{"title":"On the free energy of protein folding in optical tweezers experiments.","authors":"Christian A M Wilson, Camila G Corrêa","doi":"10.1007/s12551-025-01310-0","DOIUrl":null,"url":null,"abstract":"<p><p>Free energy is a critical parameter in understanding the equilibrium in chemical reactions. It enables us to determine the equilibrium proportion between the different species in the reaction and to predict in which direction the reaction will proceed if a change is performed in the system. Historically, to calculate this value, bulk experiments were performed where a parameter was altered at a gradual rate to change the population until a new equilibrium was established. In protein folding studies, it is common to vary the temperature or chaotropic agents in order to change the population and then to extrapolate to physiological conditions. Such experiments were time-consuming due to the necessity of ensuring equilibrium and reversibility. Techniques of single-molecule manipulation, such as optical/magnetic tweezers and atomic force microscopy, permit the direct measurement of the work performed by a protein undergoing unfolding/refolding at particular forces. Also, with the development of non-equilibrium free energy theorems (Jarzynski equality, Crooks fluctuation theorem, Bennett acceptance ratio, and overlapping method), it is possible to obtain free energy values in experiments far from equilibrium. This review compares different methodologies and their application in optical tweezers. Interestingly, in many proteins, discrepancies in free energy values obtained through different methods suggest additional complexities in the folding pathway, possibly involving intermediate states such as the molten globule. Further studies are needed to confirm their presence and significance.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1007/s12551-025-01310-0.</p>","PeriodicalId":9094,"journal":{"name":"Biophysical reviews","volume":"17 2","pages":"231-245"},"PeriodicalIF":4.9000,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12075763/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biophysical reviews","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1007/s12551-025-01310-0","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/4/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"BIOPHYSICS","Score":null,"Total":0}
引用次数: 0
Abstract
Free energy is a critical parameter in understanding the equilibrium in chemical reactions. It enables us to determine the equilibrium proportion between the different species in the reaction and to predict in which direction the reaction will proceed if a change is performed in the system. Historically, to calculate this value, bulk experiments were performed where a parameter was altered at a gradual rate to change the population until a new equilibrium was established. In protein folding studies, it is common to vary the temperature or chaotropic agents in order to change the population and then to extrapolate to physiological conditions. Such experiments were time-consuming due to the necessity of ensuring equilibrium and reversibility. Techniques of single-molecule manipulation, such as optical/magnetic tweezers and atomic force microscopy, permit the direct measurement of the work performed by a protein undergoing unfolding/refolding at particular forces. Also, with the development of non-equilibrium free energy theorems (Jarzynski equality, Crooks fluctuation theorem, Bennett acceptance ratio, and overlapping method), it is possible to obtain free energy values in experiments far from equilibrium. This review compares different methodologies and their application in optical tweezers. Interestingly, in many proteins, discrepancies in free energy values obtained through different methods suggest additional complexities in the folding pathway, possibly involving intermediate states such as the molten globule. Further studies are needed to confirm their presence and significance.
Supplementary information: The online version contains supplementary material available at 10.1007/s12551-025-01310-0.
期刊介绍:
Biophysical Reviews aims to publish critical and timely reviews from key figures in the field of biophysics. The bulk of the reviews that are currently published are from invited authors, but the journal is also open for non-solicited reviews. Interested authors are encouraged to discuss the possibility of contributing a review with the Editor-in-Chief prior to submission. Through publishing reviews on biophysics, the editors of the journal hope to illustrate the great power and potential of physical techniques in the biological sciences, they aim to stimulate the discussion and promote further research and would like to educate and enthuse basic researcher scientists and students of biophysics. Biophysical Reviews covers the entire field of biophysics, generally defined as the science of describing and defining biological phenomenon using the concepts and the techniques of physics. This includes but is not limited by such areas as: - Bioinformatics - Biophysical methods and instrumentation - Medical biophysics - Biosystems - Cell biophysics and organization - Macromolecules: dynamics, structures and interactions - Single molecule biophysics - Membrane biophysics, channels and transportation