MIT-SCIENCE-Lectures-lec7_xu

Lecture Notes - 37.24/7.88J/5.48JThe Protein Folding Problem• Reprise RNase refolding• General Features of Globular proteins• Interior PackingRnase RefoldingWe can cartoon the experiments:Assume that reduced form is unfolded and populates an ensemble of unfolded states in 8M urea:[U]Rapidly exchanging statistical ensemble of random coils[I]???[N]Native fold, $ S-S bonds, 1 out of 105 possible sets of S-S bonds [Aggregated]Non-native (no enzymatic activity), non-native (scrambled) disulfide bonds.At the end of the reaction we have native stateSoluble inactive – misfolded or perhaps small oligomersPrecipitated: Anfinsen showed that these were S-S bonded network = he called “scrambled”Critical question is nature of the intermediates as go from here to there:If there was a sequence in which these bonds formed, and one could figure out the sequence or pathway, it might give you considerable information on the steps in the folding pathway.So certainly we need to understand native-fold, and interactions that determine it, stabilize it once formed;However this does not solve problemWe will return to the question after reviewing the general anatomy of globular proteins, which will occupy most of next week.General features of 3-D structures of solved proteins; Globular, Soluble proteins:A. Isolation and CrystallizationThe first crystals of proteins sufficiently large and ordered to diffract X-rays were prepared of the digestive enzyme pepsin:Bernal, J.D., and Dorothy Crowfoot. Nature (1934): 133, 794.This was followed by crystals of insulin, Lactalbumin, hemoglobin, andchymotrypsin:Bernal, Fankuchen, and Perutz (1938)The rise to power of the Nazis in Germany and Italy and the outbreak ofwar brought all these studies to a halt. Bernal led the world scientificcommunity into opposition to the fascists. This was not only with respect to the their persecution of Jewish scientists, but that the notion of Aryanscience different from the general growth of knowledge, represented abroader attack and the growth and communication of knowledge.The rebuilding and repopulating of the civilian biomedical infrastructuretook about ten years, and re-launched in the 1950’s. In England - BritishMedical Research Council, in US - National Institutes of Health.Solution of Myoglobin structure by John Kendrew in 1958 and hemoglobin structure in 1962 by Max Perutz - it became clear that alpha helix actually present in globular proteins. With the solution of the lysozyme structure by David Phillips (1966), revealed that anti-parallel beta-pleated sheet alsopresent in globular proteins.B. Review Criteria (Biases) for the selection of proteins forcrystallization and perhaps into the success or failure ofcrystallization1. Available in large quantities - usually extracellular protein fromextracellular fluids• Hemoglobin from blood• Myoglobin from muscle• Lysozyme from egg white/ tears??• Ribonuclease from stomach??• Insulin from pancreas??• Lactalbumin from milk ??• Chymotrypsin; from saliva, stomach??2. Stable during purification• Resistant to Residual proteases• Does not aggregate• Resistant to Cold denaturation3. Very soluble in aqueous buffersThis excludes:• Fibrous proteins; evolved to interact with each otherto form structures; keratin, collagen• Other Structural proteins; ribosome structuralproteins, histones, actins, tubulins• Membrane proteins4. Must crystallize• Proteins which behave differently than the dogma;take multiple conformations in solutionProtein database sharply under-represents – fibrous proteins, structural proteins, membrane proteinsC. Structures whose atoms have been located at high resolution- Globular aqueous soluble: 1,100 domains(- Membrane: 12)(- Fibrous: 5)This is the class of proteins for which single crystals have been obtained.Clearly the most extensive database is for proteins soluble in aqueoussolutions and which form crystals.D. General Features of Crystallized Globular Proteins1. Surface/Volume RelationshipsGlobular proteins have relatively sharply defined:• Exterior Surface = H20 Interface• Interior volume - Is the interior of a native proteincrystalline or oily?A quantitative answer to this was given by Richards:"The Interpretation of Protein Structures: Total Volumes, Group Volumes and Packing Density" Frederic M. Richards (1974) J. Mol. Biol., 82, 1 - 14.Problem comes down to calculating how densely packed atoms are inprotein molecules?We can define the packing density of a molecule as:Volume of its van der Waals envelopes of its constituent atoms / actual volume of the intact folded moleculeThis value is dimensionless- Close packed hard spheres = 0.74- Close packed infinitely long cylinders = 0.91- Crystals of various small organic molecules 0.70 - 0.78 values (<0.6very rare)The van der Waals radii - from interatomic distances of nonbondedatoms in organic crystals.However calculation of the volume of irregular structures not trivial.Richards’ procedure made use of a geometrical construct calledVoronoi polyhedra.• Draw vector from each atom to closest surroundingatoms.• Draw a plane through it corresponding to intersectionof van der waals radii. This will define a polygon.