A PHP Error was encountered

Severity: Warning

Message: file_get_contents(https://...@gmail.com&api_key=61f08fa0b96a73de8c900d749fcb997acc09): Failed to open stream: HTTP request failed! HTTP/1.1 429 Too Many Requests

Filename: helpers/my_audit_helper.php

Line Number: 143

Backtrace:

File: /var/www/html/application/helpers/my_audit_helper.php
Line: 143
Function: file_get_contents

File: /var/www/html/application/helpers/my_audit_helper.php
Line: 209
Function: simplexml_load_file_from_url

File: /var/www/html/application/helpers/my_audit_helper.php
Line: 994
Function: getPubMedXML

File: /var/www/html/application/helpers/my_audit_helper.php
Line: 3134
Function: GetPubMedArticleOutput_2016

File: /var/www/html/application/controllers/Detail.php
Line: 574
Function: pubMedSearch_Global

File: /var/www/html/application/controllers/Detail.php
Line: 488
Function: pubMedGetRelatedKeyword

File: /var/www/html/index.php
Line: 316
Function: require_once

An optimized purification protocol for enzymatically synthesized S-adenosyl-L-methionine (SAM) for applications in solution state infrared spectroscopic studies. | LitMetric

AI Article Synopsis

  • S-adenosyl-L-methionine (SAM) is crucial for various biological functions, particularly as a substrate for methyltransferases that influence gene expression and metabolism.
  • Designing selective small molecule inhibitors for methyltransferases remains challenging due to off-target effects, but understanding SAM's structural changes in different environments can help improve inhibitor specificity.
  • The study introduces a revised purification method for isolating isotopically labeled CD-SAM, minimizing contaminants that interfere with infrared spectroscopy, thus aiding in determining the specific atomic movements within SAM during enzymatic processes.

Article Abstract

S-adenosyl-L-methionine (SAM) is an abundant biomolecule used by methyltransferases to regulate a wide range of essential cellular processes such as gene expression, cell signaling, protein functions, and metabolism. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off-target effects. Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between several states when free in solution. Infrared spectroscopy can detect different conformers of molecules if present in appreciable populations. When SAM is noncovalently bound within enzyme active sites, the nature and the number of different conformations of the molecule are likely to be altered from when it is free in solution. If there are unique structures or different numbers of conformers between different methyltransferase active sites, solution-state information may provide promising structural leads to increase inhibitor specificity for a particular methyltransferase. Toward this goal, frequencies measured in SAM's infrared spectra must be assigned to the motions of specific atoms via isotope incorporation at discrete positions. The incorporation of isotopes into SAM's structure can be accomplished via an established enzymatic synthesis using isotopically labeled precursors. However, published protocols produced an intense and highly variable IR signal which overlapped with many of the signals from SAM rendering comparison between isotopes challenging. We observed this intense absorption to be from co-purifying salts and the SAM counterion, producing a strong, broad signal at 1100 cm. Here, we report a revised SAM purification protocol that mitigates the contaminating salts and present the first IR spectra of isotopically labeled CD-SAM. These results provide a foundation for isotopic labeling experiments of SAM that will define which atoms participate in individual molecular vibrations, as a means to detect specific molecular conformations.

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.saa.2023.123816DOI Listing

Publication Analysis

Top Keywords

purification protocol
8
sam
8
s-adenosyl-l-methionine sam
8
free solution
8
active sites
8
isotopically labeled
8
optimized purification
4
protocol enzymatically
4
enzymatically synthesized
4
synthesized s-adenosyl-l-methionine
4

Similar Publications

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!