[ccp4bb] PhD Position in Structural Biology

[Clemens Grimm]

A PhD position in structural biology is available in the group of Prof. Utz
Fischer at the Institute of Biochemistry, Biocentre of the Julius
Maximilans-University, Wuerzburg/Germany.

The successful applicant will work on the crystallographic structure
determination of the Survival Motor Neuron (SMN) complex, the key player during
the pathogenesis of Spinal Muscular Atrophy (SMA). Protocols for bacterial
expression and reconstitution of the SMN complex are established and crystals
including a first synchrotron dataset are already available.

Please send your application including a letter of interest, curriculum vitae,
academic certificates and the names and contact details of two referees to:




Dr. Clemens Grimm
Institute for Biochemistry
Biocentre of the University
Am Hubland
D-97074 Wuerzburg
Germany

e-mail: [EMAIL PROTECTED]

[ccp4bb] PhD Opportunities in Structural Biology

[Wojtek Rypniewski]

Exciting PhD Programme in Structural Biology

The Institute of Bioorganic Chemistry of the Polish Academy of Sciences
(IChB PAN) in Poznan, in cooperation with the Max Planck Institute
for Plant Breeding Research (MPIZ) in Cologne, announce the opening
of an International PhD Programme in "Structural biology of plants
and microbes". The research projects will focus on the application
of protein engineering and protein crystallography to the study of 
structural

aspects of plant biology and of host-microbe interactions. The programme
is supported by the Foundation for Polish Science through structural funds
of the European Union. The monthly fellowship is equivalent to about
1000 euros.

Successful applicants will be enrolled in a four-to-five-year programme,
expected to conclude with the presentation and public defence
of a PhD thesis, for obtaining a doctoral degree in chemistry
or biochemistry from the Institute of Bioorganic Chemistry, Polish
Academy of Sciences. The programme is open to all candidates who have
a university MSc degree (or equivalent) in chemistry, biology, or a related
field. Candidate selection will be conducted according to the regulations
for the Graduate School of IChB PAN and will include an oral examination
in chemistry or biochemistry. The entire program is in English.

The recruitment will be conducted in two rounds, with the following
application deadlines: September 30, 2008 and July 18, 2009. Successful
candidates are expected to start the programme on November 1, 2008
and September 1, 2009. For more information about the application 
procedure,

please visit

http://www.man.poznan.pl/CBB/MPD

or contact Dr. Anna Urbanowicz ([EMAIL PROTECTED]);
Institute of Bioorganic Chemistry, Polish Academy of Sciences,
Noskowskiego 12/14, 61-704 Poznan, Poland).

The Institute of Bioorganic Chemistry, Polish Academy of Sciences,
is an equal opportunity employer and encourages applications
from disabled persons.

[ccp4bb] Protein Color

[Matthew Alan Bratkowski]

Hello.

I am working with a protein that turns a yellowish-brown color when it is
concentrated to around 2 mg/ml or higher in a small volume (a few hundred
uL).  I was wondering if the protein bound a metal or other prosthetic
group that would give it this color?  The protein's color somewhat
resembles iron binding proteins, but there is no peak in the 400 nm range
that would suggest heme, and an iron sulfur cluster is not that likely
since there are only five cysteines in the protein.  Proteins with
structures homologous to the one I am studying bind magnesium, but are not
know to bind other metals.  Any information about what this color might
suggest about the protein or how I could analyze possible bound metals or
prosthetic groups using only a small amount of protein would be helpful.

Matt

Re: [ccp4bb] Protein color

[Palm]

Hi Matt, 
to check, if the color comes from a metal ion, you can get an AAS 
analysis done. You need something in the range of 1 mg of well dialyzed 
protein (metal free buffer!). We got an analysis done for the most 
important metals (Mn, Fe, Co, Ni, Cu, Zn) for ca. 20 Eur per element 
from a local analytic company. 
greetings
  Gottfried


Dr. Gottfried Palm
Ernst-Moritz-Arndt-Universität 
Inst. für Biochemie (MNF)
Abt. Biochemie I
Felix-Hausdorff-Straße 4
17489 Greifswald

