The Role of Conformational Strains in Enzyme Catalysis

Mahaa Albusharif Author

08/03/2020 Added

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[00:00:02.250]Hello, my name is Maha Albusharif.
[00:00:04.470]I'll be presenting on the role
[00:00:05.820]of conformational strain in enzyme catalysis.
[00:00:08.750]I work with Dr. Mark Wilson in the Department
[00:00:10.850]of Biochemistry at the University of Nebraska-Lincoln.
[00:00:14.171]Extra crystallography is a structure determination tool.
[00:00:18.110]It's a technique used to determine the atomic
[00:00:21.150]and molecular structure of a crystal.
[00:00:23.150]And we use it to solve the structures of proteins
[00:00:25.721]and to determine functions of residues,
[00:00:28.993]observe interactions with substrates
[00:00:31.330]and interactions with other proteins or nucleic acids.
[00:00:34.770]And serial crystallography is a new technique
[00:00:37.690]to time resolved crystallography.
[00:00:39.486]It was impossible for us to look
[00:00:41.440]at a protein though is actually turning over, which means
[00:00:43.770]that it's going through a continuous cycle of catalysis.
[00:00:46.412]And serial crystallography can be used
[00:00:52.060]to observe time deepen the X-ray diffraction
[00:00:54.580]using brief pulses of X-rays generated
[00:00:58.380]at a free electron lasers.
[00:00:59.763]And the main problem with X-ray crystallography
[00:01:02.680]is that causes radiation damage to the crystal.
[00:01:05.251]And serial crystallography is different
[00:01:09.270]because we have many crystals and they're very, very tiny,
[00:01:12.220]and each crystal is only exposed once.
[00:01:14.460]So there's no opportunity for the accumulation
[00:01:16.850]of radiation damage to happen, which has been a problem
[00:01:20.570]for these types of these types of experiments.
[00:01:23.261]The protein that we started here is Isocyanide hydratase,
[00:01:26.560]also known as ICH.
[00:01:28.500]which is one of the few enzymes
[00:01:30.147]whose catalysis has been characterized in real time
[00:01:33.057]using mixed and inject zero crystallography,
[00:01:35.530]also known as misc.
[00:01:37.670]It's an antibiotic deviating enzyme.
[00:01:39.800]And from previous studies, we know that ICH
[00:01:42.910]from bacteria in between, and one is fluorescence
[00:01:45.500]that it showed a helix motion, your 16 active site.
[00:01:49.980]which is important during catalysis and Ralston ICH
[00:01:54.100]is the specific protein that we use in this research.
[00:01:56.620]It's a newly characterized member of the ICH family.
[00:02:00.750]It diffracts x-ray to an exceptional,
[00:02:02.870]0.74 angstrom resolution.
[00:02:05.270]This is a very, ultra high resolution crystal structure
[00:02:08.640]of all Sonia.
[00:02:09.473]which reveals several unusually strange amino acid
[00:02:12.890]side chains that cluster unimportant parts of the structure.
[00:02:15.961]And most proteins diffractive
[00:02:17.478]are two angstrom resolution on average,
[00:02:19.790]and ours is 0.74 angstrom resolution.
[00:02:22.560]So this is a better resolution because we get to see
[00:02:24.840]everything in detail that is unusual
[00:02:27.110]for a macro molecular crystal structure.
[00:02:30.900]And these deviations from ponarity are wolves
[00:02:34.060]are most of them are well determined by the data.
[00:02:36.255]And therefore residue geometry is confidentially known,
[00:02:39.820]but the causes of these strands are still unknown.
[00:02:42.527]So just a quick overview
[00:02:47.177]of the methodology.
[00:02:49.210]We determined the structure of all Sonia I C H to 0.7
[00:02:52.779]angstrom resolution resolution
[00:02:54.920]using a synchrotron dataset collected from a crystal
[00:02:57.740]at 100 degrees, kelvin.
[00:02:59.194]At the Stanford synchrotron radiation light source
[00:03:03.810]also know as SSRL.
[00:03:05.827]And the data was then collected using shorter list
[00:03:08.280]data collection, which is a developed way
[00:03:10.580]for high speed, precise data collection
[00:03:12.430]and protein crystallography.
[00:03:13.902]And then it was solved using molecular replacement,
[00:03:17.350]which is a method used to solve face problems,
[00:03:19.592]in extra crystallography.
[00:03:23.520]Then it was auto built, using CCB4.
[00:03:27.190]it was then manually improved using coot,
[00:03:29.208]and then it was refined.
[00:03:32.870]We're using ref Mac, which is a refinement program
[00:03:35.210]that incorporates various tools
[00:03:36.618]to do with some crystal abnormalities.
[00:03:39.740]And right here, the first table and x-ray diffraction,
[00:03:42.960]data statistics, and the data about it shows,
[00:03:46.237]the quality of the data,
[00:03:48.290]which is exceptionally high resolution.
[00:03:50.200]which is 0.74 angstrom resolution.
[00:03:52.209]And there in the resolution cutoff was determined
[00:03:55.330]using the correlation coefficient for the data
[00:03:58.520]for, sorry, the correlation coefficient for one half
[00:04:01.740]of the data, which shows the correlation between