• When you do this for all atoms space is completelyfilled.• Thus obtain geometrically legitimate volume. Cancompute this from atomic coordinates.For the following solved proteins, the average packing density:• Hen egg white lysozyme = 0.75• Ribonuclease S = 0.74Conclusion: The interior of globular proteins is as tightly packed as areamino acids in organic crystals.Subsequent investigations have supported this general result for thatset of proteins included in crystallized set.Counter argument; rigidity necessary for crystallization is only found inthose proteins which are continuously dense and lack hinges.2. Are there interior cavities or entrained solvent bobs?Careful examination of a larger set of proteins was carried out byA.A. Rashin, Michael Iofin, and Barry Honig (1986) "Internal Cavities and BuriedWaters in Globular Proteins" Biochemistry, 25, 3619-3625.These procedures involve in general:• Construction of Van der Waals radii;• Find places where these radii not buried or in contact with other radii;• Use a spherical probe (water model = sphere radius 1.4A) walkalong van der Waals radii with smaller and smaller probe to findplaces that can walk alongAccessible surface area is defined by center of water molecule rollingover van der Waals surface. Thus there exists inaccessible surface area.Gives an estimate of volume of cavity and accessible surface area.H2Os# Protein MW#VolA3Pancreatic Trypsin Inhibitor 6,000 2 70 4cytochrome c 11,000 5 34 24115ribonuclease 13,000egg lysozyme 14,000 8 190 54019erythrocruorin 14,0001391Myoglobin 17,0002317494trypsin 23,00013chymotrypsin 26,000 26 571 10• Cavities = less than 2% of the volumes of theseproteins.• No single cavity can host a probe of 2A or more.• Almost all cavities become connected to proteinsurface as probe diameter goes down to 0.4 A.What does this suggest?Suggests that these represent channels or hinges.Volume of internal water molecule for larger cavities is about 30AVan der waals radius of water is about 1.4 ASome cavities which appear non-polar can contain a water moleculeforming three hydrogen bonds. This is because the hydrogen bond canbe longer than the sum of the van der waals radius of a polar atom anda probe., and thus some polar atoms with which water forms hydrogenbonds may not contribute to the accessible surface.Note: those cavities that seem to be empty could be contributing boundwater, but if water crystallographically not fixed, smeared out.3. Functional Significance of small cavitiesLargest empty cavity in their sample located between helices B, E, andG, between two domains of the globin.Conserved in six other myoglobin type proteins (Very diverse). May beimportant for relative motions of helices.More than half of cavities in multi-domain proteins are at domaininterfaces.He considers similar, linkage explanation, rather than packing problem.Prediction; Those sequences that have evolved can close pack?Question: WHAT FRACTION OF amino acid sequences is there noclose packed globular structure?For retinal containing pigments models suggest that cavity of 100Awould allow for rotation of chromophore.Further studies carried out by:"Interior and Surface of Monomeric Proteins" Susan Miller, Joel Janin Arthur Lesk and Cyrus Chothia (1987) J. Mol. Biol., 196, 641-656.For example lysines and arginines almost always at surface (<4%buried)4. Additional Features of Inside OutsideGeneral Features of Globular proteins• No Knots• More than half of the polypeptide sequence isorganized into regular secondary structure,predominantly alpha helices and beta sheets• Interiors are densely packed - Extremely rare to burybulk solvent• Buried charges very rareo In rare cases, ion pair, metal complex, oractive site• Interiors are hydrophobic (carbonyls and amides areH-bonded)• A large fraction of the accessible surface of theextended polypeptide chain is buried in the proteininterior.E. Conclusions re protein folding1) Buried side chains truly removed from water: Major energetic contribution toprotein folding; but driving force, as originally proposed by Walter Kauzmann, is entropic effect on water structure; hydrophobic residues cannot H bondwith water; organizes water molecules with respect to own partners.2) These side chains almost always hydrophobic: extremely rare to burycharged side chains; If the inside of a protein is closely packed and excludes water, becomes quite clear that in fact the inside and the outside are verydifferent environments. Therefore the partitioning of residues into the chainbetween interior and surface positions may be very important in defining fold.3) Contradiction: Burying side chains also requires burying backbone; butbackbone has Carbonyl oxygens and amide nitrogens which are hydrophilic, polar groups;4) But in actual fact, largest number of peptide bonds in proteins are partof regular secondary structures, alpha helices and beta sheets: partialcharges are neutralized by maximal hydorgen bond formation:5) SO naively folding processes may have at least two level of organizationFormation of secondary structure, docking against each other to formtertiary structure; Framework model:Now