{"title":"Time-resolved IR spectroscopy for monitoring protein dynamics in microcrystals.","authors":"Wataru Sato, Daichi Yamada, Minoru Kubo","doi":"10.1016/bs.mie.2024.10.006","DOIUrl":null,"url":null,"abstract":"<p><p>Analysis of protein dynamics is crucial for understanding the molecular mechanisms underlying protein function. To gain insights into the structural changes in proteins, time-resolved X-ray crystallography has been greatly advanced by the development of X-ray free-electron lasers. This tool has the potential to trace structural changes at atomic resolution; however, data interpretation and extrapolation to the solution state is often not straightforward as the in crystallo environment is not the same as it is in solution. On the other hand, time-resolved spectroscopy techniques, which have long been used for tracking protein dynamics, offer the advantage of being applicable irrespective of whether the target proteins are in crystalline or solution phase. Time-resolved IR spectroscopy is a particularly powerful technique, as it can be used on various proteins, including those that are colorless, and provides information on the chemical structures of functional sites of proteins and ligands which complements X-ray crystallography. This chapter presents the protocol for time-resolved IR microspectroscopic measurements of protein microcrystals. It includes an overview of the measurement system assembly, sample preparation, setting of experimental conditions, and time-resolved data analysis. It also describes, with examples, the usefulness of time-resolved IR measurements for comparing the dynamics between crystalline and solution conditions.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"709 ","pages":"161-176"},"PeriodicalIF":0.0000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in enzymology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mie.2024.10.006","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/10/22 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
Abstract
Analysis of protein dynamics is crucial for understanding the molecular mechanisms underlying protein function. To gain insights into the structural changes in proteins, time-resolved X-ray crystallography has been greatly advanced by the development of X-ray free-electron lasers. This tool has the potential to trace structural changes at atomic resolution; however, data interpretation and extrapolation to the solution state is often not straightforward as the in crystallo environment is not the same as it is in solution. On the other hand, time-resolved spectroscopy techniques, which have long been used for tracking protein dynamics, offer the advantage of being applicable irrespective of whether the target proteins are in crystalline or solution phase. Time-resolved IR spectroscopy is a particularly powerful technique, as it can be used on various proteins, including those that are colorless, and provides information on the chemical structures of functional sites of proteins and ligands which complements X-ray crystallography. This chapter presents the protocol for time-resolved IR microspectroscopic measurements of protein microcrystals. It includes an overview of the measurement system assembly, sample preparation, setting of experimental conditions, and time-resolved data analysis. It also describes, with examples, the usefulness of time-resolved IR measurements for comparing the dynamics between crystalline and solution conditions.
期刊介绍:
The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.