Tel. +49(03834)-864393 Fax. +49(03834)-864373
email [EMAIL PROTECTED]




===
WEB-Mailer der Uni-Greifswald ( http://www.uni-greifswald.de/ )
===

Re: [ccp4bb] Protein Color

[Nathaniel Echols]

>
> I am working with a protein that turns a yellowish-brown color when it is
> concentrated to around 2 mg/ml or higher in a small volume (a few hundred
> uL).  I was wondering if the protein bound a metal or other prosthetic
> group that would give it this color?  The protein's color somewhat
> resembles iron binding proteins, but there is no peak in the 400 nm range
> that would suggest heme, and an iron sulfur cluster is not that likely
> since there are only five cysteines in the protein.  Proteins with
> structures homologous to the one I am studying bind magnesium, but are not
> know to bind other metals.


This does not rule out other metals, though.  For instance, protein kinases
in eukaryotes always use magnesium, but the ones in TB - which are nearly
identical in global structure (with > 30% sequence identity) and appear to
be enzymatically similar - use manganese instead, as do several other TB
homologs of magnesium-binding proteins.  This is a quirk of TB's physiology
but there are certainly other protein families (glyoxalases, dioxygenases)
that vary in metal preference depending on organism and substrate
preference.  I don't know if Mn would cause that color (although some Mn
compounds are brown or orange-brown); however, there are many ligands that
could.  Heme will also change color depending on iron oxidation state, and
possibly local chemical environment.  Endogenously purified full-length E.
coli catalase, for instance, is green, but a common proteolytic fragment
(from the same prep) is yellow-brown.

Any information about what this color might
> suggest about the protein or how I could analyze possible bound metals or
> prosthetic groups using only a small amount of protein would be helpful.
>

Mass spec, obviously; if it's a metal, ICP-AES may be more effective, but
I'm not sure how much protein you need.  You can also use X-ray fluorescence
to identify metals, but as far as I know this requires a synchrotron.
 Sometimes the visual light spectrum alone is enough to tell you what's
there, but it can be difficult to cross-reference this with published data
since (AFAIK) there is no online database of spectra.

Re: [ccp4bb] Protein Color

[Roger Rowlett]

Matthew Alan Bratkowski wrote:

Hello.

I am working with a protein that turns a yellowish-brown color when it is
concentrated to around 2 mg/ml or higher in a small volume (a few hundred
uL).  I was wondering if the protein bound a metal or other prosthetic
group that would give it this color?  The protein's color somewhat
resembles iron binding proteins, but there is no peak in the 400 nm range
that would suggest heme, and an iron sulfur cluster is not that likely
since there are only five cysteines in the protein.  Proteins with
structures homologous to the one I am studying bind magnesium, but are not
know to bind other metals.  Any information about what this color might
suggest about the protein or how I could analyze possible bound metals or
prosthetic groups using only a small amount of protein would be helpful.

Matt
  
Many proteins will turn yellowish if concentrated enough, due to the UV 
absorption tail. However, it usually takes >>10 mg/mL of homogeneous, 
cofactor-free protein to see this, so your supposition there may be a 
cofactor or metal involved warrants further investigation. The most 
straightforward way to analyze proteins for metal is to use ICP-OES, and 
it will not require a large amount of protein to do this. We routinely 
quantify zinc-metalloenzymes (and metal-substituted enzymes) using 
ICP-OES. For most first-row transition elements, ICP-OES sensitivies are 
such that you can get reasonable signal for 0.1 ppm (100 ppb) metal ion. 
We normally take protein that is 100-500 uM and dilute it in 
high-quality (18 Mohm/cm2) deionized water 30-100X, depending on the 
starting protein concentration. Using our ICP-OES (Perkin-Elmer 3000) we 
can get very reasonable readings from 1.5 mL of diluted solution (2 
replicates). If you can spare more protein, you can get more accuracy 
and S/N. Calibration can be done with commercial standards diluted to 1 
ppm. We use a mixed metal standard so we can screen for many metals at 
once. Dilution of your sample is important, as large viscosity 
differences between samples will dramatically affect the nebulizer 
efficiency of the ICP, and your results. In practice, we have found that 
a dilution of 20X or more is usually sufficient to make the viscosity 
differences between the calibration standard and the sample negligible 
in practice. (If you are a real stickler, you should do a standard 
addition to a diluted protein sample, but this is not usually necessary, 
and consumes double the protein.)