clearly, given the large fraction of chain that is in regular secondarystructure, a substantial fraction of the internal packing of globular proteins is the...So at least two processes that we need to investigate:• Formation of secondary structure;• Next level; packing of secondary structure into tertiary structure: Might be tightly coupled;F. Packing of Secondary StrcuturesOne of the determinants of the shapes of proteins and the folds is thepacking of the units of secondary structure against each other.Short amino acid sequences = peptides > At room temperature in constantmotion; flickering in and out between a variety of conformations;Homopolymers; In solution also flickering in and out between non-regularconformations and mixture of these with alpha helices;Two levels of Packing:• Within units of secondary structure; alpha helix, beta strands• Between units of secondary structure- helix/helix, helix /sheet• Packing Interactions critical for formation of native structure.。

24.962 Features-2Flemming gives contrast a formal role in shaping sound patterns.The markedness of a segment can be effected by the sounds it contrasts with in agiven system/context.e.g. central vowels are more marked than peripheral vowels (*i,* >> *,i,u) butwhen the peripheral contrast is suspended then central vowels are preferable ongrounds of articulatory economy.1. Formalization:i. maximize distinctiveness of contrastii. minimize articulatory effortiii. maximize the number of contrasts2. Application to vowels: organized into three-dimensional acoustic space (7).Mindist = F1:1 >> Mindist = F1:2 >> … >> Mindist = F1:4(ranks binary oppositions from worse to best on scale of distance in acoustic space)Maximize contrasts: number of contrasting vowels(9) shows that if we rank MaxContrasts low then Mindist will favor greater dispersion of vowels. As the number of contrasts increases they must be crowded into a fixed articulatory-acoustic space entailing loss of distinctiveness: under such pressure there are various outcomes: accept narrower space, enhance the contrast by reinforcing maneuvers, lose a contrast. These are modeled by different constraint rankings in an OT grammar.Considerations of vocabulary size and perceptual limitations discourage grammars with extreme rankings: many-fine grained contrasts or just a couple of broad contrasts.3. Exemplification:Tuscan Italian: [e,ϯ,o,ѐ ] contrast neutralized to [e,o] in unstressedsyllablesF states that in unstressed syllables less duration is available so thatmaximally open articulations requiring greater effort/duration areeliminated. If the Min-Dist constraint ranking is maintained with respectto Maximize Contrasts then *short low V forces [a] to be realized as [3].The space is now too crowded to maintain a four-way contrast in F1 with amin-dist of 2 and so the number of contrasts is reduced minimally.Mindist=F1:2 MaxContrasts Mindist=F1:3-> [i,e,E,a] #### **[i,e,a] ###![i,a] ##!*short-low Mindist=F1:2 MaxContrasts Mindist=F1:3 [i,e,E,a] *! #### **[i,e,a] *![i,e,E,3] *! #### *** -> [i,e,3] ### **S. Italian [i,u,e,o,a]; in unstressed position, mid vowels become highIn stressed position, min-dist threshold set at F1:3: Mindist=F1:3 >>MaxContrastsMindist=F1:2 Mindist=F1:3 MaxContrasts[i,e,E,a] **!-> [i,e,a] ###[i,a] ##!*short-low Mindist=F1:2 Mindist=F1:3 MaxContrasts [i,e,E,a] *! *** ####[i,e,E,3] *!** ####[i,e,a] *![i,e,3] *!* ###-> [i,3] ##[i,a] *!4. Vertical vowel systems of Marshallese and Kabardian lack articulatory/acoustic specifications on F2 dimension—value determined by transitions from surrounding consonants. In Kabardian these vowels are also shorter than full vowels (phonemicized with glides): 58 ms. for [i], 84 for schwa, vs. 140 for full vowels.5. Vowel reduction in Dutch and English suspends F1 and F2 contrasts; result is an F1 that is comparable to high vowels—low F1 reflecting the constrictions of surrounding consonants (see Kondo).6. Enhancement and reinforcement strategies: articulatory effort expended to maintain a contrastPre- and post-oralized nasals consonants shield the oral vowel from contextual nasalization: found only in languages that contrast oral and nasal vowels (Herbert ’86) Voiced stops replaced by implosives or prenasalized consonants only when incontrast with voiceless stops.Analysis: VOT 0 1 2 Voice 0 1 2d t th t d D, n d*implosive, prenasal >> *voiced stopMindist-VOT-1+ #Contrasts *implosive Mindist-VOT-2+Voice:1 Voice:2__________________________________________________-> t-d ## *t-D ## *!t #!Mindist-VOT-1 Mindist-VOT-2 #Contrasts *implosiveVoice:1 Voice:2_____________________________________________________ t-d *! ##->t-D ## * t #!Implosives and prenasals only enhancements; not found in position ofneutralization;Walker’s typology of nasal harmony:Vowels, laryngeals, > glides > liquids > fricatives > obstruent stopsBroadly derives markedness scale for nasalizable segments: m > ~a > ~l > ~z; but nasalized glottal stop does not arrest nasal harmony yet is never the site of a contrast. So *~? should be high ranked but then this is incompatible with harmony.Fl: block where nasalization would endanger contrast with nasal stops [m,n,N]。

