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MIT 10 37 - Lecture 14- Kinetics of Non-Covalent Biomolecular Interactions

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Noncovalent InteractionsBiosensor10.37 Chemical and Biological Reaction Engineering, Spring 2007 Prof. K. Dane Wittrup Lecture 14: Kinetics of Non-Covalent Biomolecular Interactions This lecture covers : Significance, typical values and diffusion limit, approach to equilibriu, and multivalency Noncovalent Interactions Protein Ligand Complex P + L C kkonoffU Figure 1. Protein-ligand binding. Association rate = kC on pCLDissociation rate = kC off c @ equilibrium, kC= on pCLkCoff c 1 s CCpLk==offK CkdconL mol s In general, for protein-protein interactions, km51− −1 on≈ 10 ols half-time for complex dissociation ln 2τ12= koff Kd τ 12types mM milliseconds non-specific stickinessμM (micromolar) milliseconds-seconds cell surface, multi valent nM minutes-hours antibodies, enzymespM hours-weeks growth factorsfM (femtomolar) weeks-months hycholase inhibitors stronger interactions Fractional saturation CCY==cc CCpo,c+CpCite as: K. Dane Wittrup, course materials for 10.37 Chemical and Biological Reaction Engineering, Spring 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].CCpLCKYLd=→=CCcL+Kd CL Y Kd log CL Y Figure 2. Left: Graph of fractional saturation versus ligand concentration. Right: Graph of fractional saturation versus the logarithm of ligand concentration. If Cpo,≈ C, then at equilibrium, Lo,CCLL≠ ,o CyLo,,− CY =po CyLo,,−+CpoKd KCd++ C2L,,oCpo−()Kd++CL,o po,−4CYpo,C=L,o 2Cpo, If instead CC, C≈ C Lo,,po LL,o CY=Lo, CKLo,+d How quickly is equilibrium reached? dCc=−kCdton LCpkoffC c If CC “pseudo-1st order” Lo,,po kC on L= kCon L,o CC po,=+pCcc(complexed) CC pp=−,oC 10.37 Chemical and Biological Reaction Engineering, Spring 2007 Lecture 14 Prof. K. Dane Wittrup Page 2 of 5 Cite as: K. Dane Wittrup, course materials for 10.37 Chemical and Biological Reaction Engineering, Spring 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].dCc=−kCon L,,oCpkoffCc=kCon L o()Cp,o−Cc−kdtoffCc =−kCon p,,oCL o(konCL,o+koffCc C⇒=Ct() CLo,(1−e−ktobscp,o) CKLo,+d kk obs=+onCL,okoff )ln 2=half-time for reaching equilibrium kobs t cC ln 2obsk equlibrium Figure 3. Concentration of complex versus time. Equilibrium is approached at long times. Biosensor Surface plasmon resonance (label-free) flow thin gold film hν amount of reflected light is a function of complex formation Figure 4. Schematic of how surface plasmon resonance works. 10.37 Chemical and Biological Reaction Engineering, Spring 2007 Lecture 14 Prof. K. Dane Wittrup Page 3 of 5 Cite as: K. Dane Wittrup, course materials for 10.37 Chemical and Biological Reaction Engineering, Spring 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].t signal flow signal stop signal onoffkk dK offk association dissociation equilibrium Figure 5. Signal of detector versus time. redundant estimates: k in both association & dissociation, offkKoffd= in equilibriukonphase best approach: fit one set of parameters to three phases of experiment. (global lesquares) Multivalency (Avidity) cell cell m ast surface cell cell multivalent label Figure 6. Three examples of multiple protein-ligand binding. How does multivalency effect apparent interaction strength? 10.37 Chemical and Biological Reaction Engineering, Spring 2007 Lecture 14 Prof. K. Dane Wittrup Page 4 of 5 Cite as: K. Dane Wittrup, course materials for 10.37 Chemical and Biological Reaction Engineering, Spring 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].free U bound U Keffdetected same (high effective local concentration) Figure 7. Multivalent binding equilibrium. 10.37 Chemical and Biological Reaction Engineering, Spring 2007 Lecture 14 Prof. K. Dane Wittrup Page 5 of 5 Cite as: K. Dane Wittrup, course materials for 10.37 Chemical and Biological Reaction Engineering, Spring 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month


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MIT 10 37 - Lecture 14- Kinetics of Non-Covalent Biomolecular Interactions

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