BioMoo Held on the 13th May

ClareS turns the ClareS_recorder on.

ClareS says "Hi, welcome to our BioMOO meeting on secondary structure of proteins..."

ClareS [to Tobias] would you mind repeating your question (you can cut & paste) so that it's on the record?#

Tobias says "How is it possible to determin the elements of the secondary"

Tobias says "How is it possible to determin the elements of the secondary strucutre (sheets, etc.) "

Tobias says "By ciricular dichroism spectroscopy. Because the optical rotation is indeucd by every amino acid"

Ajj materializes out of thin air.

Tobias says "How do you determin which part of the messured optical rotation is caused by secondary structuer elements and which is by asymmetrical C-atoms ?"

ClareS must warn everyone that this isn't exactly her field, but will try...

Tobias says "sorry the copy / paste command somehow didnot work properly"

ClareS [to ajj] welcome - we're discussing circular dichroism

ClareS says "each amino acid will absorb circularly polarised light slightly differently depending on its environment..."

ClareS says "each class of secondary structure (helix, sheet and turn) has a different and characteristic pattern of CD absorbance"

ClareS says "& the absorbances characteristic of secondary structure patterns are in a particular region of the ultra-violet, different from those characteristic of amino acid side chains"

ClareS says "in the course material ( it is instructive to compare figures 22 & 23"

ClareS says "you will see that the spectrum for an all-alpha protein like myoglobin is very similar to that expected for "pure" alpha-helical structures"

ClareS says "in fact, CD is particularly reliable for predicting alpha-helical proteins"

ClareS says "does this make more sense to you?"

Tobias says "yes, this makles it clear"

ClareS says "good"

ClareS is learning something new from the course material on CD as well

Ajj says "on an unknown protein how can you deconvolute the contributions of the different secondary structural components"

Ajj says "Let's say from a complex spectrum?"

ClareS [to ajj] you mean a CD spectrum?

ClareS says "it's difficult, if not impossible, to determine the *fold* of a protein from a CD spectrum"

Ajj says "What i mean is: Suppose your protein has alpha helicies, beta sheets and othr components and it gives a complex CD spectrum How han you identify the contribution from each"

ClareS says "what you should be able to do, quite easily (given accurate experimentation) is determine the class of secondary structure present - mostly alpha, mostly beta or mixed"

Pawel [guest] says "Are there some numerical parameters of CD spectrum ? I hope that these parameters might predict eg. the percent of alpha helical structure."

Ajj says "So you couldn't subtract out the alpha and see what's left?"

ClareS says "you can, of course, compare the CD spectrum of your protein with that if proteins of known folds"

ClareS says "the reference cited in the course material for a statistical method of determining contributions is:0"

ClareS [to Manalavan] P. & Johnson, W. C. jr. Variable Selection Method Improves Prediction of Protein Secondary Structure from Circular Dichroism Apectra. Anal. Biochem. 167, 76-85.

ClareS . o O ( that's Manalavan, P. etc... the perils of cut & paste ;) )

ClareS says "(and of course it's CD Spectra not Apectra at the end)"

Ajj says "Thank you, Clare"

ClareS says "unfortunately Anal. Biochem. is not a very common journal"

Ajj says "In the US it's pretty popular"

ClareS looks at that reference & discovers the year is missing

ClareS . o O ( 1987 )

Ajj says "I think there is enough information to get it. Thnks"

ClareS consults BIDS

ClareS has found it, but there's no abstract

ClareS says "CD is extremely useful for studying secondary structure transitions, particularly of peptides"

ClareS looks round expectantly

ClareS wonders if there are any other questions

Pawel [guest] says "Can we say something about a parameter I often use ?"

ClareS [to Pawel] go on...

Pawel [guest] says "I think about a HPLC index."

(Tobias has disconnected.)

(Tobias has connected.)

ClareS [to Pawel] can you say more about it, please?

Tobias says "Sorry, my connection broke"

Pawel [guest] says "this parameter I calculate in many programs (eg. MacBioSpec) trying to predict the retention of my peptides from C18 column"

Pawel [guest] says "and I observed thet ALWAYS the calculated HPLC index is too big"

ClareS says "I'm no HPLC expert, I'm afraid... has anyone else here had the same problem?"

Ajj says "Well we do some Hplc but have not used that parameter. is it based on the hydrophobic indexbaseed on"

ClareS says "how is the index calculated?"

Pawel [guest] says "Yes, the HPLC index is strictly connected with the hydrophobic properties of the peptide"

ClareS [to Pawel] do you know what hydrophobicity scale is used (I presume the h'phobic properties are just calculated from the sequence)?

