Macromolecular Structure BCH 701
Popular in Course
Popular in Biochemistry
This 24 page Study Guide was uploaded by Kian Berge on Thursday October 15, 2015. The Study Guide belongs to BCH 701 at North Carolina State University taught by Staff in Fall. Since its upload, it has received 27 views. For similar materials see /class/223863/bch-701-north-carolina-state-university in Biochemistry at North Carolina State University.
Reviews for Macromolecular Structure
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 10/15/15
Interactions Biological role is almost always dependent on a physical interaction with something else a complex Bind to LIGANDS without ligand apo with ligand holo Use NMR Xray and Mass spec to determine structures of complexes General Usually a specific unique binding site for a ligand Sometimes say for metal binding similar sites There are exceptions however ex Cytochrome C3 binds 4 heme s in different sites 1Binding sites often are sizable depressions on the surface 2The interactions surface between a protein and ligand tends to be maximized a Small ligands don t perturb the dimensions of a protein much b Larger ligands tend to bind in surface depressions where they can associate and dissociate c Nondissociators tend to be band in deep cavities near protein interior to become integral parts of the structure d Long linear ligands ex Polysaccharides bind in clefts on the surface e If the protein and ligand are approx same size say 2 associating proteins their interface tends to be flat and large f With large ligands such as nucleic acids the protein tends to bind to depressions on the surface of the ligand Need steric and physical complementarity between each Approx same rules we ve already seen Electrostatics Hydrophobics Polar groups on surface Hbonded Charges neutralized Water molecules can act as intermediates Sometimes charges can be solvated by multiple Hbonds The structure of a protein domain generally does not change substantially when it binds a ligand exceptions are usually of functional importance Small movements of atoms of the protein do occur in every case but they are often comparable to the experimental errors in crystallographic structure analysis The most extreme changes in domains generally involve movements of flexible loops on the protein surface On the other hand some small adjustments are probably important in general to permit rapid rates of association and dissociation totally rigid complex structures in which atoms interlock and interdigitate would e unlikely to be able to come together readily But small subtle motions are seen However this is not always the case some proteins go through wild changes upon binding some metal binders ex Calmodulin Basic Binding Energetics What s the affinity of the protein for the ligand a measure of the free energy of the interaction The magnitude of the affinity determines whether a particular interaction is relevant under a given set of conditions 2 parts to affinity does it like to naturally bind protein ligand concentration AG AH TAS Tells you how much protein will like its ligand Binding Affinities The affinity between a protein P and a ligand A is measured by the association constant Ka for the binding reaction at equilibrium PA L PA PA a PllAl All species are presumed to be present as sufficiently low concentrations for thermodynamic ideality to apply if not activities rather than concentration must be measured Ka is a constant under a given set of conditions and is measured experimentally by the dependence of binding on the free ligand concentration The ratio of bound to free protein should be directly proportional to the freeligand concentration P A P KaiA An experimentally more useful measure of binding is the fraction y of protein molecules with bound ligands A B PAe w hB P AK3 P B1103 P A B P A g m P 39 B KG B The greater the value of Ka the greater the affinity The value of Ka has units of concentration391 however and it is often intuitively easier to consider the dissociation constant Kd which is simply the reciprocal of Ka with units of concentration With concentrations of free ligand below Kd little binding to the protein occurs With a concentration equal to Kd half the protein molecules have bound ligand An occupancy of 90 requires a nine times greater concentration of free ligand whereas 99 occupancy requires that the concentration be 99 times Kd Binding equilibria are simplest when the ligand is present at a concentration much greater than that of the protein binding sites Sped cbuu ngbyawou nofonehgandandnotanothendependsontheu relative affinities their concentrations and whether they bind at the same site Two ligands are present at a concentration of 10395 but have different values of Kd Only the ligand with the lower Kd is bound significantly If both are present at much higher concentrations both are bound to the protein to the maximum extent if they bind at separate sites In this case the higher affinity of one Hgandisahnostinnnate al chetmmHgandscon1peteforthesarneshethe ligand with the higher affinity is bound to a correspondingly greater extent when the ligands are present at the same concentration Weaker affinity can always be overcome by a higher concentration of that ligand so binding a h esshouk ahNaysbecon deredreb vetotheconcen a on the ligand Therefore If you increase the affinity the stronger the Kd the tighter the binding complex less dissociation A lower Kd means a tighter binding Increase the concentration to overcome the Kd Energetics AGbmd Rt In Ka RT In Kd substitute in AG AH TAS DNA binding proteins Generally involved in replication or expression of genetic info Proteins mainly bind to very specific sites on DNA defined by a specific sequence of the 4 nucleotides A T C and G at a few adjacent positions For a protein to distinguish among different nucleotide sequences in double stranded