You should be aware that all dilution operations for preparing samples 
should be carried out in glass, preferably acid-washed glass, and NOT in 
plastic tubes. Samples should be analyzed immediately. Glass can slowly 
leach metal ions into your sample, including Fe and the ubiquitious Zn. 
Plastic is even worse. At  low protein concentrations, plastic eppendorf 
tubes can rapidly and irreversibly adsorb some of all of your sample. In 
addition, nearly all plasticware notoriously leaches Fe into the 
solution over time, and is thus especially unsuitable for this metal. 
Even clean, non-acid-washed glass is better than plastic. Dialysis of 
your protein against metal-free buffer or distilled water (if it can 
tolerate it) may be required to remove adventitious metal ions. 
(However, loosely protein-bound metals may be removed by this process, 
too.) Running the dialysis buffer through the ICP at the same dilution 
as the sample is advised to assess your background level of contamination.


Cheers,


--

Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [EMAIL PROTECTED]

Re: [ccp4bb] Protein Color

[Kornelius Zeth]

you can do a simple wavelength scan at the synchrotron of the protein solution 
frozen in a loop.

Best wishes

Kornelius

On Fri, 5 Sep 2008 12:21:29 -0400
 Matthew Alan Bratkowski <[EMAIL PROTECTED]> wrote:
> Hello.
> 
> I am working with a protein that turns a yellowish-brown color when it is
> concentrated to around 2 mg/ml or higher in a small volume (a few hundred
> uL).  I was wondering if the protein bound a metal or other prosthetic
> group that would give it this color?  The protein's color somewhat
> resembles iron binding proteins, but there is no peak in the 400 nm range
> that would suggest heme, and an iron sulfur cluster is not that likely
> since there are only five cysteines in the protein.  Proteins with
> structures homologous to the one I am studying bind magnesium, but are not
> know to bind other metals.  Any information about what this color might
> suggest about the protein or how I could analyze possible bound metals or
> prosthetic groups using only a small amount of protein would be helpful.
> 
> Matt

 --
 Kornelius Zeth
 Max Planck Institute for Developmental Biology
 Dept. Protein Evolution
 Spemannstr. 35
 72076 Tuebingen, Germany
 [EMAIL PROTECTED]
 Tel -49 7071 601 323
 Fax -49 7071 601 349

[ccp4bb] pKa for protein C-terminus?

[Patrick Loll]

What value do we expect for the pKa of a protein's C-terminal  
carboxylate?  pKa values for free amino acids are quite low (2-3),  
but it seems to me that this may have something to do with the  
proximity of a free amine group; I'd expect a higher value (4-ish?)  
for the peptide's C-terminus.


A quick & lazy search using the term "pka" was stymied by a gazillion  
references to protein kinase A...


Thanks,
Pat


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry & Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

Re: [ccp4bb] pKa for protein C-terminus?

[Roger Rowlett]

You are correct. See */Protein Sci/* Thurlkill et al. 15 (5): 1214, 
http://www.proteinscience.org/cgi/reprint/15/5/1214, which is a recent 
effort to codify "typical" pKa values for protein ionizable groups.


Cheers,

--

Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [EMAIL PROTECTED]
Patrick Loll wrote:
What value do we expect for the pKa of a protein's C-terminal 
carboxylate?  pKa values for free amino acids are quite low (2-3), but 
it seems to me that this may have something to do with the proximity 
of a free amine group; I'd expect a higher value (4-ish?) for the 
peptide's C-terminus.


A quick & lazy search using the term "pka" was stymied by a gazillion 
references to protein kinase A...


Thanks,
Pat


---

Patrick J. Loll, Ph. D.  


Professor of Biochemistry & Molecular Biology

Director, Biochemistry Graduate Program

Drexel University College of Medicine

Room 10-102 New College Building

245 N. 15th St., Mailstop 497

Philadelphia, PA  19102-1192  USA


(215) 762-7706

[EMAIL PROTECTED]