MIT Sloan Communication Pr ogram Teaching Note JoAnne Yates Active Listening and Reflective ResponsesOne of the basic buil di ng blocks of communication--and one of the most difficult skillsto learn and practice--is effective listening. We all spend much of our time hearingother people speak, but not necessarily listening to what they are really saying.Instead, for example, we daydream, start formulating responses before hearing thewhole point, and interrupt to make a tangential comment. Meanwhile, we have failed to understand the speaker's real point and the thoughts or feelings underl yi ng t hat point.As a consultant or manager, you will be called on to listen in many different contextsand for many purposes. You will need to gather data about a problem in order to help . solve it. You will need to listen to a subor di nate's career problems in order to help hm develop. You will need to understand the point of view of another person in order to carry on effective negotiations for somethng you want from her. You will need to interview cand i dates f or positions in your group. Before you can communicateeffectively as managers you need to learn some useful approaches and techniques for effective l istening. Th i s teachng note describes active listening, a comprehensive approach to the task of listening It also describes reflective responses, a pa rti cular responding technique that is based upon Western concepts of the role of feelings in interpersonal relationsh i ps. Reflective r esponses can be especially useful in cert ai ntypes of listening situations.Active ListeningActive listening is a term often used to describe a general approach to listening thathelps you gain more information, improve your understanding of other p oi nts of view, and work cooperatively with superiors, subordinates, and peers. This approachrequires not just that you learn and remember more of what the other party has said, but also that you communicate your interest and involvement to that party, as well. Active listening requires effective use of verbal and nonverbal communication, as well asmental and emotional discipline.An active listener:Looks and sounds interested in the speaker. By convey i ng your interest,you can encourage the speaker to communicate more extensively and to clarifyand expand on thoughts and feelings. Communicate your interest bymaintaining good eye contact. (In A merican culture and many Westerncultures, this means loo ki ng into the other person's eyes much, though not all,of the time. Too much eye contact may make the speaker feel self-conscious,but too little will make h i m f eel ignored). Maintain a body position and facialexpression that indicate attentiveness, not boredom. Nod encouragingly toshow understanding and interest. Avoid drawing, playing with your pen, orother distracting behaviors. (Conversely,t ry not to be distracted by thespeaker's mannerisms.) Use vocalizations such as "uh-huh" and "yes" toencourage him to continue.Adopts the speaker's point of view. You will understand and rememberthe speaker's points most effectively if you try to see things from her point ofview, at least initially. Try to listen, not to interrupt, finish sentences, or rushthe speaker. Most of all, try to suppress your initial reactions and to hear andunderstand the speaker's perspective. Try to listen and respond from thespeaker's frame of reference, not your own. Listen for her feelings, not just herwords. Try t o e mpathize with her position. Depending on the context andpurpose of your communication, you may later shft m odes into a discussion inwhich y ou also present your own point of view, but to b e a good active listener,you shouldn't do t hat until you thoroughly understand the speaker's point ofview.Clarifies the speaker's thoughts and feelings. You will listen better ifyou are not talking too much yourself. When you are in active listening mode,limit your tal k i ng to t hings that will contribute to getting the fullest informationaland emotional content from the s peaker. Avoid inserting your own marginallyrelated experiences and minimize inte rru ptions. When the speaker pauses, askopen-ended questions (e.g., "How do y ou feel about X?" "Tell me about X.""What concerns you about X?") rather than questions that can be answered in asingle word or phrase ("Are you satisfied with X?" "Is X on schedule?'). Usereflective response techniques (described in more detail below) to check theaccuracy of your understanding of the speaker's ideas and especial1y feelings(e.g., "So you are frustrated at your inability to progress on X?").You may adapt active listening techniques to different types of listening, with your role ranging from minimal intervention to m ore active solicitation and even into giving advice, depending on your needs.Reflective ResponsesReflection, or reflective response technique, borrowed from certain types of counseling tech ni ques,i s d esigned to e licit as f ull a sense as possible of the speaker's thoughts and especially feelings. It is a way of helping someone explore her own personal meanings. This technique involves reflecting back to the speaker what you believe she has said in order to v erify (or clarify) your understanding and to encourage the speakerto c ontinue elaborating on her point of view.