[00:04:04.520]a reflection in one half of the data
[00:04:06.840]and its counterpart in the other half of the data,
[00:04:10.800]In the next table, the model statistics.
[00:04:13.437]The most important part of that table
[00:04:15.060]is the, our free volume, which to 0.122,
[00:04:17.849]indicating the agreement between the model
[00:04:20.520]and the experimental data.
[00:04:24.290]That we have excluded from the refinement optimization.
[00:04:27.560]Which is high.
[00:04:30.130]And more pre more privity was used to validate the geometry
[00:04:34.280]of the model and indicate generally good agreement
[00:04:37.040]with the dictionary values.
[00:04:39.220]So one of the things that, the first thing that we actually
[00:04:41.830]observed in the Protein structure is that, it has three
[00:04:45.220]outliers.
[00:04:46.810]We observed that using the Ramachandran plot,
[00:04:49.738]as you can see right here, a, B and C.
[00:04:52.500]A's isoleucin B is aspartic and C is alanine.
[00:04:56.639]As you can see here in A and B the electron density,
[00:05:00.280]which is the blue mesh is well determined by the data.
[00:05:02.810]There aren't any orders or weird,
[00:05:06.290]structure right here, or weird electron densities
[00:05:09.440]right here.
[00:05:10.273]But comparing that to C you see how the electron density
[00:05:13.320]is disordered.
[00:05:15.060]You have red blobs, green blobs indicating
[00:05:17.870]that there's something wrong here that needs to be further,
[00:05:20.410]that needs to be investigated and needs to be fixed.
[00:05:23.780]We also observed, residue, or we observed,
[00:05:27.920]ponarity deviation in the structure.
[00:05:31.770]And we observed that using, the look by looking at triple
[00:05:34.800]defined 85, and we're focused on the aromatic ring
[00:05:37.560]right here.
[00:05:38.850]This is a top view of the aromatic ring .
[00:05:42.210]As You can see the aromatic ring is actually bent.
[00:05:44.880]which is very, very unusual because,
[00:05:48.620]all aromatic ones are supposed to be flat
[00:05:51.060]because of the pybon conjugation
[00:05:52.790]and the electron delocalisation.
[00:05:54.790]They required, the planarity to line up those P orbitals
[00:05:58.350]in order for them to share electrons.
[00:06:01.220]These are also right here.
[00:06:02.820]These are extra or additional,
[00:06:05.614]examples of strained residues.
[00:06:08.810]We have 163 tyrosine we observed two confirmations
[00:06:12.750]of the aromatic ring, and the Z score is 5.09.
[00:06:16.300]We also observed a strained,
[00:06:19.770]aromatic ring in tyrosine right here, which is also a bent,
[00:06:23.606]just like the trick to find 85.
[00:06:25.990]And the D score is 6.38,
[00:06:29.050]and the Z score is the number of standard deviation
[00:06:31.880]and the residue perimeter that differs
[00:06:33.630]from its average mean.
[00:06:35.370]It is calculated by subtracting the value we observed
[00:06:38.190]and expected mean value divided by the standard deviation.
[00:06:41.970]And if the Z score is a big number,
[00:06:43.930]so five, six, seven, or eight, it is considered a serious
[00:06:47.160]outlier cause it's far from the normal distribution,
[00:06:49.460]which is about two.
[00:06:50.810]And if it is supported by the electron density,
[00:06:53.110]then it's meaningful and important.
[00:06:56.780]So in conclusion, we observed, three outliers,
[00:07:00.640]so much on a plot two of which were well determined
[00:07:03.540]by the data, one of them showed a disorder
[00:07:05.450]in its electron density.
[00:07:07.010]We also observed a ponarity deviation,
[00:07:09.540]multiple residues of Ralston, in the ICH protein structure,
[00:07:14.030]and the exceptionally high resolution data collection
[00:07:16.730]allows us, or data allows us to test whether strain residues
[00:07:20.610]like to defend 85.
[00:07:22.370]Changed their catalysis in a mess experiment.
[00:07:25.000]And residue clusters around Ralston ICH show,
[00:07:28.610]geometric strains indicating relevance of functionally
[00:07:31.310]important motions during catalysis.
[00:07:34.230]Moving forward, we would like to determine
[00:07:35.724]the type of social environments that caused these aromatic
[00:07:39.030]residues to bend and caused ponarity deviation.
[00:07:43.460]We would also like to characterize non-equilibrium,
[00:07:47.540]during catalysis or non-equilibrium motions during catalysis
[00:07:51.300]by ICH using serial crystallography.
[00:07:54.480]Finally I would like to acknowledge dr. Mark Wilson,
[00:07:58.640]which is my UNL faculty mentor,
[00:07:59.473]UNL McNair scholars program.
[00:08:03.110]Dr. Maganjoli Disputo which was a PhD student
[00:08:06.090]that worked on the data collection,
[00:08:09.970]for us on ICH Stanford synchrotron radiation,
[00:08:13.310]light source, as well as dr. Anna Cohen.
[00:08:15.873]Thank you.

The Role of Conformational Strains in Enzyme Catalysis

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The Role of Conformational Strains in Enzyme Catalysis

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