Re: [ccp4bb] Looking for proteins for undergraduate biochemistry lab

[James Holton]

What about proteins where the crystals don't diffract well?  I've found 
in my teaching experience that students take your advice much more 
seriously when they see it work on 3.5A data than they do when they see 
it work on 1.5 A data.


-James Holton
MAD Scientist

On 6/17/2021 7:40 AM, Tanner, John J. wrote:


We developed a senior undergraduate biochemistry lab around an acid 
phosphatase from Francisella tularensis (FtHAP).


https://pubmed.ncbi.nlm.nih.gov/27980518/ 



The enzyme is easy to purify and crystallize, and the crystals 
diffract well. L-tartrate and phosphate ion are inexpensive 
inhibitors, which can be included in crystallization (3IT0/1). We have 
the students grow crystals and collect X-ray diffraction data 
remotely. Then they view maps in Coot. The enzyme assay is a simple 
colorimetric test with p-nitrophenylphosphate as the substrate.


--

John J. Tanner

Professor of Biochemistry and Chemistry

Associate Chair of Biochemistry

Department of Biochemistry

University of Missouri
117 Schweitzer Hall

503 S College Avenue
Columbia, MO 65211
Phone: 573-884-1280

Email: tanne...@missouri.edu 
https://cafnrfaculty.missouri.edu/tannerlab/ 



Lab: Schlundt Annex rooms 3,6,9, 203B, 203C

Office: Schlundt Annex 203A

*From: *CCP4 bulletin board  on behalf of P. H 


*Date: *Wednesday, June 16, 2021 at 5:19 PM
*To: *CCP4BB@JISCMAIL.AC.UK 
*Subject: *[ccp4bb] Looking for proteins for undergraduate 
biochemistry lab


*WARNING:*This message has originated from an External Source. This 
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Hello All,

We are looking for some candidate proteins for an undergraduate level 
advanced biochemistry lab. They should be expressed in bacteria, 
simple enough to purify and it will be nice to perform some simple 
characterization experiments(binding assays, enzymatic assays).


Any suggestions?

Thank you in advance.

Prerna gupta



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[ccp4bb] Postdoctoral fellowship available to study the structure and evolution of viral proteins at Linköping University, Sweden

[Eleonore von Castelmur]

Dear colleagues,

I have an opening for a postdoctoral fellowship in my group at Linköping 
university, Sweden. The aim of the project is to study the evolutionary 
relationship and structural and functional repurposing of human proteins 
acquired by picornaviruses, building on research already ongoing in the lab. 
Please see the attached document for more details and feel free to spread it to 
anyone you think might be interested and suitable for the position. The 
deadline for applications is 31st of July 2021.

Best wishes,
Eleonore

Eleonore von Castelmur, PhD
Assistant Professor
Wallenberg Center for Molecular Medicine
Department of Physics, Chemistry and Biology (IFM)
Linköping University
SE-581 83 Linköping
phone: +46 13 28 19 66
e-mail: eleonore.von.castel...@liu.se





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postdoctoral_fellowship_2021.pdf
Description: postdoctoral_fellowship_2021.pdf

Re: [ccp4bb] Enzyme Vmax and Km

[Artem Evdokimov]

Is your enzyme monomeric or multimeric?

What time exactly does it mean when you say unprocessed substrate versus
processed?

What are your error margins (no error bars evident on your plots)?
Depending on your errors, your second plot is not a 'steep decline' but
instead could be a constant line.

So, if your affinity for processed substrate is higher, it does not have to
mean that catalytic efficiency will also be higher
 For example RNA polymerase have to eventually let go of the promoter
region in order to elongate, but if the promoter binds too tightly they
will instead abort translation and fall off. Nature is complicated :)

In general it would help us a lot to help you if we understand the details
of your issue.

There is also a common issue that can be encountered with weakly associated
multimeric enzymes that act on polymeric substrate (in your case, DNA) -
namely when you add more substrate than enzyme the monomers of enzyme can
become 'smeared' along the DNA and this results in very odd kinetic
behavior.

Artem


On Fri, Jun 18, 2021, 12:05 AM Prem Prakash  wrote:

> Dear all,
> Sorry for this off topic. I am working on an enzyme that has an
> exonuclease activity. The enzyme preferentially cleaves an unprocessed
> substrate at a faster rate than the processed one (known by qualitative
> analysis). Recently, I calculated the Vmax, Km and kcat of the enzyme for
> unprocessed substrate which are 18.2 pmol/min, 182 nM and 7.1 sec-1
> respectively. However, the Processed substrate has apparently a lower range
> of Km (not calculated) as reflected from the curve (because the same
> increasing concentration range which is used for unprocessed, shows a steep
> decline in the initial velocity of the enzyme with processed substrate.
> The latter suggests that Km is way lower than expected. In this case, the
> question is, if the Km of processed substrate is way lower than the
> Unprocessed, how can we see a faster rate with the former substrate than
> later. i.e lower Km and slower rate of cleavage. If it's possible please
> give some insights. I have attached the plot comparison between two kinetic
> assays.
>
> With kind regards,
>
> Prem
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>



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Re: [ccp4bb] Enzyme Vmax and Km

[Jon Cooper]

Hello, you could also try:

http://www.ic50.tk/kmvmax.html

Or

http://ic50.org/kmvmax.html

The former has an exponential decay thing that might help with your substrate 
inhibition and other things if you have biphasic behaviour ;-0

Cheers, Jon.C.