*An active listener is already using aspects of this technique, but reflection requ i res tak in g even greater care in the following areas:Reflect the speaker's thoughts and feelings. Restate what you believethe speaker has said to check for the accuracy of your understanding (e.g., "Soyou couldn't finish the assignment on time." "Then you think the time allottedwas inadequate?"). Even more importantly, reflect back the speaker's feelingsas you have heard or inferred them (e.g., "You seem to feel anxious becauseyou couldn't finish the assignment on time."). This interpretation of feelings is,of course, more tricky in that it often requires you to read between the lines, toinfer feelings underlying what has been said (e.g., "You seem angry about thereorganization," rather than "So the department was reorganized."). Thus youmay want to use wording or voice tone make your inferences into q uestions,rather than statements (e.g., "So you feel anxious because you couldn't finishthe assignment on time?" "Do you feel anxious beca u se you couldn't finish theassignment on time?").Respond rather than lead the conversation. Let the speaker's thoughts'and feelings be your guide in the conversation. Don't guide the conversation by asking questions or interjecting ideas or suggestions that take the speaker into*This description of the technique is based on Chapters 9 and 10 of Interpersonal Behavio r: Communication and Understanding in Relationships,by Anthony G. Athos and John J. Gabarro (Prentice Hall).new areas of interest to you (e.g., "Have you thought about X?" "Maybe youshould ask about Y ."). Instead, respond to and reflect back w hat he actuallysaid or what you sense is implied by what he said (e.g., "So you feel t ra ppedby this project?"). Try to stay wi t h i n t he speaker's frame of reference, ratherthan asking questions or making suggestions that come from your own frame ofreference. Even asking "Why?" may distract the speaker from one line ofthoughts or feelings into defending and justifying feelings or actions.Respond to feelings, rather than content. As suggested earlier, feelingsare generally a better indication of personal meanings than content is. T hus youwill help the speaker's self-exploration more by responding to her feelings(e.g., "So you resent Susan's frequent absences?") than to the content (e.g.,"How often was Susan out of the office?") There is a corollary to thisguideline: You can get at a person's feelings better by responding to the morepersonal aspects of what she says. Whenever possible, choose the specific,personal points (e.g., "So your subordinate, John, tends to challenge you infront of others.") rather than the abstract generalizations (e.g., "So the firm'shierarchy is weak ") to respond to and reflect back at the speaker. Thisencourages her to explore those personal feelings more thoroughly and to makeclear her own assumptions.Reflective vs. Directive ResponsesObviously, this reflective t ech ni que is not always appropriate to the circumstances a nd to your needs or purposes. At times you may want to be more directive and lessref e ctive in your interactions. You may want to argue, advise, or confront. Thus once you have learned to use the reflective mode of listening, you need to consider when to use it, when to sh i ft f rom that mode to a more directive mode, and when not to be reflective at all. Here are some reasons and times for using this reflective technique:When you need or want to understand the other person's feelings morecompletelyWhen you sense that the other person has not yet revealed his thoughts andfeelings about the situationWhen you sense that the other person is not sure of his true feelingsThus active listening with reflective responses is often the first stage of an interaction. Then, once you feel you really understand the person's perspective, you can switch to a more directive or confrontational or persuasive stance. Here, you can lead as well as respond and speak from your own frame of reference as well as the other person's.ConclusionListening is a critical communication skill for managers and consultants, as well as f or all of us in our personal lives. Advising someone well on a career, personal, or organizational issue requires that you understand that person's point of view. Youcan't negotiate effectively until you understand what the other person wants. Effective persuasion depends on a clear understanding o f the other person's perspective. In a llof these situations, active listening, often beginning with reflective responses, is crucial to achieving your ultimate communication objectives.Active listening is a skill that, like other communication skills, must be developed. It does not come naturally to most of us. Practicing active listening and particularly reflective responses can feel artificial when these skills are isolated from a real communication need. Still, only by practicing can you develop these skills and then integrate them with your other communication s ki lls.。