1 Module 2 overview lecture lab 1 Introduction to the module 1 Start up protein eng 2 Rational protein design 2 Site directed mutagenesis 3 Fluorescence and sensors 3 DNA ampli cation 4 Protein expression 4 Prepare expression system SPRING BREAK 5 Review gene analysis 5 Gene analysis induction 6 Puri cation and protein analysis 6 Characterize expression 7 Binding af nity measurements 7 Assay protein behavior 8 High throughput engineering 8 Data analysis 2 Lecture 2 Rational protein design I Blob level protein design A Engineered fusion proteins B Knowledge required for blob level engineering II Protein engineering at high resolution A Modifying existing proteins B De novo protein engineering C Knowledge needed for high resolution design D Computational modeling 3 Rational protein design Knowldege based deterministic engineering of proteins with novel characteristics Irrational high throughput protein engineering Selection for desired properties from libraries of random variants 4 Blob level protein design Basic idea is to combine protein units of de ned function domains to engineer a fusion protein with novel functionality Examples include sensors signal transduction components transcription factors therapeutics etc 5 GFP based approaches extend to other sensors Ting et al 2001 Proc Natl Acad Sci USA 98 15003 8 Can you think of other sensors one could construct based on this design strategy See Ting A Y et al Genetically Encoded Fluorescent Reporters of Protein Tyrosine Kinase Activities in Living Cells PNAS 98 no 26 2001 15003 8 6 An early synthetic biology project signal transduction triggered by a small molecule dimerizing agent Image from Spencer D M et al Controlling Signal Transduction with Synthetic Ligands Science 262 no 5136 1993 1019 24 DOI 10 1126 science 7694365 AAAS All rights reserved This content is exc luded from our Creative Commons license For more information see http ocw mit edu fairuse Spencer et al 1993 Science 262 1019 24 Engineered antibodies as therapeutic agents 7 single chain nanobodies bispeci c antibodies Jain et al 2007 Trends Biotechnol 25 307 16 Courtesy of Elsevier Inc http www sciencedirect com Used with permission 8 What knowledge is required for blob level protein engineering rough geometry of protein domains low resolution structure secondary structure if insertions or disruptions are planned desired linker properties length exibility hydrophilicity Example CaM based calcium sensors Nagai et al 2004 Proc Natl Acad Sci USA 101 10554 9 Courtesy of National Academy of Sciences U S A Used with permission Source Nagai T et al Expanded Dynamic Range of Fluorescent Indicators for Ca2 by Circularly Permuted Yellow Fluorescent Proteins PNAS 101 no 29 July 20 2004 10554 9 DOI 10 1073 pnas 0400417101 Copyright 2004 National Academy of Sciences U S A What we ve called blob level design is useful for combining functionalities associated with individual protein domains but what if we want to create new functionalities or make subtle manipulations 9 Image removed due to copyright restrictions Cartoon A business meeting in which the presenter using an overhead transparency projector puts up the slide Attention to Detail and the slide is projecting upside down The presenter says Which Brings me to my Next Point See http www cartoonstock com cartoonview asp search site catref mban740 10 Protein engineering at high resolution Alter tune properties of proteins by making structurally or computationally informed changes at the amino acid level In some cases produce entirely new proteins based on predictions of structure and function from amino acid sequence Can be rational when combined with structural information and or computational modeling approaches Can be irrational when combined with high throughput screening and random mutagenesis to be discussed later in the module This is what we are doing in the lab for this module 1 We looked at the CaM GFP structures and made predictions about which point mutations would shift the calcium af nity of pericam 2 We are now going to produce the mutant genes and proteins and assay puri ed molecules for desired properties 3 If we had more time we might then go on and make a new round of predictions mutant proteins to continue the process of tuning the calcium af nity Classic example tyrosyl tRNA synthetase engineered to study mechanism of catalysis 11 Leatherbarrow et al 1985 Proc Natl Acad Sci USA 82 7840 4 Courtesy of Robin Leatherbarrow Used with permission Rational design can also be used to stabilize proteins general route to improvement of function utility Images from Perry L J and R Wetzel Disulfide Bond Engineered into T4 Lysozyme Stabilization of the Protein toward Thermal Inactivation Science New Series 226 no 4674 1984 555 557 AAAS All rights reserved This content is excluded from our Creative Commons license For more information see http ocw mit edu fairuse 12 The holy grail of rational engineering is to design entire proteins de novo to fold into a de ned shape and ideally carry out a function Simplest task is to design peptides with de ned 2 structure Chou Fasman 1974 Biochemistry 13 222 45 American Chemical Society All rights reserved This content is excluded from our Creative Commons license For more information see http ocw mit edu fairuse Related task is to predict 2 structure from sequence MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADG helix sheet EEEEEEEE EEEEEEEEEEE EEEEE turns T TT T T TT T TT T NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE helix sheet EEEEEEEE EEEEEEEEEEE turns TT T T T T T T T TT EVDEMIREADIDGDGQVNYEEFVQMMTAK helix sheet EEEEEEEEE turns T T 13 De novo design can be extended to 3 and 4 structure Example is design of a functional enzyme from so called coil coil peptides 14 Kaplan Degrado 2004 Proc Natl Acad Sci USA 101 11566 70 Courtesy of National Academy of Sciences U S A Used with permission Source Kaplan J and W F DeGrado De Novo Design of Catalytic Proteins PNAS 101 no 32 2004 11566 70 DOI 10 1073 pnas 0404387101 Copyright 2004 National Academy of Sciences U S A 15 Courtesy of Protein Structure Prediction Center Used with permission Reference Kryshtafovych A et al New Tools and Expanded Data Analysis Capabilities at the Protein Structure Prediction Center Proteins 69 2007 Suppl 8 19 26 16 What knowledge is required for high resolution protein engineering determination of 3D structure for mutagenesis based engineering knowledge of protein folding rules