DNA is not easy since the nucleotides of the 2 antiparallel strands are basepaired A T T A C G G C in the interior of the double helix Hard to find specific spot because similar environment all way thru H Binding region 338 A The exterior of the DNA double helix is almost independent of its sequence being composed of the constant phosphate sugar backbone Only the edges of the nucleotides are accessible to the solvent and to the protein primarily in the major groove The nucleotides are distinguished mainly by the accessible polar groups How a protein recognizes which major groove to bind to If a protein is to discriminate among DNA base pairs by interacting with their edges in the major groove it needs to have interacting groups that protrude substantially from its surface to be able to contact the nucleotides at the base of the groove The best characterized structural motif that accomplishes this is the helixturnhelix which protrudes from the protein surface It is observed in a number of proteins that have no other structural similarities This motif seems to have sufficient intrinsic stability to be able to exist as a protuberance with few interactions with the rest of the protein structure in order to penetrate the DNA major groove xi repressor To stabilize the helixturnhelix hydrophobics pack between the helices You can predict HTH s from sequence info based on this HTH come in pairs and sit adjacent in major grooves There are lots of hydrophobic in middle to stabilize The specificities of the various HTH motifs for binding to different DNA sequences arise mainly from the different amino acids side chains that protrude from the amino end of the 2nd helix known as the recognition helix These enter the major groove ofthe DNA The other or helix lies across the major groove making nonspecific contacts Recognition helix 2nd of HTH The residues that interact with the DNA are primarily polar especially those with multiple hydrogen bonding side chains Asn Gln Arg Asp Glu and LYS These direct interactions involve flexible side chains Different HTH s interact with DNA in a variety of geometrics there is no simple code relating the amino acid sequence to the nucleotide sequence it recognizes Specific affinities and interactions seem to be determined by the ability of the DNA to undergo specific structural changes so that complementary surfaces are formed between the proteins and the DNA Interactions between the DNA sugar phosphate backbone and the proteins are important for establishing such structural changes and so for positioning the recognition helix correctly in the major groove Often it s Hbonds between the DNA phosphate groups and the peptide backbone NH groups that help establish the recognition helix for specific interactions Non recognition helix Peptide bond of 1st helix binds to phosphate backbone weakly inducing structural changes Use backbone NH groups to H bond to phosphate group of DNA specific recognition lSt helix doesn t go into groove Makes non specific bonds with recognition helix 1 Another structural source of putting an 0i helix in the right place to interact with nucleotides in the major groove is the Zinc Finger The most conspicuous feature of which Zn2and chelated by 2 His 2 Cys Characteristic motif X3 C X24 C X12 H X34 H X4 X can be anything Very big in gene regulation in eukaryotes Hydrophobics In middle a COZH helix If hairpin XFlN 31 Multiple Zn fingers are usually present in tandem along a polypeptide chain linked by a few residues frequently Thr Gly Glu Lys An individual finger only binds to DNA weakly but many together bind very well Each Zn finger contacts 3 adjacent nucleotide bp s in the major groove bp1 bp2 bp3 39 Finger 2 Finger 1 Direct interaction between the protein and the DNA nucleotides is a major factor in the specificity of binding but another factor is thought to be the deformability of the DNA double helix DNA is observed to bind to some proteins in conformations that are distorted to varying extents from the classical linear double helix The DNA structure not that of the protein is perturbed in these cases indicating that the DNA is more pliable structurally than is the protein An exception is the Cro protein in which binding to DNA causes the two monomers to rotate 400 relative to each other by twisting of the two Bstrands that connect one monomer with the other There is considerable evidence for the plasticity of the DNA doublehelix structure and the extent and nature of this plasticity vary with the nucleotide sequence Some DNAbinding proteins are thought to discriminate among different nucleotide sequences by binding their specific sequence in a distorted conformation that is energetically favorable for that sequence but not for others to which the protein might otherwise bind Proteins that bind to DNA irrespective of it nucleotide sequence or with little sequence specificity recognize primarily the DNA backbone of phosphate and sugar groups Bind nonspecifically Electrostatic interactions and the release of counter ions and bound water molecules appear to be the driving force for binding DNA to proteins like this DNA is polyarionic with one ionized phosphate group per nucleotide Even with proteins that bind specifically the initial binding is onspecific the protein binds loosely to the DNA and it can search along the essentially 1D molecule for its specific binding site to which it binds more tightly Kd of 10393 10396 is nonspecific mainly 10396 and lower specific Interactions between DNA and proteins are usually measured by bandshift electrophoresis and footprinting in which the band protein protects the DNA from chemical modification The presence of DNA binding proteins tightly bound causes the electrophoretic mobility of the DNA segment to be shifted good assay
Are you sure you want to buy this material for
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'