ClareS says "it may be that the scale you are using is inappropriate for your peptides"

Pawel [guest] says "The program that counts HPLC index uses almost all known hydrophobicity scales byt I observed that the result varies only slightly"

ClareS says "I presume that it calculates a separate result using each hydrophobicity scale - is that right? - and they all give values that are too high?"

Pawel [guest] says "It is surprise for my, because I thought that the program can well predict the hydrophobic properties of the peptides I use"

ClareS says "do your peptides have unusual sequences?"

Pawel [guest] says "-- there are short peptides - about 10 a.a. and are from alpha-helix-rich proteins"

Pawel [guest] says "eg. myoglobin or hemoglobin"

Ajj says "Could it be that they are very amphpathic?"

Pawel [guest] says "No"

Ajj says "helically that is"

ClareS says "would you be able to list one of your sequences, or is it confidential?"

Pawel [guest] says "Moment, please..."


ClareS can't see anything particularly unusual just by looking at them

ClareS . o O ( they're certainly not very hydrophobic - or hydrophilic - or amphipathic )

Pawel [guest] says "There are fragments of alpha globin"

ClareS says "are they predicted to be alpha-helical?"

Pawel [guest] says "No, there are the fragments of alpha-helical domains"

Ajj says "are you sure they are not dimerizing?"

ClareS says "I've got the PredictProtein server up on the web: would it be worthwhile my running one of your sequences through it to see what comes up?"

Ajj says "are you sure they are not dimerizing?"

Pawel [guest] says "YES !!!! The dimerization is my often problem"

Jim Pitts finds his way in.

Jim Pitts says "Hi, I am sorry I was called away"

Ajj says "the D and K could be forming ionic bonds and holding them so that these charges cancel "

Ajj says "making the dimer more hydrophobic as a unit."

Pawel [guest] says "Yes, it is interesting idea..."

ClareS says "certainly interesting..."

Ajj says "i've forund this to be true with some of the peptides we have been working with"

Ajj says "it really changes their biological activity"

Pawel [guest] says "but, in other way, the drastic conditions of HPLC should brake these bonds..."

Ajj says "completely"

Ajj says "i'm not so sure"

Jim Pitts [to ClareS] sorry a student came to see me earlier than he should

Pawel [guest] says "and I should not have the problems"

ClareS says "and in VGAHAGEYGAEALER one E is about one turn away from the R: might it not even be possible for these two residues to form a salt bridge together, "cancelling" the charges?"

Ajj says "you should try to separate them by dtemperature and see if the retention shanges after high temperature treatment or ho"

Ajj says "sorry"

Pawel [guest] says "heat the sample and then resolve on HPLC ???"

Ajj says "you should try to separate them by size exclusion perhaps and see if their elution changes with high ionic strength or temperature."

Pawel [guest] says "Hmmmm...."

Ajj says "i'm sorry but i have to go to work now i'll be back at tonight's session"

Ajj says "bye folks"

(Ajj has disconnected.)

ClareS will have another meeting to go to soon

Jim Pitts [to ClareS] I hope so, 22.00?, If i do not fall asleep first

ClareS says "so thank you all for your valuable contributions"

JudyH says "I also make peptides and purify them by hplc; I have not used"

ClareS says "there will be another meeting on this subject tonight, 22:00GMT (23:00BST)"

ClareS says "you're all very welcome to carry on discussing HPLC - it's what BioMOO is for ;) - but I shall be turning the recorder off very soon"

Pawel [guest] says "to JudyH have you some problems ?"

Tobias says "One ,last question. Is itin NMR only possible to determin secondary strucutre via the 'messurement ' of the torsion angels or are there other possibilities"

Tobias says "I meant the Karplus relationship"

JudyH says "sorry hit return by mistake! I was going to say that a lot of factors besides hydrophobicity affect retention time"

JudyH says "eg size, make of column and so a program may not be able to predict very accurately for any peptide,"

ClareS [to Tobias] you can calculate secondary structures, and localise them to pieces of the polypeptide chain, using NMR without calculating a full 3D structure

ClareS says "see"

ClareS [to pavel] have you tried running a set of peptide "standards" through your column to see whether the hydrophobicity values are being calculated wrong for *all* peptides?

The housekeeper arrives to cart ajj off to bed.

Pawel [guest] says "to ClareS - yes,of course !"

ClareS [to Pawel] and what was the result?

Pawel [guest] says "I can generalize:"

Pawel [guest] says "the HPLC index predict rather the order of the peptides retention"

Pawel [guest] says "but the JudyH is right - there are many factors that can affect the practical retention times"

Tobias says "OK, Goodbye everyone"

(Tobias has disconnected.)

ClareS will turn the recorder off - thanks for such a lively discussion

ClareS turns the ClareS_recorder off.