BCHM 317/ 310 - Experiment 11

Lab Report Instructions and Marking Rubric 2022

Please ensure you have read the document "BCHM 317/310_Guidelines_and_FAQs" posted on OnQ. These instructions are meant to supplement the information contained in that document and provide some notes that are specific to Experiment 11.

Titl e Page

•    Do not include names. Include the student IDs of you and your partner.

•    Include the report number, experiment name, section/group, and date of submission.

O bjectives [1 mark ]:

•    250 character limit, roughly 2-3 sentences.

•   A brief statement detailing the experiments that were performed and the purpose of doing so.

•    Include descriptions of data collection methodologies, names of proteins, etc.

•    Do not mention specific details regarding results achieved or methodology.

Materials and Methods [ 0.5 marks]:

•    400 character limit.

•    Reference the lab manual with an in-text citation.

•    State any deviations made from the protocol.

•    If you and your partner collaborated with another pair, list their student #'s here.

Results [4 marks]:

•    4000 character limit, roughly 2.5 pages.

•    Here, you will walk the reader through a series of figures and tables, connecting figures/tables through bodies of text that highlight and compare important trends and values.

•    For each figure and table, you must:

1. Introduce the figure/table before it is presented.

2. State how the figure was obtained (without overtly detailing materials and methods).

3 . Highlight/compare important trends/values.

4 . Present the figure/table and its respective title/caption.

• Body of text [1.5]

• Specific to experiment 11:

• Figure 1 - Sequence alignment [0.25]

•   present a multiple sequence alignment of four class A β-lactamases (TEM- 1, KPC-2, Bla- 1, SHV- 1) and four class D β- lactamases (OXA-48, OXA-24/40, OXA-23, OXA-51)

•    annotate the figure to highlight regions of interest (the loop-helix region (residues 98- 114 for KPC-2), residues important for the β-lactamase reaction mechanism,   your assigned mutation, and anything else you feel is relevant)

• Figure 2 – PyMOL images comparing the global folds of KPC-2 and OXA-48 [0.5]

•    This will be a multipart figure consisting of:

•    A. An image of the overall KPC-2 structure (PDB: 5UL8)

•    B. An image of the overall OXA-48 structure (PDB: 3HBR)

•    C. The aligned structures showing regions of the structures that differ

•    Show the two structures overlaid – use colour to indicate the regions that are not aligned by PyMOL

• Figure 3 – PyMOL images of the KPC-2 and OXA-48 active sites [0.5]

•    This will be a multipart figure consisting of:

•    A. KPC-2 active site bound to hydrolyzed cefotaxime (PDB: 5UJ3)

•    B. OXA-48 active site bound to hydrolyzed cefotaxime (PDB: 6PQI)

•    C. Tabulate interactions between each sidechain and its antibiotic substrate

•    For Figure 3 A and B, only show relevant sidechains and display their interactions with the hydrolyzed cefotaxime

• Figure 4 – Mutational analysis of KPC-2 (PDB: 5UJ3) [0.5]

•    This will be a multipart figure consisting of:

•    A. Environment of the wild-type residue

•    B. Environment of the mutant residue

•    For Figure 4 A and B, only show relevant sidechains and interactions. Display interactions with the substrate when present.

• Figure 5 – PyMOL images of the β-lactamase-βLIP-II interface [0.5]

•    This will be a multipart figure consisting of:

•    A. Images of the TEM- 1-βLIP-II interface (PDB: 1JTD)

•    illustrate how the inhibitory loop of βLIP-II (residues 50-57) occupies the TEM- 1 active site

•    colour the inhibitory loop a separate colour and show its sidechains

•    display TEM- 1 as a surface representation to show the topology of the active site

•    B. Images of KPC-2 (5UJ3) aligned onto TEM- 1 in the TEM- 1-βLIP-II structure

•    illustrate why KPC-2 is highly compatible with the βLIP-II interface

•    colour the inhibitory loop of βLIP-II (residues 50-57) a separate colour

•    color the loop-helix regions of TEM- 1 (residues 98- 114) and KPC-2 (residues 98- 114) separate colors

•    C. Images of OXA-48 (6PQI) aligned onto TEM- 1 in the TEM- 1-βLIP-II structure

•    illustrate why OXA-48 is less compatible with the βLIP-II interface

•    colour the inhibitory loop of βLIP-II (residues 50-57) a separate colour

•    color the loop-helix regions of TEM- 1 (residues 98- 114) and OXA-48 (residues 96- 107) separate colors

Discussion [ 3 marks]:

•    7000 character limit, roughly 4 pages

•   Using the suggested subheadings , compartmentalize your discussion based on each major portion of the experiment

•   You do not need to strictly adhere to the order of discussion prompts, but ensure each point is addressed somewhere in your discussion.

Conservation between class A and D serine β -lactamases [1.0 mark]

•    Refer the reader back to Fig. 1 and Fig. 2 using in-text citations.

•    Explain the theories underlying multiple sequence alignments and structural alignments.

•    Describe where class A and D β-lactamases demonstrate sequence and structural similarities, and where they diverge.

•    When is it valuable to use sequence and structural alignments, respectively? Explain using examples from Fig. 1 and Fig. 2.

Mechanistic overview of β- lactamases [1.0 mark ]

•    Refer the reader back to Fig. 3 and Fig. 4 using in-text citations.

•    Compare and contrast the proposed enzymatic mechanisms for class A and D β - lactamases.

•    Describe in detail the evidence you observe for these mechanisms from Fig. 3.

•    KPC-2 and OXA-48 both have broad substrate profiles. Based on your results in Fig. 3, as well as published literature, what other classes of β-lactams are these enzymes capable of hydrolyzing?    Explain your answers in detail.

•    How did your simulated mutation affect the active site chemistry of KPC-2? Does this agree with the published data pertaining to your assigned mutation (see: Ian M. Furey et al. (2021) Journal of Biological Chemistry , 296 100799).

Structural interrogation of the β LIP-II-β-lactamase interface [1.0 mark]

•    Refer the reader back to Fig. 5 using an in-text citation.

•    Explain how βLIP-II inhibits the activity of class-A β- lactamases such as TEM- 1. Rationalize, based on your structural data, why β LIP-II is such a remarkably strong inhibitor.

•   Using your Fig. 1 sequence alignment as a reference, describe where the β LIP-II-binding         interface of KPC-2 and TEM- 1 is similar and where it diverges. Speculate how any sequence  changes would impact the binding of βLIP-II to KPC-2. Do these finding agree with published literature regarding KPC-2 inhibition by βLIP-II?

•    Speculate whether βLIP-II would be an effective inhibitor of OXA-48. Reinforce your claims by referencing the multiple sequence alignment in Fig. 1 (specifically the loop-helix region).

•    If you have performed Experiment 10, do these structural results agree with your kinetic data for KPC-2 and OXA-48 inhibition by β LIP-II? Rationalize your results.

C onclusion [1.0 mark ]:

•    750 character limit

•    State whether you achieved the experimental objectives

•    If you didn't achieve what you set out to do, what were the main differences?

•    Give a brief summary of the most important values/trends obtained.

•    How do your results compare to literature values?

•    Character count permitting, suggest future directions.

References [0 .5]:

•    Format your references and in-text citations in the style of JBC