of words you post. A minimum of TWO references should be used for your initial post. For your response post, you do not need references as personal experiences would count as response posts. If you however cite anything from the literature for your response post, it is required that you cite your reference. You should include a minimum of THREE references for papers in this course. Please note that references should be no more than 5 years old except recommended as a resource for the class. Furthermore, for each discussion board question, you need ONE initial substantive response and TWO substantive responses to either your classmates or your instructor for a total of THREE responses. There are TWO discussion questions each week, hence, you need a total minimum of SIX discussion posts for each week. I usually post a discussion question each week. You could also respond to these as it would count towards your required SIX discussion posts for the week.

I understand this is a lot of information to cover in 5 weeks, however, the Bible says in Philippians 4:13 that we can do all things through Christ that strengthens us. Even in times like this, we are encouraged by God’s word that we have that ability in us to succeed with His strength. I pray that each and every one of you receives strength for this course and life generally as we navigate through this pandemic that is shaking our world today. Relax and enjoy the course!

Hi Class,

Please read through the following information on writing a Discussion question response and participation posts.

Contact me if you have any questions.

Important information on Writing a Discussion Question

• Your response needs to be a minimum of 150 words (not including your list of references)
• There needs to be at least TWO references with ONE being a peer reviewed professional journal article.
• Include in-text citations in your response
• Do not include quotes—instead summarize and paraphrase the information
• Follow APA-7th edition
• Points will be deducted if the above is not followed

Participation –replies to your classmates or instructor

• A minimum of 6 responses per week, on at least 3 days of the week.
• Each response needs at least ONE reference with citations—best if it is a peer reviewed journal article
• Each response needs to be at least 75 words in length (does not include your list of references)
• Responses need to be substantive by bringing information to the discussion or further enhance the discussion. Responses of “I agree” or “great post” does not count for the word count.
• Follow APA 7th edition
• Points will be deducted if the above is not followed

• Remember to use and follow APA-7th edition for all weekly assignments, discussion questions, and participation points.
• Here are some helpful links
• The is a great resource