Sent from ProtonMail mobile

 Original Message 
On 18 Jun 2021, 05:04, Prem Prakash wrote:

> Dear all,
> Sorry for this off topic. I am working on an enzyme that has an exonuclease 
> activity. The enzyme preferentially cleaves an unprocessed substrate at a 
> faster rate than the processed one (known by qualitative analysis). Recently, 
> I calculated the Vmax, Km and kcat of the enzyme for unprocessed substrate 
> which are 18.2 pmol/min, 182 nM and 7.1 sec-1 respectively. However, the 
> Processed substrate has apparently a lower range of Km (not calculated) as 
> reflected from the curve (because the same increasing concentration range 
> which is used for unprocessed, shows a steep decline in the initial velocity 
> of the enzyme with processed substrate.
> The latter suggests that Km is way lower than expected. In this case, the 
> question is, if the Km of processed substrate is way lower than the 
> Unprocessed, how can we see a faster rate with the former substrate than 
> later. i.e lower Km and slower rate of cleavage. If it's possible please give 
> some insights. I have attached the plot comparison between two kinetic assays.
>
> With kind regards,
>
> Prem
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1



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Re: [ccp4bb] Enzyme Vmax and Km

[Roger Rowlett]

For sure, you will need to collect data at lower substrate concentration to
determine what is going on for processed substrate. One should also
consider how the "initial rates" are measured. Ideally, the amount of
substrate depleted during the initial rate measurement should be less that
5%. When it is larger than this, or when the product is a strong inhibitor
of the enzyme, complicated or even artifactual results can be obtained. We
have seen similar, time-dependent kinetic behavior for an enzyme that is
strongly allosterically inhibited by its product. For complex kinetic
behavior, it may be necessary to have a finer grid of measurements to allow
for accurate data fitting.

As others have pointed out, linear transforms of the Michaelis-Menten
equation are statistically flawed without applying proper y-weighting. It
is much better to do nonlinear least squares fiting of rate vs. substrate
data. For true product inhibition, there are several possible models that
could be considered, based on the behavior of the data. Consultation with
an enzyme kineticist might be warranted for for complex behavior.

Roger Rowlett


On Fri, Jun 18, 2021, 3:53 AM Harmer, Nicholas 
wrote:

> Dear Prem,
>
>
>
> I agree entirely with Tristan’s conclusion that the processed substrate is
> also acting as an inhibitor at higher concentrations. You would need to run
> an experiment with a wider range of concentrations used (especially lower
> concentrations) to get a better feel for the range of the reaction. There
> is a well described substrate inhibition equation (see e.g.
> https://www.graphpad.com/guides/prism/latest/curve-fitting/reg_substrate_inhibition.htm)
> that you can try. I have a recent chapter on experiment design covering
> such cases (
> https://www.researchgate.net/publication/331806855_Reaction_Chemical_Kinetics_in_Biology)
> that I can share with you if you need.
>
>
>
> I would strongly recommend to avoid the Lineweaver-Burk plot to calculate
> your Km and kcat. There are known issues with this (especially, as in your
> image, overweighting the lowest rate and usually least accurate point).
> Better is to fit directly to the equation with non-linear fitting, for
> example in *R*. This will also give you a better estimate of the error
> and confidence intervals.
>
>
>
> Hope this helps,
>
>
>
> Nic
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Tristan
> Croll
> *Sent:* 18 June 2021 08:19
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Enzyme Vmax and Km
>
>
>
> Hi Prem,
>
>
>
> The immediate problem here is that the curve for the processed substrate
> simply cannot be described by simple Michaelis-Menten kinetics. Assuming
> the assay has worked as expected, the declining rate with increasing
> substrate concentration suggests to me that this substrate also acts as an
> allosteric inhibitor, so assays at high [substrate] will make it *look*
> like the unprocessed substrate is preferred even though the processed one
> is cleaved faster by the uninhibited enzyme.
>
>
>
> Hope this helps,
>
> Tristan
>
>
> On 18 Jun 2021, at 05:05, Prem Prakash  wrote:
>
> Dear all,
>
> Sorry for this off topic. I am working on an enzyme that has an
> exonuclease activity. The enzyme preferentially cleaves an unprocessed
> substrate at a faster rate than the processed one (known by qualitative
> analysis). Recently, I calculated the Vmax, Km and kcat of the enzyme for
> unprocessed substrate which are 18.2 pmol/min, 182 nM and 7.1 sec-1
> respectively. However, the Processed substrate has apparently a lower range
> of Km (not calculated) as reflected from the curve (because the same
> increasing concentration range which is used for unprocessed, shows a steep
> decline in the initial velocity of the enzyme with processed substrate.
>
> The latter suggests that Km is way lower than expected. In this case, the
> question is, if the Km of processed substrate is way lower than the
> Unprocessed, how can we see a faster rate with the former substrate than
> later. i.e lower Km and slower rate of cleavage. If it's possible please
> give some insights. I have attached the plot comparison between two kinetic
> assays.
>
>
>
> With kind regards,
>
>
>
> Prem
>
>
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
> 
>
> 
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe

Re: [ccp4bb] Enzyme Vmax and Km

[Harmer, Nicholas]

Dear Prem,

I agree entirely with Tristan's conclusion that the processed substrate is also 
acting as an inhibitor at higher concentrations. You would need to run an 
experiment with a wider range of concentrations used (especially lower 
concentrations) to get a better feel for the range of the reaction. There is a 
well described substrate inhibition equation (see e.g. 
https://www.graphpad.com/guides/prism/latest/curve-fitting/reg_substrate_inhibition.htm)
 that you can try. I have a recent chapter on experiment design covering such 
cases 
(https://www.researchgate.net/publication/331806855_Reaction_Chemical_Kinetics_in_Biology)
 that I can share with you if you need.

I would strongly recommend to avoid the Lineweaver-Burk plot to calculate your 
Km and kcat. There are known issues with this (especially, as in your image, 
overweighting the lowest rate and usually least accurate point). Better is to 
fit directly to the equation with non-linear fitting, for example in R. This 
will also give you a better estimate of the error and confidence intervals.

Hope this helps,

Nic

From: CCP4 bulletin board  On Behalf Of Tristan Croll
Sent: 18 June 2021 08:19
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Enzyme Vmax and Km

Hi Prem,

The immediate problem here is that the curve for the processed substrate simply 
cannot be described by simple Michaelis-Menten kinetics. Assuming the assay has 
worked as expected, the declining rate with increasing substrate concentration 
suggests to me that this substrate also acts as an allosteric inhibitor, so 
assays at high [substrate] will make it *look* like the unprocessed substrate 
is preferred even though the processed one is cleaved faster by the uninhibited 
enzyme.

Hope this helps,
Tristan

On 18 Jun 2021, at 05:05, Prem Prakash 
mailto:prem...@gmail.com>> wrote:
Dear all,
Sorry for this off topic. I am working on an enzyme that has an exonuclease 
activity. The enzyme preferentially cleaves an unprocessed substrate at a 
faster rate than the processed one (known by qualitative analysis). Recently, I 
calculated the Vmax, Km and kcat of the enzyme for unprocessed substrate which 
are 18.2 pmol/min, 182 nM and 7.1 sec-1 respectively. However, the Processed 
substrate has apparently a lower range of Km (not calculated) as reflected from 
the curve (because the same increasing concentration range which is used for 
unprocessed, shows a steep decline in the initial velocity of the enzyme with 
processed substrate.
The latter suggests that Km is way lower than expected. In this case, the 
question is, if the Km of processed substrate is way lower than the 
Unprocessed, how can we see a faster rate with the former substrate than later. 
i.e lower Km and slower rate of cleavage. If it's possible please give some 
insights. I have attached the plot comparison between two kinetic assays.

With kind regards,

Prem





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Re: [ccp4bb] Enzyme Vmax and Km

[Tristan Croll]

Hi Prem,

The immediate problem here is that the curve for the processed substrate simply 
cannot be described by simple Michaelis-Menten kinetics. Assuming the assay has 
worked as expected, the declining rate with increasing substrate concentration 
suggests to me that this substrate also acts as an allosteric inhibitor, so 
assays at high [substrate] will make it *look* like the unprocessed substrate 
is preferred even though the processed one is cleaved faster by the uninhibited 
enzyme.

Hope this helps,
Tristan

On 18 Jun 2021, at 05:05, Prem Prakash 
mailto:prem...@gmail.com>> wrote:

Dear all,
Sorry for this off topic. I am working on an enzyme that has an exonuclease 
activity. The enzyme preferentially cleaves an unprocessed substrate at a 
faster rate than the processed one (known by qualitative analysis). Recently, I 
calculated the Vmax, Km and kcat of the enzyme for unprocessed substrate which 
are 18.2 pmol/min, 182 nM and 7.1 sec-1 respectively. However, the Processed 
substrate has apparently a lower range of Km (not calculated) as reflected from 
the curve (because the same increasing concentration range which is used for 
unprocessed, shows a steep decline in the initial velocity of the enzyme with 
processed substrate.
The latter suggests that Km is way lower than expected. In this case, the 
question is, if the Km of processed substrate is way lower than the 
Unprocessed, how can we see a faster rate with the former substrate than later. 
i.e lower Km and slower rate of cleavage. If it's possible please give some 
insights. I have attached the plot comparison between two kinetic assays.

With kind regards,

Prem





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